NZ760654B2 - Compositions comprising a bacterial strain of the genus megaspahera and uses thereof - Google Patents

Abstract

The invention provides compositions comprising bacterial strains, in particular Megasphaera massiliensis (strain NCIMB 42787) for treating and preventing a neurodegenerative disorder or brain injury. Neurodegenerative disorders to be treated include Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration. Brain injuries to be treated include stroke, such as cerebral ischemia, focal cerebral ischemia, ischemic stroke or hemorrhagic stroke. clear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration. Brain injuries to be treated include stroke, such as cerebral ischemia, focal cerebral ischemia, ischemic stroke or hemorrhagic stroke.

Description

COMPOSITIONS COMPRISING A BACTERIAL STRAIN OF THE GENUS HERA AND USES THEREOF TECHNICAL FIELD This invention is in the field of compositions sing bacterial strains isolated from the mammalian digestive tract and the use of such compositions in the treatment of disease.

BACKGROUND TO THE INVENTION The human intestine is thought to be e in utem, but it is exposed to a large y of al and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1]. The human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2]. The successful symbiotic relationships g from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial ons. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of ducts providing an important nutrient source for the host. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an ed immune system that is functionally reconstituted ing the introduction of commensal bacteria [3-5].

Dramatic s in microbiota composition have been documented in intestinal ers such as inflammatory bowel disease (IBD). For example, the levels of Clostridium cluster XIVa bacteria are d in IBD patients whilst numbers of E. 6012' are increased, suggesting a shift in the e of symbionts and pathobionts within the gut [6-9].

In recognition of the potential positive effect that certain bacterial strains may have on the animal gut, s strains have been proposed for use in the treatment of various diseases (see, for example, [10- 13]). Also, certain strains, ing mostly Lactobacillus and Bifidobacterium strains, have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [14] and [15] for reviews). However, the relationship between different diseases and different bacterial strains, and the precise effects of ular bacterial strains on the gut and at a systemic level and on any particular types of diseases are poorly characterised, particularly for neurodegenerative disorders.

Recently, there has been increased interest in the art regarding alterations in the gut microbiome that may play a hysiological role in human brain diseases [16]. Preclinical and clinical evidence are strongly suggesting a link between brain development and microbiota [17]. A growing body of nical literature has demonstrated bidirectional signalling between the brain and the gut microbiome, involving multiple neurocrine and endocrine signalling systems. Indeed, increased levels of Clostridium species in the microbiome have been linked to brain disorders 18], and an imbalance of the Bacteroidetes and Firmicutes phyla has also been implicated in brain development disorders [19]. Suggestions that altered levels of gut commensals, including those of Bifidobacterium, Lactobacillus, Sutterella, Prevotella and Ruminococcus genera and of the genaceae family are ed in immune-mediated central nervous system (CNS) disorders, are questioned by studies suggesting a lack of alteration in the microbiota between patients and healthy subjects [19]. There have also been suggestions that the administration of probiotics may be cial in the treatment of neurological disorders. r, these studies failed to conclude that probiotic compositions per se can achieve therapeutic benefits with respect to the treatment of neurodegeneration and did not show any useful effects for any particular bacteria [20, 21]. This tes that, at present, the practical effect of the link between the microbiome and human brain diseases is poorly characterised.

Accordingly, more direct analytical studies are required to identify the therapeutic impact of altering the microbiome on neurodegenerative disorders.

There is a requirement in the art for new methods of treating neurodegenerative disorders. There is also a requirement for the potential s of gut bacteria to be characterised so that new ies using gut bacteria can be developed.

SUMMARY OF THE INVENTION The inventors have developed new therapies for treating and preventing neurodegenerative ers. The inventors have identified that bacterial s from the genus Megasphaera may be effective for treating neurodegenerative diseases. As described in the examples, administration of itions comprising Megasphaera massiliensis can protect against reactive oxygen species and prevent inflammation, thus acting as a neuroprotectant. The inventors have also identified that treatment with Megasphaera massiliensis can reduce the activation of proinflammatory molecules, such as NFκB and IL-6, by LPS and mutant clein. The inventors have fied that treatment with Megasphaera massiliensis can reduce histone deacetylation activity and lipid peroxidation in vitro, which can help to reduce cell death and apoptosis. The inventors have also fied that Megasphaera massiliensis can produce indole that can attenuate inflammation and oxidative stress. Furthermore, the inventors have demonstrated that treatment with Megasphaera massiliensis can increase kynurenine levels.

The ors have also identified that Megasphaera massiliensis es certain organic acids including ic acid, valeric acid and 4-hydroxyphenylacetic acid. The inventors have also found that Megasphaera massiliensis can increase the activation of the flammatory cytokine IL-8, which can help to promote neuron ation. The inventors have also identified that ent with a combination of Megasphaera massiliensis and retinoic acid can increase the secretion of brain-derived neurotrophic factor (BDNF), which can help promote neurogenesis and neuritogenesis and/or prevent cell death. The inventors have also identified that treatment with Megasphaera massiliensis, which can produce valeric acid, can reduce histone deacetylation, which can help to reduce cell death and apoptosis. Furthermore, the inventors have also found that Megasphaera massiliensis can produce hexanoic acid, which can be neuroprotective or neurorestorative, for e by promoting neurite outgrowth. The inventors have found that Megasphaera massiliensis that can produce hexanoic acid increase the expression of MAP2 (Microtubule –associated protein 2), which is thought to be essential for ubule formation in neuritogenesis. Therefore, the inventors have found that Megasphaera massiliensis that can produce hexanoic acid can be used to promote neurite outgrowth. Megasphaera massiliensis and other bacteria that produce organic acids like hexanoic acid, valeric acid and 4- hydroxyphenylacetic acid may therefore be useful for treating neurodegenerative disorders.

Accordingly, in one aspect, the t invention es the use of a composition comprising a bacterial strain of the genus Megasphaera , wherein the bacterial strain has a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2, in the manufacture of a medicament for treating or preventing a neurodegenerative disorder in a patient.

In another aspect, the present invention provides the use of a composition comprising a bacterial strain of the genus Megasphaera , wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, identical to SEQ ID NO:2, in the manufacture of a medicament for treating a brain injury in a patient.

In another aspect, the present invention provides a cell of the Megasphaera massiliensis strain deposited under ion number NCIMB 42787.

In another aspect, the present invention provides a composition sing the cell of the invention.

In another aspect, the present invention provides a biologically pure culture of the haera massiliensis strain deposited under accession number NCIMB 42787.

In r aspect, the present invention provides the use of a cell of the Megasphaera massiliensis strain ted under accession number NCIMB 42787, in the cture of a medicament.

Disclosed herein is a composition comprising a bacterial strain of the genus Megasphaera , for use in therapy, such as for use in a method of treating or preventing a neurodegenerative disorder.

Also disclosed herein is a composition comprising a bacterial strain of the genus Megasphaera , for use in a method of ng or preventing a e or ion selected from the group consisting of: Parkinson’s e, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, ar or arteriosclerotic parkinsonism and drug-induced parkinsonism; mer’s disease, including 's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic l sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive a; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration.

Also disclosed herein is a composition sing a bacterial strain of the genus Megasphaera , for use in a method of treating or preventing Parkinson’s disease, such as environmental, familial or Parkinson’s associated with l inflammatory . The inventors have identified that treatment with Megasphaera strains can reduce the activation of proinflammatory molecules, such as NFκB and IL-6, by LPS and mutant α-synuclein in in vitro models of nmental and familial Parkinson’s. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Megasphaera massiliensis, for use in the treatment of Parkinson’s disease. Compositions using Megasphaera iensis may be particularly effective for treating Parkinson’s.

The compositions disclosed herein are for use in a method of treating or preventing early-onset neurodegenerative disease. The compositions disclosed herein are for use in a method of preventing or delaying onset or progression of a neurodegenerative disorder.

In preferred embodiments, the bacterial strain in the ition sed herein is of Megasphaera massiliensis. Closely related strains may also be used, such as bacterial strains that have a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% cal to the 16S rRNA sequence of a bacterial strain of haera massiliensis. Preferably, the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% cal to SEQ ID NO:1 or 2. Preferably, the sequence identity is to SEQ ID NO:2.

Preferably, the bacterial strain disclosed herein has the 16S rRNA sequence represented by SEQ ID NO:2.

In certain embodiments, the ition of the invention is for oral administration. Oral administration of the strains of the invention can be effective for egenerative disorders.

Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or total colonisation of the ine.

In certain embodiments, the composition of the invention comprises one or more pharmaceutically acceptable excipients or rs.

In certain embodiments, the composition of the invention comprises a bacterial strain that has been lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.

In certain embodiments, the disclosure provides a food product comprising the composition as described above.

In certain embodiments, the disclosure provides a vaccine composition comprising the composition as described above. 4A followed by page 5 Additionally, disclosed herein is a method of treating or preventing neurodegenerative disorders, comprising administering a composition comprising a ial strain of the genus Megasphaera.

In developing the above invention, the inventors have identified and characterised a bacterial strain that is particularly useful for therapy. The Megasphaera massiliensis strain of the invention is shown to be effective for treating the diseases described herein, such as neurodegenerative diseases. Therefore, in another aspect, the invention provides a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also es a cell of the haera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof, for use in y, in ular for the es described .

In certain embodiments, the composition disclosed herein may be for use in treating brain .

The neuroprotective activity of the compositions of the invention and their ability to reduce levels of histone deacetylase activity (HDAC) may make them useful for treating brain injury. In preferred ments, the compositions disclosed herein may be for use in treating stroke, such as treating brain injury resulting from a stroke.

BRIEF DESCRIPTION OF DRAWINGS Figure 1: Cell viability of neuroblastoma cells Figure 2: Down-regulation of IL-6 secretion [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Figure 3 Secretion of IL-8 Figure 4: Inhibition of or- synuclein IL-6 and IL-8 secretion Figure 5: Inhibition of or- ein induced NFKB promoter activation Figure 6: Inhibition of LPS induced NFKB promoter tion Figure 7: Change in antioxidant capacity Figure 8: Change in total anti-oxidant capacity (lipid oxidation) Figure 9: Change in histone ylase (HDAC) activity Figure 10: Level of Indole production Figure 11: Level of Kyrunenine production Figure 12: Mean Dopamine (DA) levels (Figure 12A), DOPAC levels (Figure 12B) and HVA levels (Figure 12C) in striatum. Data is displayed as Mean + SEM.

Figure 13: Promoting neurite outgrowth: light copy and MAP2 gene expression (Figure 13A), idin immunofluorescence microscopy (Figure 13B) Figure 14: Change in ROS levels in (a) U373 cells and (b) Y cells Figure 15: Neuroprotection — cell viability. Figure 15 shows the same data as Figure 1.

Figure 16 -induced changes in whole cell and cell lysate histone deacetylase activity (Figure 16A), acid-induced changes in histone deacetylase activity (Figure 16B), metabolite production by strains (Figure 16C) Figure 17 HDACl inhibition (Figure 17A), HDAC2 inhibition (Figure 1?B), HDAC3 inhibition e 17C) Figure 18 Inhibition of Class I HDACs (Figure 18A); inhibition of HDACl (Figure 18B); inhibition of HDAC2 e 18C); inhibition of HDAC3 (Figure 18D) Figure 19: Level of BDNF production Figure 20: Levels of metabolite production — neurotransmitters in the brain Figure 21: Levels of metabolite production — organic acids in the supernatant Figure 22: Effect on inal barrier function.

Figure 23: Production of neurotransmitters in the brain Figurn Changes in Hippocampal or Expression — A) Oxytocin Receptor, B) Vasopressin Receptor, C) Glucocorticoid Receptor and D) Mineralocorticoid Receptor [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Figure 25: Changes in Hippocampal sion of A) Corticotropin-Releasing Hormone (CRH), B) BDNF Expression and C) TLR4 Figure 26: A) Changes in Hippocampal Corticotropin Releasing Hormone Receptor 1 (CRFRl) Expression and B) Corticotropin Releasing Hormone Receptor 2 (CRFR2) Expression Figure 27: Changes in Hippocampal sion of A) Tumour Necrosis Factor, B) Interleukin lb and C) IL-6 Figure 28: A) s in Hippocampal Integrin Alpha M (CD1 lb) Expression and B) Changes in Hippocampal Serotonin lA Receptor (5-HT1A receptor) Expression Figure 29: A) Changes in Hippocampal ate Ionotropic Receptor NMDA Type Subunit 2A A) and B) Glutamate Ionotropic Receptor NMDA Type Subunit 2B (Grin2B) sion Figure 30: Changes in Hippocampal Expression of A) Gamma-Aminobutyric Acid A or 2 (GABA A2), B) Gamma-Aminobutyric Acid B Receptor 1 (GABA BRl) and C) Dopamine Receptor 1 (DRDl) Figure 31: Changes in Amygdala Receptor Expression — A) Oxytocin Receptor, B) Vasopressin Receptor, C) Glucocorticoid Receptor and D) Mineralocorticoid Receptor Figure 32: Changes in Amygdala Expression of A) Brain Derived Neurotrophic Factor (BDNF), B) Toll-like or 4 (TLR-4), C) Corticotropin Releasing e Receptor 1 (CRFRl) and D) Corticotropin Releasing Hormone Receptor 2 (CRFR2) Figure 33: Changes in Amygdala sion of A) Integrin Alpha M (CD1 lb), B) Interleukin-6 (IL- 6), C) Glutamate Ionotropic Receptor NMDA Type Subunit 2A (Grin2A) and D) Glutamate Ionotropic Receptor NMDA Type Subunit 2B (Grin2B) Figure 34: Changes in Amygdala Expression of A) GABA-A Receptor Alpha 2 Subunit (GABRA2), B) GABA-A Type B Receptor 1 Subunit (GABBRl) and C) Dopamine Receptor 1 (DRDl) Figure 35: s in Prefrontal Cortex Expression of A) Oxytocin Receptor, B) Brain Derived Neurotrophic Factor , C) Mineralocorticoid Receptor and D) Glucocorticoid Receptor Figure 36: Changes in Prefrontal Cortex Expression of A) ike Receptor 4 (TLR-4), B) Corticotropin Releasing e Receptor 1 (CRFRl), C) Corticotropin Releasing e Receptor 2 (CRFR2) and D) Integrin Alpha M (CD1 1b) Figure 37: Changes in Prefrontal Cortex Expression of A) Interleukin-6 (IL-6), B) Glutamate Ionotropic Receptor NMDA Type Subunit 2A (Grin2A), C) Glutamate opic Receptor NMDA Type Subunit 2B (Grin2B) and D) GABA-A Receptor Alpha 2 Subunit (GABRA2) [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Figure 38: Changes in Prefrontal Cortex Expression of A) GABA-A Receptor Type B Receptor t 1 (GABBRl) and B) Dopamine Receptor 1 (DRDl) Figure 39: Changes in Colon Expression of A) Tryptophan Hydroxylase-l (Tphl) and B) Indoleamine2,3-Dioxygenase-1 (IDOl) Figure 40: s in Ileum sion of A) Tryptophan Hydroxylase-l (Tphl) and B) Indoleamine2,3-Dioxygenase-1 (IDOl) Figure 41: s in Circulating Tryptophan Metabolite Levels A) Kynurenine, B) Tryptophan and C) Kynurenine/ Tryptophan Index of metabolism Figure 42: Effect on Interferon-y Production from mouse Splenocytes from mice fed with MRx0029 Figure 43: Effect on Interleukin-1B Production from Splenocytes Figure 44: Effect on Interleukin-6 Production from cytes Figure 45: Effect on Tumour Necrosis Factor tion from Splenocytes Figure 46: Effect on Interleukin-10 Production from cytes Figure 47: Effect on Chemoattractant CXCLl Production from Splenocytes Figure 48: Changes in Caecal Short Chain Fatty Acid Levels Figure 49: MRx0029 and -induced changes in gene expression levels of Actin, Villin, Occludin TJPl, TJP2, MAP2, DRD2, GABRB3, SYP, PINKl, PARK7 and NSE.

Figure 50: SHSYSY cell differentiation induced by MRx0005 and MRx0029. (A-C) entative images of immuno labelled cells with Phalloidin and MAP2. (D—F) images of A-C merged with DAPI images. (G—I) [33 tubulin immunolabelled cells. (J-L) merged with DAPI images.

Magnification x630. Western blot analysis of effects of MRx0005 and MRx0029 treatment on SHSYSY cells. Western blot membranes were probed with antibodies to MAP2 (M) and b3 tubulin (N). Actin was used as a loading control. Lower panels: representative blots from one of six separate experiments; upper panels: relative densitometric intensity.

DISCLOSURE OF THE INVENTION Bacterial strains The compositions ofthe invention comprise a bacterial strain ofthe genus Megasphaera. The examples demonstrate that bacteria of this genus are useful for treating or ting egenerative disordDThe preferred bacterial strains are of the species Megasphaera massiliensis.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm Examples of Megasphaem species for use in the invention include Megasphaera elsdem'z', Megasphaera cerevisiae, Megasphaera iensis, Megasphaera indica, Megasphaerapaucivorans, Megasphaera sueciensis and Megasphaera micronucz'formis. A r e of a Megasphaera species for use in the invention is Megasphaera hexanoica, The Megasphaera are obligately anaerobic, lactate-fermenting, gastrointestinal microbe of ruminant and non-ruminant mammals, including humans.

The type strain of M. massiliensis is NP3 (=CSUR P245=DSM 26228)[22]. The GenBank accession number for the 16S rRNA gene sequences of M. iensis strain NP3 is JX424772.1 (disclosed herein as SEQ ID N021).

The Megasphaera massiliensis bacterium tested in the Examples is referred to herein as strain MRx0029. A 16S rRNA sequence for the MRx0029 strain that was tested is provided in SEQ ID NO:2.

Strain MRx0029 was deposited with the ational tary authority NCIMB, Ltd. son Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Cornhill Road, Aberdeen, AB25 2ZS, Scotland) on 13th July 2017 as “Megasphaera massiliensis MRx0029” and was assigned accession number NCIMB 42787.

Bacterial strains closely related to the strain tested in the examples are also expected to be effective for treating or preventing neurodegenerative ers. In certain embodiments, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA ce of a bacterial strain ofMegasphaera massiliensis. Preferably, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID N021 or 2. ably, the sequence identity is to SEQ ID NO:2. Preferably, the bacterial strain for use in the invention has the 16S rRNA ce represented by SEQ ID N022.

Bacterial strains that are biotypes of strains MRx0029 or NP3 are also ed to be effective for treating or preventing neurodegenerative disorders. A biotype is a closely related strain that has the same or very r physiological and biochemical characteristics.

Strains that are es of strains MRx0029 or NP3 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for strains MRx0029 or NP3. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% ce identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive ces such as BOX, ERIC, (GTG)5, or REP or [23]. Biotype strains may have sequences with at least 95%, 96%, 97%, 0 99.5% or 99.9% sequence identity to the corresponding sequence of the strains , 99%, MRXOEOI‘ NP3.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Alternatively, strains that are biotypes of strains MRx0029 or NP3 and that are suitable for use in the invention may be identified by using strains MRx0029 or NP3 and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified nt length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein ng, or partial 168 or 23S rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Megasphaera massiliensis strains.

In n embodiments, strains that are biotypes of strains MRx0029 or NP3 and that are suitable for use in the invention are strains that provide the same n as strains MRx0029 or NP3 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for e when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example,[24]). Alternatively, biotype s are identified as strains that have the same carbohydrate fermentation patterns as strains MRx0029 or NP3.

Other Megasphaera strains that are useful in the itions and methods of the invention, such as biotypes of strains MRx0029 or NP3, may be identified using any appropriate method or strategy, including the assays described in the examples. For instance, strains for use in the invention may be identified by culturing with neuroblastoma cells and then assessing cytokine levels and levels of rotection or neuroproliferation. In particular, bacterial strains that have similar growth patterns, metabolic type and/or surface ns to strains MRx0029 or NP3 may be useful in the invention. A useful strain will have comparable immune modulatory activity to strains MRx0029 or NP3. In particular, a biotype strain will elicit able s on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols bed in the Examples.

A particularly preferred strain of the invention is the Megasphaera massiliensis MRx0029 . This is the exemplary strain tested in the examples and shown to be ive for treating disease. Therefore, the invention provides a cell, such as an isolated cell, of the Megasphaera massiliensis strain 9, or a derivative thereof. The invention also provides a composition comprising a cell of the Megasphaera iensis strain MRx0029, or a derivative thereof. The invention also provides a biologically pure culture of the Megasphaera massilz'ensis strain 9. The invention also provides a cell of the Megasphaera massiliensis strain MRx0029, or a derivative thereof, for use in therapy, in particular for the diseases bed herein.

A particularly preferred strain of the invention is the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. This is the exemplary MRx0029 strain tested in the examples and shown to be effective for treating disease. Therefore, the invention provides a cell, such as an isolated cell, of the haera massiliensis strain deposited under accession number NCIMB 42787, or a massilderivamthereof. The invention also provides a composition comprising a cell of the Megasphaerais strain deposited under ion number NCIMB 42787, or a derivative thereof. The [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ion also provides a biologically pure culture of the Megasphaera massilz'ensz's strain deposited under accession number NCIMB 42787. The invention also provides a cell of the Megasphaera massilz‘ensz's strain deposited under accession number NCIMB 4278?, or a derivative f, for use in therapy, in particular for the es described .

A derivative of the strain of the invention may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A derivative of a strain of the ion may be modified, for example at the genetic level, without ablating the biological activity. In particular, a tive strain of the ion is therapeutically active. A tive strain will have comparable therapeutic activity to the MRx0029 strain. In particular, a derivative strain will elicit able effects on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples. A derivative of the MRx0029 strain will generally be a biotype of the MRx0029 References to cells of the Megasphaera massilz'ensz's MRx0029 strain encompass any cells that have the same safety and therapeutic efficacy characteristics as the strain MRx0029, and such cells are encompassed by the invention.

In preferred embodiments, the bacterial strains in the compositions of the invention are viable and capable of partially or totally colonising the intestine.

The inventors have found that haera massiliensis strains reduce the activation of atory cytokines such as IL-6 and se the activation of the inflammatory cytokine IL-8. IL-8 has been implicated in myelin sheath formation [25]. Chronic inflammation induced by IL-6 can ultimately lead to cell death. Therefore, the ial strains of the ion are particularly useful in the treatment or prevention of neurodegenerative disorders. In some embodiments, the ial strains are useful in the ent of conditions characterised by the enhanced activation of IL-6. In some embodiments, the compositions of the invention are for use in the treatment or prevention of neurodegenerative es characterised by demyelination. Many neurodegenerative diseases are characterised by demyelination.

Demyelination impedes the propagation of action potentials within neurons, impairing effective communication within the nervous system. IL-8 has been shown to contribute positively to myelin sheath ion and repair. Therefore, the compositions of the invention are particularly beneficial in the treatment or prevention of neurodegenerative disorders characterised by demyelination, such as Multiple Sclerosis.

The inventors have found that the Megasphaera massiliensis strains of the invention alleviate symptoms of neurodegenerative diseases in models of the disease. For example, the inventors have found that the Megasphaera iensis strains promote neurite outgrowth in vitro, and may therefore be useapromoting neuron restoration for the treatment or prevention of neurodegenerative diseases.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Thus, bacterial strains of the ion are for use in the ent or prevention of neurodegenerative The ors have also found that the bacterial strains of invention increase the activation of BDNF.

BDNF is a neurotrophic growth factor that has been shown to enhance neuron differentiation and survival. Thus, the compositions of the invention can be used in a method of enhancing nerve cell survival in the treatment or prevention of neurodegenerative diseases.

A further bacteria that may be used in the compositions of the invention is the species Parabacteroides distasonis. The examples demonstrate that Parabacteroides distasom's and haera massilz'ensz's both have rotective activities, but produce different metabolites and may have different mechanisms of action and specific neuroprotective activities. Therefore, these species may be particularly ive when used in ation. In preferred embodiments, the composition comprises a strain ofthe species Parabacteroides distasonis and a strain ofthe species Megasphaera massilz'ensis.

The Parabacteroides distasonis bacterium deposited under accession number NCIMB 42382 was tested in the Examples and is also referred to herein as strain MRx0005. MRX0005, MRX005, MRx005 and MRx0005 are used herein interchangeably. A 16S rRNA sequence for the 5 strain that was tested is provided in SEQ ID NO:17. Strain 5 was deposited with the international depositary authority NCIMB, Ltd. son Building, Aberdeen, AB21 9YA, Scotland) by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 12th March 2015 as “Parabacteroides sp 755” and was assigned accession number NCIMB 42382. GT Biologics Ltd. Subsequently changed its name to 4D Pharma Research d.

In preferred embodiments, the invention provides a composition comprising the strain deposited at NCIMB under accession number NCIMB 42787, or a derivative or e f, and the strain deposited at NCIMB under accession number NCIMB 42382, or a tive or biotype thereof, preferably for use in therapy, preferably for use in treating a neurodegenerative disease such as Parkinson’s disease.

Therapeutic uses As demonstrated in the examples, the bacterial compositions of the invention are effective for treating egenerative disorders. In particular, treatment with compositions of the invention increase neuro-proliferation and act as a neuroprotectant against agents that destroy dopaminergic neurons.

Therefore, the compositions of the invention may be useful for ng or preventing neurodegenerative disorders that are the result of neuron death. itions of the invention can decrease the activation of the NFKB promoter, which activates cytokine production, for example IL-1 [3, IL-loc, IL-18, TNFoc and IL-6. Treating cells with mutant 0t- synucfis a model for familial Parkinson’s. A point mutation at on 53 from adenine to threonineleads -synuclein mis-folding. The incorrectly folded oc-synuclein subsequently aggregates into [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm insoluble fibrils which form Lewy bodies. Therefore, the itions of the invention may be useful for treating or preventing neurodegenerative disorders that are the result of neuroinflammation, protein misfolding and/or environmental exposure. Compositions of the invention can be used for treatment of familial Parkinson’s. Activation ofthe NFKB promoter is mediated through the TLR4 ligand. TLR4 is known to mediate cell death in the mouse model MPTP, which simulates Parkinson’s disease.

Compositions of the invention can be used to inhibit the ability of TLR4 signalling to activate the NFKB promoter. Of particular relevance for PD, both TLR2 and TLR4 were found to be upregulated in brains of PD patients [26]. Moreover (it-syn has been described as a ligand for TLR2 [27] and we have trated that a-syn is also a ligand for TLR4 using HEK-TLR4 cells [28]. itions of the invention decrease the secretion of pro-inflammatory cytokines such as IL-6, which can be induced by lysaccharide (LPS). Treatment of cells with LPS simulates Parkinson’s caused by environmental s. Compositions of the invention can be used to decrease IL-6 secretion.

Compositions of the invention can be used for treatment of environmental Parkinson’s.

Examples of neurodegenerative diseases to be treated by compositions of the invention include: Parkinson’s disease, including progressive uclear palsy, progressive uclear palsy, Steele- Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone e; prion e; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment, and corticobasal degeneration. A further disease to be treated by compositions of the invention is progressive inflammatory athy.

In certain embodiments, the compositions of the invention are for use in reducing neuron death, in particular, in the treatment of neurodegenerative disorders. In certain ments, the compositions of the ion are for use in protecting neurons, in ular in the treatment of neurodegenerative disorders.

In certain embodiments, the compositions of the invention are for use in reducing or ting loss of dopaminergic cells in the substantia nigra. In certain embodiments, the compositions of the invention are for use in reducing or ting the degeneration of dopaminergic neurons in the substantia nigra pars compacta. In certain ments, the itions of the invention are for use in reducing or preventing the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the consequent loss of their projecting nerve fibers in the striatum. In certain embodiments, the compositions of the invention are for use in reducing or preventing loss of nigrostriatal dopaminergic neurons.

In certaembodiments, the compositions of the invention are for use in increasing dopamine .

In certain embodiments, the compositions of the ion are for use in increasing DOPAC (3,4- [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Dihydroxyphenylacetic acid) . In certain embodiments, the compositions of the invention are for use in increasing dopamine and DOPAC levels. In certain ments, the ne and/or DOPAC levels are increased in the striatum. Dopamine and DOPAC levels may be measured using any appropriate method known in the art, such as a radioenzymatic , for example in plasma or CSF (for example as described in [29]), or a reverse-phase HPLC method, perhaps with electrochemical detection, for example in plasma or CSF (for e as described in [30]).

The neuroprotective properties of the compositions of the ion, as shown in the es, mean that the compositions may be particularly effective for preventing or delaying onset or progression of neurodegenerative disorders. In certain embodiments, the compositions of the invention are for use in delaying onset or ssion of neurodegenerative disorders.

Compositions of the invention can increase the secretion of IL-8. IL-8 has been shown to play a role in neuron myelination. In some ments, compositions of the invention can be used to increase IL-8 secretion.

The therapeutic compositions of the invention can increase the tion of BDNF. BDNF acts on certain neurons of the central nervous system to support the survival of existing neurons and help the growth and development of new neurons and synapses. BDNF is active in the hippocampus, cortex and basal forebrain, and is important for long-term memory. The compositions of the invention can therefore be used to increase the secretion of BDNF. The compositions may therefore be used in the treatment of neurodegenerative diseases associated with the impairment of long-term . The compositions of the invention may be used for improving long-term memory, in particular for improving long-term memory that is impaired by a neurodegenerative disease.

In certain embodiments, the compositions ofthe invention increase the mitochondria metabolic activity in neuronal cells.

Modulation ofthe microbiota-gut—brain axis Communication between the gut and the brain (the microbiota-gut-brain axis) occurs via a bidirectional neurohumoral communication system. Recent ce shows that the microbiota that resides in the gut can modulate brain pment and e behavioural phenotypes via the microbiota-gut-brain axis. Indeed, a number of s suggest a role of the microbiota-gut—brain axis in maintaining central nervous system functionality and implicate dysfunction of the microbiota-gut-brain axis in the development of central nervous system disorders and conditions [l6],[l9],[3 l].

The ctional communication between the brain and the gut (Le. the-gut-brain axis) includes the central nervous system, neuroendocrine and neuroimmune systems, including the alamus- pituitary-adrenal (HPA) axis, sympathetic and mpathetic arms of the autonomic nervous system (ANS'nluding the enteric nervous system (ENS) and the vagus nerve, and the gut microbiota.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm As demonstrated in the examples, the compositions of the present invention can te the microbiota—gut-brain axis and reduce cell death ated with neurodegenerative ers.

Accordingly, the compositions of the invention may be useful for treating or preventing neurodegenerative ers, in particular those disorders and conditions associated with dysfiinction of the microbiota—gut-brain axis.

In particular embodiments, the compositions of the invention may be useful for treating or preventing a disease or condition selected from the group consisting of: Parkinson’s disease, ing progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson—Olszewski syndrome, normal re hydrocephalus, vascular or osclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia; including Lewy body; vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; lated cognitive impairment and corticobasal degeneration.

The compositions of the invention may be particularly useful for treating or preventing chronic disease, treating or preventing disease in patients that have not responded to other therapies (such as treatment with Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, ate antagonists, and/or anticholinergics), and/or treating or preventing the tissue damage and symptoms associated with dysfunction of the microbiota—gut-brain axis.

In certain embodiments, the compositions of the invention modulate the CNS. In some embodiments, the compositions of the invention modulate the autonomic nervous system (ANS). In some ments, the compositions of the invention te the enteric nervous system (ENS). In some embodiments, the compositions of the invention te the hypothalamic, pituitary, adrenal (HPA) axis. In some embodiments, the compositions of the invention modulate the neuroendocrine pathway.

In some ments, the compositions of the invention modulate the neuroimmune pathway. In some embodiments, the compositions of the ion modulate the CNS, the ANS, the ENS, the HPA axis and/or the ndocrine and neuroimmune pathways. In certain embodiments, the compositions of the invention module the levels of commensal metabolites and/or the intestinal permeability of a subject.

The signalling of the microbiota—gut-brain axis is modulated by neural systems. Accordingly, in some embodiments, the compositions of the invention modulate signalling in neural systems. In certain embodiments, the compositions of the invention te the signalling of the l nervous system.

In some embodiments, the compositions of the ion modulate signalling in sensory neurons. In other embodiments, the compositions of the invention modulate signalling in motor neurons. In some embofints, the compositions of the invention modulate the signalling in the ANS. In some embo nts, the ANS is the parasympathetic s system. In preferred embodiments, the [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm compositions of the invention te the signalling of the vagus nerve. In other embodiments, the ANS is the sympathetic nervous system. In other embodiments, the compositions of the invention modulate the signalling in the enteric nervous system. In n embodiments, the signalling ofANS and ENS neurons responds directly to l contents of the gastrointestinal tract. In other embodiments, the signalling ofANS and ENS neurons responds indirectly to neurochemicals produced by luminal ia. In other embodiments, the signalling of ANS and ENS neurons responds to neurochemicals ed by luminal bacteria or enteroendocrine cells. In certain preferred embodiments, the neurons ofthe ENS activate vagal afferents that influence the functions of the CNS.

In some embodiments, the compositions of the invention regulate the activity of chromaffin cells.

Neurodegenerative diseases Parkinson ’3 disease Parkinson’s disease is a common neurodegenerative disease neuropathologically characterised by degeneration of heterogeneous populations of neural cells (dopamine-producing cells). The clinical diagnosis ofParkinson’s disease requires bradykinesia and at least one ofthe following core symptoms: resting tremor; muscle rigidity and al reflex impairment. Other signs and symptoms that may be present or develop during the progression of the disease are autonomic disturbances (sialorrhoea, seborrhoea, pation, micturition disturbances, sexual oning, orthostatic hypotension, hyperhydrosis), sleep disturbances and bances in the sense of smell or sense of temperature.

Parkinson’s disease is a neurodegenerative disease that may p or persist due to dysfunction of the microbiota—gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing Parkinson’s disease in a t.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Megasphaera, for use in a method of treating or preventing Parkinson’s disease.

Compositions comprising a bacterial strain of the genus Megasphaera may improve motor and cognitive functions in models of Parkinson’s disease. Treatment with haera strains may modulate signalling in the l, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may te the levels of commensal metabolites, atory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of Parkinson’s disease. In red embodiments, the invention provides a composition sing a ial strain of the species Megasphaera massiliensis for use in a method of treating or preventing son’s disease.

Compositions using Megasphaera massiliensis may be particularly effective for treating son’s disease.

In preud embodiments, the compositions of the ion prevent, reduce or alleviate one or more of the symptoms of Parkinson’s disease in a subject. In preferred embodiments, the compositions of [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm the invention prevent, reduce or alleviate one or more core symptoms of Parkinson’s disease in a t. In certain embodiments, the compositions of the invention prevent, reduce or alleviate bradykinesia in a t. In certain embodiments, the compositions of the invention prevent, reduce or ate resting tremor; muscle rigidity and/or postural reflex impairment in a subject. In certain embodiments, the itions of the invention prevent, reduce or alleviate one or more symptoms associated with son’s disease progression selected from autonomic bances (sialorrhoea, seborrhoea, pation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate depressive symptoms comorbid with Parkinson’s disease. In certain embodiments, the compositions of the invention improve verbal memory and/or executive functions. In certain embodiments, the compositions of the invention improve attention, working memory, verbal fluency and/or anxiety.

In other preferred embodiments, the compositions of the invention prevent, reduce or alleviate cognitive dysfunctions comorbid with Parkinson’s e.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate Parkinson’s disease progression. In n ments, the compositions of the invention prevent, reduce or alleviate later motor complications. In certain ments, the itions of the invention prevent, reduce or alleviate late motor fluctuations. In certain embodiments, the itions of the invention prevent, reduce or alleviate neuronal loss. In certain embodiments, the compositions of the invention improve symptoms of Parkinson’s disease dementia (PDD). In n embodiments, the compositions ofthe invention prevent, reduce or alleviate impairment of executive function, attention and/or working memory. In certain embodiments, the compositions of the invention improve dopaminergic neurotransmission. In certain embodiments, the compositions of the invention prevent, reduce or alleviate impaired dopaminergic neurotransmission.

In some embodiments, the compositions of the invention e the symptoms of Parkinson’s disease according to a matic or diagnostic scale. In certain embodiments, the tests for assessing symptomatic improvement ofmotor function in son’s disease is the Unified Parkinson’s Disease Rating Scale. In particular, UPDRS II considers the activity of daily life and UPDRS III considers motor-examination.

In some ments, the compositions of the ion improve the symptoms associated with PDD according to a symptomatic or diagnostic test and/or scale. In certain embodiments, the test or scale is selected from the Hopkins Verbal Learning Test — Revised (HVLT-R); the Delis-Kaplan Executive Function System S) Color-Word Interference Test; the Hamilton Depression Rating Scale (HAM-D l7; sion); the Hamilton y Rating Scale (HAM-A; anxiety) and the Unified Parkinns Disease Rating Scale (UPDRS; PD symptom severity).

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm In some embodiments, the compositions of the invention improve the Clinical Global sion — Global Improvement (CGI—I) scale for assessing psychiatric and neurological disorders. In some embodiments, the itions of the invention display a positive effect on global social and occupational impairment of the subject with Parkinson’s disease.

In certain embodiments, the compositions of the ion are for use in treating or preventing neurological disorders such as Parkinson’s disease in a subject wherein said use involves reducing or preventing loss minergic cells in the substantia nigra. In certain embodiments, the compositions of the invention are for use in treating or preventing neurological disorders such as Parkinson’s disease in a subject wherein said use involves reducing or preventing the degeneration of dopaminergic neurons in the substantia nigra pars compacta. In certain embodiments, the compositions of the ion are for use in treating or preventing neurological disorders such as Parkinson’s disease in a t wherein said use involves reducing or preventing the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the consequent loss of their projecting nerve fibers in the striatum. In certain embodiments, the itions of the invention are for use in treating or preventing neurological disorders such as son’s e in a subject wherein said use es ng or preventing loss of nigrostriatal dopaminergic neurons.

In certain embodiments, the compositions of the invention are for use in treating or preventing neurological disorders such as Parkinson’s disease in a subject n said use involves increasing dopamine levels. In certain embodiments, the compositions of the invention are for use in treating or preventing neurological disorders such as Parkinson’s disease in a t wherein said use involves increasing DOPAC levels. In n embodiments, the compositions of the invention are for use in treating or preventing neurological disorders such as Parkinson’s disease in a t wherein said use involves increasing dopamine and DOPAC levels. In certain embodiments, the dopamine and/or DOPAC levels are increased in the striatum.

Alzheimer ’s disease and dementia In DSM-5, the term dementia was replaced with the terms major neurocognitive disorder and mild neurocognitive disorder. Neurocognitive disorder is a heterogeneous class of psychiatric diseases. The most common neurocognitive disorder is Alzheimer’s disease, followed by vascular dementias or mixed forms of the two. Other forms of neurodegenerative disorders (eg. Lewy body disease, frontotemporal dementia, Parkinson’s dementia, Creutzfeldt-Jakob disease, Huntington’s disease, and ke-Korsakoff syndrome) are accompanied by dementia.

Alzheimer’s disease and dementia are also characterised by neuronal loss, so the neuroprotective and neuroproliferative effects shown in the examples for the itions of the invention indicate that they may be useful for treating or preventing these conditions.

The syDomatic criteria for dementia under DSM-5 are evidence of significant cognitive e from a previous level ofperformance in one or more ive s selected from: learning and memory; [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm language; executive function; complex attention; perceptual-motor and social ion. The cognitive deficits must interfere with independence in everyday activities. In addition, the cognitive deficits do not occur exclusively in the context of a delirium and are not better explained by another mental disorder (for example MDD or schizophrenia).

In addition to the primary symptom, subjects with neurodegenerative disorders display behavioural and psychiatric symptoms including agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances.

Neurodegenerative disorders may p or persist due to dysfunction of the microbiota—gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing neurodegenerative disorders in a t. In preferred embodiments, the neurodegenerative disorder is Alzheimer’s disease. In other embodiments, the neurodegenerative disorder is ed from vascular dementias; mixed form Alzheimer’s disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson’s dementia; Creutzfeldt-Jakob disease; Huntington’s e; and Wernicke-Korsakoff syndrome.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms ofneurodegenerative disorders in a t. In certain embodiments, the compositions of the ion prevent, reduce or alleviate the occurrence of cognitive decline in a subject. In certain embodiments, the compositions of the invention e the level of mance of a subject with neurodegenerative ers in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social ion. In some embodiments, the itions of the invention prevent, reduce or ate the occurrence of one or more behavioural and psychiatric symptoms associated with neurodegenerative disorders selected from agitation, sion, depression, anxiety, , psychosis and sleep-wake cycle disturbances.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate symptomatic e by intervention in ted pathogenic mechanisms at a preclinical stage. In certain embodiments, the compositions of the invention improve disease modification, with slowing or arrest of m progression. In some embodiments, the slowing or arrest of symptom ssion ates with evidence in delaying the underlying athological process. In preferred embodiments, the compositions of the invention improve symptoms of neurodegenerative disorders comprising enhanced cognitive and functional improvement. In preferred ments, the compositions of the invention improve the oural and psychiatric symptoms of dementia (BPSD).

In preferred embodiments, the compositions of the invention improve the ability of a subject with neurodegenerative disorder to undertake everyday activities.

In preferred embodiments, the compositions of the invention improve both cognition and functioning in a snot with Alzheimer’s disease. In some embodiments, the composition of the invention improves the cognitive nt in a subject with Alzheimer’s disease. In some embodiments, the [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm compositions of the invention improve the functional endpoint in a subject with Alzheimer’s disease.

In preferred embodiments, the compositions of the invention improve the cognitive and functional endpoint in a subject with Alzheimer’s disease. In yet further preferred embodiments, the compositions of the invention improve the l clinical se (the global endpoint) in a subject with Alzheimer’s e.

In some embodiments, the compositions of the invention improve the symptoms of neurodegenerative disorders according to a symptomatic or diagnostic test. In certain embodiments, the tests for assessing matic ement of Alzheimer’s disease (and other neurodegenerative disorders) are selected from objective cognitive, activities of daily living, global assessment of change, health related quality of life tests and tests assessing behavioural and psychiatric ms of neurodegenerative disorders.

In certain embodiments, the objective ive tests for assessment of symptomatic ement use the Alzheimer’s disease Assessment Scale ive subscale cog) and the classic ADAS scale. In certain embodiments, symptomatic improvement of cognition is assessed using the Neurophysiological Test Battery for Use in Alzheimer’s Disease (NTB).

In some embodiments, the global assessment of change test uses the Clinical Global Impression — Global Improvement (CGI—I) scale for assessing psychiatric and neurological disorders. In some embodiments, the global scale is the Clinician's Interview Based Impression of Change plus (CIBIC-plus). In some embodiments, the global scale is the mer’s Disease Cooperative Study Unit Clinician’s Global Impression of Change (ADCS-CGIC).

In certain embodiments, the health-related quality of life measures are the Alzheimer’s Disease- Related QOL ) and the QOL-Alzheimer’s Disease (QOL-AD).

In certain embodiments, the tests assessing behavioural and psychiatric symptoms of neurodegenerative ers are selected from the Behavioural pathology in Alzheimer’s Disease Rating Scale (BEHAVE-AD); the oural Rating Scale for Dementia (BRSD); the Neuropsychiatric Inventory (NPI); and the Mansfield Agitation Inventory (CMAI).

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating neurodegenerative disorders when used in combination with another therapy for treating neurodegenerative disorders. In certain embodiments, such therapies include acetylcholinesterase tors ing zil (Aricept®), galantamine yne®) and rivastigmine (Exelon ®), and memantine.

Multiple Sclerosis Multiple sclerosis (MS) is a demyelinating disease in which the myelin sheath surrounding neurons in the brand spinal cord are damaged. The exact underlying causes of MS are unknown, but are t to vary between individuals. Certain forms of MS are hereditary. Environmental factors are [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm also thought to contribute to MS. In some individuals, a combination ofboth genetic and environmental factors may r the onset of MS.

There are a wide variety of symptoms associated with MS. Subjects may t almost any neurological symptom associated with the ment of mic, , motor or sensory control.

The exact symptoms will vary depending on the site of neuronal damage/demyelination.

IL-8 has been ated in the formation of myelin s. The compositions of the invention may therefore be for use in the remyelination of neurons in subjects with MS. The compositions of the invention may also be used to protect neurons from demyelination. In other words, the compositions of the invention may be for use in a method of treating or preventing multiple sclerosis by restoring or preventing loss of neuron myelin sheaths.

In some embodiments, the compositions of the invention prevent, reduce or alleviate one or more symptoms ofMS in a subject. In some embodiments, the compositions of the invention prevent, reduce or alleviate fatigue in a t. In n embodiments, the compositions of the ion prevent, reduce or alleviate resting tremor, muscle weakness, muscle spasms, muscle stiffness, paraesthesia and/or ataxia in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more symptoms associated with MS progression selected from the list consisting of autonomic disturbances: constipation, micturition disturbances, sexual functioning, dysphagia, dysarthria, syncope, vertigo and/or dizziness; sleep disturbances; and bances in the sense of smell or sense of ature. In some embodiments, the compositions of the invention prevent, reduce or alleviate one or more ocular symptoms associated with MS. In some embodiments, the ocular m is selected from the list consisting of loss of vision, eye pain, colour blindness, double vision and/or involuntary eye movements in a subject.

In some embodiments, the itions of the invention prevent, reduce or alleviate dizziness, vertigo, neuropathic pain, musculoskeletal pain, ive dysfunction, bowel incontinence, dysphagia, dysarthria, or any combination thereof.

In some embodiments, the itions of the invention prevent, reduce or alleviate depressive symptoms or anxiety comorbid with MS.

In some embodiments, the improvement of symptoms are determined using the 2017 McDonald criteria for diagnosing MS.

In certain embodiments, treatment with the compositions of the invention results in a reduction in MS incidence or MS severity. In n embodiments, the compositions of the ion are for use in reducing relapse incidence or relapse severity. In certain embodiments, treatment with the itions of the invention prevents a decline in motor function or s in improved motor functicnssociated with MS. In certain embodiments, the compositions of the invention are for use in preventing a decline in motor function or for use in improving motor function in the treatment of MS.

[Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In certain embodiments, treatment with the compositions of the invention ts the development of paralysis in MS. In certain embodiments, the compositions of the invention are for use in preventing paralysis in the treatment of MS.

In certain embodiments the compositions of the invention are for use in preventing multiple sis in a patient that has been identified as at risk of multiple sclerosis, or that has been diagnosed with early—stage multiple sclerosis or “relapsing-remitting” multiple sis. The compositions of the invention may be useful for preventing the development of MS. The compositions of the invention may be useful for preventing the ssion of MS. In certain embodiments, the compositions of the invention are for use in a patient identified as having a genetic predisposition to MS, such as major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2 or HLA- DR4.

The compositions of the invention may be useful for ng or ating multiple sclerosis. The compositions of the invention may be particularly useful for reducing symptoms associated with multiple sclerosis. ent or prevention of multiple sclerosis may refer to, for example, an alleviation of the severity of ms or a reduction in the frequency of exacerbations or the range of triggers that are a problem for the patient. In certain embodiments, the compositions of the invention slow or stop progression of the disease.

In certain ments, the compositions of the invention are for use in treating relapsing-remitting MS. In alternative ments, the compositions of the ion are for use in treating ssive MS, such as secondary progressive MS (SPMS), which develops over time following diagnosis of RRMS, primary progressive MS (PPMS) which exhibits gradual continuous neurologic oration and progressive relapsing MS (PRMS), which is similar to PPMS but with pping relapses.

In certain embodiments, the compositions of the invention are for use in treating one or more of symptoms ofMS selected from the group consisting of: fatigue, vision problems, numbness, tingling, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain, problems with thinking, learning and planning, depression and anxiety, sexual problems, bladder problems, bowel problems, speech and swallowing difficulties.

Neurochemical factors, neuropeptides and neurotransmitters and the microbiota-gut—brain axis As outlined above, the microbiota—gut-brain axis is modulated by a number of different physiological systems. The microbiota—gut—brain axis is modulated by a number of signalling molecules. Alterations in the levels of these signalling les s in neurodegenerative diseases. The experiments performed by the inventors indicate that administration of haera species, and in particular Megasphaera massiliensis, can modulate levels of indole and kynurenine. Dysregulation of these lites can lead to neurodegenerative diseases, such as Parkinson’s disease.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm In certain embodiments, the compositions of the invention modulate the levels of brain monoamines and metabolites thereof. In preferred embodiments the metabolite is kynurenine. In certain embodiments, the compositions of the invention modulate kynurenine, which is the main route of tryptophan metabolism, which serves as a route to nicotinamide adenine dinucleotide (NAD+) tion. nine can be metabolized to neuroactive compounds such as nic acid (KYNA) and 3-hydroxy-l-kynurenine (3-OH-l-KYN), and in further steps to quinolinic acid (QUIN).

Dysregulation of the kynurenine pathway can lead to activation of the immune system and the accumulation of potentially neurotoxic compounds. Alterations in the kynurenine metabolism may be involved in the development of Parkinson’s diseases. Kynurenine levels have been demonstrated to be decreased in the frontal cortex, putamen and substantia nigra pars ta of patients with PD [32].

Therefore, in certain embodiments the compositions of the invention are for use in increasing the levels of kynurenine in the treatment of Parkinson’s disease.

In certain embodiments of the invention the compositions of the invention can increase the levels kynurenin. Increased levels of kynurenine have been shown to attenuated MPP+-induced neuronal cell death in vitro in a human dopaminergic neuroblastoma cell line [33]. In certain embodiments kynurenine and kynurenic acid, can activate GI aryl hydrocarbon receptor (Ahr) and GPR35 ors.

Activation ofAhr receptor induces IL-22 production, which can inhibit local inflammation. Activation of GPR35 inducing the production of inositol triphosphate and Ca2+ mobilization.

In certain ments, the compositions of the invention modulate the levels of indole. In preferred embodiments the lite is kynurenine. In certain embodiments, the compositions of the invention modulate nine, which is the main route of tryptophan metabolism.

The ling of the microbiota—gut-brain axis is modulated by levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain embodiments, the compositions of the invention modulates levels of neurochemical factors, neuropeptides and neurotransmitters.

Accordingly, in certain preferred embodiments, the compositions of the invention directly alter CNS biochemistry.

The signalling of the microbiota—gut-brain axis is modulated by levels of y-aminobutyric acid . ingly, in preferred embodiments, the compositions of the invention modulate the levels of GABA. GABA is an inhibitory neurotransmitter that s neuronal excitability. In certain embodiments, the compositions of the invention increase the levels of GABA. In n ments, the compositions of the invention decrease the levels of GABA. In certain embodiments, the compositions of the invention alter GABAergic neurotransmission. In certain embodiments, the compositions of the invention modulate the level of GABA transcription in different regions of the central nervous system. In certain embodiments, the commensal derived GABA s the blood- brain fier and affects neurotransmission directly. In certain embodiments, the compositions of the invent lead to a reduction of GABA in the hippocampus, amygdala and/or locus coeruleus. In [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm certain embodiments, the compositions of the invention lead to an increase of GABA in cortical regions.

Immune res onse The signalling of the microbiota-gut—brain axis is modulated by alterations in the immune response and inflammatory factors and s. Accordingly, in certain embodiments, the compositions of the invention may modulate the immune response. In certain embodiments, the compositions of the invention modulate the systemic levels of circulating neuroimmune signalling molecules. In certain preferred embodiments, the compositions of the invention modulate pro-inflammatory cytokine production and inflammation. In certain embodiments, the compositions of the invention modulate the inflammatory state. In certain embodiments, the compositions of the invention decrease IL-6 production and secretion. In n embodiments, the compositions of the invention se the tion of the NFKB promoter. In certain embodiments, the compositions of the invention are able to modulate the activation of IL-6 production by the potent pro-inflammatory endotoxin lipopolysaccharide (LPS). In certain ments, the compositions of the invention are able to modulate the activation of the NFKB promoter by LPS and or-synuclein mutant proteins such as A53T.

Increased circulating levels of cytokines are closely associated with various neurodegenerative ers, including Parkinson’s, dementia and Alzheimer’s. In certain embodiments, the compositions of the invention are for use in reducing IL-6 levels and/or NFKB levels in the treatment of a neurodegenerative disorder.

In some embodiments, the compositions of the invention increase the secretion of IL-8. IL-8 has been shown to induce myelin sheath formation and restore or ve ive neuronal communication.

Thus, in some embodiments, the compositions of the invention are for use in inducing myelin sheath formation in the treatment of neurodegenerative diseases. In some ments, the compositions of the ion are for use in restoring al communication. In some embodiments, the itions of the invention are for use in ving neuronal communication.

The signalling of the microbiota-gut—brain axis is modulated by levels of commensal metabolites.

Accordingly, in certain embodiments, the compositions of the invention modulate the systemic levels of microbiota metabolites. In certain preferred embodiments, the compositions of the invention te the level of short chain fatty acids (SCFAs). In certain embodiments the level of SCFAs is increased or sed. In some embodiments, the SCFA is butyric acid (BA) (or butyrate). In some embodiments, the SCFA is propionic acid (PPA). In some embodiments, the SCFA is acetic acid. In certain embodiments, the compositions of the invention modulate the ability of SCFAs to cross the blood—brain barrier.

Histone acetylation and ylation are important epigenetic regulators of gene expression. An imbala in histone acetylation and ylation can result in apoptosis. ulation of such histone acetyltransferases has been implicated in the pathogenesis associated with age-associated [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm neurodegenerative diseases, such as Parkinson’s disease, gton's disease, mer's disease, amyotrophic lateral sis and ive decline [34]. Accordingly, in certain embodiments, the compositions of the invention can modulate histone deacetylase activity. In n embodiments, the compositions of the invention can reduce histone deacetylase activity. In certain embodiments, the compositions of the invention can reduce histone ase activity.

Patients with neurodegenerative diseases, including Parkinson’s disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis, exhibit high levels of lipid peroxidation. Lipid are vulnerable to oxidation by reactive oxygen species, and the brain is rich in polyunsaturated fatty acids. Accordingly, in certain embodiments, the compositions of the invention can modulate lipid peroxidation. In certain embodiments, the compositions of the invention can reduce lipid peroxidation.

Reducing the oxidative damage caused by reactive oxygen s can be used to target early the stages neurodegenerative diseases. Accordingly, in certain embodiments, the compositions of the invention are for use in ng early stage neurodegeneration. Also accordingly, in certain embodiments, the compositions of the invention are for use in preventing the development of a neurodegenerative disorder. In such embodiments, the compositions of the invention may be for use in a patient that has been identified as at risk of developing a neurodegenerative er.

The signalling of the microbiota—gut-brain axis is modulated by levels of gastrointestinal bility.

Accordingly, in some embodiments, the compositions of the ion alter the integrity of the gastrointestinal tract epithelium. In certain embodiments, the compositions of the invention modulate the bility of the gastrointestinal tract. In certain embodiments, the compositions of the invention modulate the barrier function and integrity of the gastrointestinal tract. In certain embodiments, the compositions of the invention modulate gastrointestinal tract motility. In certain ments, the itions of the invention modulate the translocation of commensal metabolites and inflammatory signalling molecules into the bloodstream from the intestinal tract lumen.

The signalling of the microbiota—gut—brain axis is modulated by microbiome composition in the intestinal tract. Accordingly, in certain embodiments, the compositions of the invention modulates the microbiome composition of the intestinal tract. In certain ments, the compositions of the invention prevents microbiome dysbiosis and associated increases in toxic metabolites (e.g. LPS). In certain embodiments, the compositions of the invention modulate the levels of Clostridium in the gastrointestinal tract. In preferred embodiments, the compositions of the invention reduce the level of Clostridium in the gastrointestinal tract. In certain embodiments, the compositions of the invention reduce the levels of Campylobacter jejuni. In certain embodiments, the compositions of the invention te the proliferation of harmful anaerobic bacteria and the production of neurotoxins produced by these bacteria. In n ments, the compositions of the invention modulate the microbiome levels of Lactobacillus and/or Bifidobacterium. In certain embocants, the compositions of the invention modulate the microbiome levels of Sutterella, Prevotella, Ruminococcus genera and/or the Alcaligenaceae family. In certain embodiments, the [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm compositions of the invention increase the level of Lactobacillus plantarum and/or Saccharomyces boulardii.

Brain injury The examples demonstrate that the itions of the invention are neuroprotective and have HDAC tory activity. HDAC2 is a l target for functional recovery from stroke [35] and HDAC inhibition can prevent white matter injury [36], so the compositions of the invention may be useful in the treatment of brain injury.

In certain embodiments, the compositions of the invention are for use in treating brain injury. In some embodiments, the brain injury is a traumatic brain injury. In some embodiments, the brain injury is an acquired brain injury. In some embodiments, the compositions of the ion are for use in treating brain injury resulting from trauma. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from a tumour. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from a . In some embodiments, the compositions of the invention are for use in treating brain injury resulting from a brain rhage.

In some embodiments, the compositions of the invention are for use in treating brain injury resulting from alitis. In some embodiments, the compositions of the invention are for use in treating brain injury ing from cerebral hypoxia. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from cerebral .

In preferred embodiments, the compositions of the invention are for use in treating stroke. The effects shown in the examples are particularly relevant to the treatment of stroke. Stroke occurs when blood flow to at least a part of the brain is interrupted. Without an adequate supply of blood to e oxygen and nutrients to the brain tissue and to remove waste products from the brain , brain cells rapidly begin to die. The symptoms of stroke are dependent on the region of the brain which is affected by the inadequate blood flow. Symptoms include paralysis, numbness or weakness of the muscles, loss of balance, ess, sudden severe headaches, speech impairment, loss of memory, loss of ing ability, sudden confusion, Vision impairment, coma or even death. A stroke is also referred to as a brain attack or a cerebrovascular accident (CVA). The symptoms of stroke may be brief if adequate blood flow is restored within a short period of time. However, if inadequate blood flow continues for a significant period of time, the symptoms can be ent.

In some embodiments, the stroke is cerebral ischemia. al ischemia results when there is cient blood flow to the tissues of the brain to meet metabolic demand. In some embodiments, the cerebral ischemia is focal cerebral ischemia, i.e. confined to a specific region of the brain. In some embodiments the cerebral ischemia is global cerebral ischemia, i.e. encompassing a wide area of the brain tissue. Focal cerebral ischemia commonly occurs when a cerebral vessel has become blocked, either 'ally or tely, reducing the flow of blood to a specific region of the brain. In some embodiments the focal cerebral ischemia is ischemic stroke. In some embodiments, the ischemic [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm stroke is thrombotic, i.e. caused by a thrombus or blood clot, which develops in a cerebral vessel and restricts or blocks blood flow. In some ments the ischemic stroke is a thrombotic stroke. In some embodiments, the ischemic stroke is embolic, i.e. caused by an embolus, or an unattached mass that travels through the bloodstream and restricts or blocks blood flow at a site t from its point of origin. In some embodiments the ischemic stroke is an embolic stroke. Global cerebral ischemia commonly occurs when blood flow to the brain as a whole is blocked or reduced. In some embodiments the global cerebral ia is caused by hypoperfusion, i.e. due to shock. In some embodiments the global cerebral ischemia is a result of a cardiac arrest.

In some embodiments the subject diagnosed with brain injury has suffered cerebral ischemia. In some ments, the subject diagnosed with brain injury has suffered focal cerebral ischemia. In some embodiments, the subject sed with brain injury has suffered an ischemic . In some embodiments, the subject diagnosed with brain injury has suffered a thrombotic stroke. In some embodiments, the subject diagnosed with brain injury has suffered an embolic stroke. In some embodiments, the subject sed with brain injury has suffered global cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered hypoperfusion. In some embodiments, the subject diagnosed with brain injury has suffered a cardiac arrest.

In some embodiments, the compositions of the invention are for use in treating cerebral ischemia. In some embodiments, the compositions of the invention are for use in treating focal cerebral ischemia.

In some embodiments, the compositions of the invention are for use treating ic stroke. In some embodiments, the compositions of the invention are for use in treating thrombotic stroke. In some embodiments, the compositions of the invention are for use in treating embolic stroke. In some ments, the itions of the invention are for use in treating global cerebral ischemia. In some embodiments, the itions of the invention are for use in treating hypoperfusion.

In some embodiments, the stroke is hemorrhagic stroke. hagic stroke is caused by bleeding into or around the brain ing in swelling, pressure and damage to the cells and tissues of the brain.

Hemorrhagic stroke is commonly a result of a weakened blood vessel that ruptures and bleeds into the surrounding brain. In some embodiments, the hemorrhagic stroke is an intracerebral hemorrhage, i.e. caused by bleeding within the brain tissue itself. In some embodiments the intracerebral hemorrhage is caused by an intraparenchymal hemorrhage. In some embodiments the intracerebral hage is caused by an intraventricular hemorrhage. In some ments the hemorrhagic stroke is a subarachnoid hemorrhage i.e. bleeding that occurs e of the brain tissue but still within the skull.

In some embodiments, the hemorrhagic stroke is a result of cerebral amyloid angiopathy. In some embodiments, the hemorrhagic stroke is a result of a brain aneurysm. In some embodiments, the hemorrhagic stroke is a result of cerebral arteriovenous malformation (AVM).

In sorfiibodiments the subject diagnosed with brain injury has suffered hemorrhagic . In some diments, the subject diagnosed with brain injury has suffered an intracerebral hemorrhage.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In some embodiments, the subject diagnosed with brain injury has suffered an intraparenchymal hemorrhage. In some embodiments, the subject diagnosed with brain injury has ed an entricular hemorrhage. In some ments, the subject diagnosed with brain injury has suffered a subarachnoid hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with brain injury has suffered a brain aneurysm. In some embodiments, the subject diagnosed with brain injury has suffered cerebral AVM.

In some ments, the compositions of the invention are for use in treating hemorrhagic stroke. In some embodiments, the compositions of the invention are for use in treating an intracerebral hemorrhage. In some ments, the compositions of the invention are for use in treating an intraparenchymal hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraventricular hage. In some embodiments, the compositions of the invention are for use in treating a subarachnoid hemorrhage. In some embodiments, the itions of the invention are for use in treating a cerebral amyloid angiopathy. In some embodiments, the compositions of the invention are for use in treating a brain sm. In some embodiments, the itions of the invention are for use in treating cerebral AVM. ation of adequate blood flow to the brain after a period of interruption, though effective in alleviating the symptoms associated with stroke, can paradoxically result in further damage to the brain . During the period of interruption, the affected tissue suffers from a lack of oxygen and nutrients, and the sudden ation of blood flow can result in inflammation and oxidative damage through the induction of oxidative stress. This is known as reperfusion injury, and is well documented not only following , but also following a heart attack or other tissue damage when blood supply returns to the tissue after a period of ischemia or lack of oxygen. In some embodiments the t diagnosed with brain injury has suffered from reperfusion injury as a result of stroke. In some embodiments, the compositions of the invention are for use in treating reperfusion injury as a result of stroke.

A transient ischemic attack (TIA), often referred to as a mini-stroke, is a recognised g sign for a more serious stroke. Subjects who have suffered one or more TIAs are therefore at greater risk of stroke. In some embodiments the subject diagnosed with brain injury has suffered a TIA. In some embodiments, the compositions of the invention are for use in treating a TIA. In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has ed a TIA.

High blood pressure, high blood cholesterol, a familial history of stroke, heart disease, diabetes, brain aneurysms, arteriovenous malformations, sickle cell disease, vasculitis, bleeding disorders, use of nonsteroidal anti-inflammatory drugs (NSAIDs), smoking o, drinking large amounts of alcohol, illegalfig use, obesity, lack of physical activity and an unhealthy diet are all considered to be riskfactor stroke. In ular, lowering blood pressure has been conclusively shown to prevent both [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ischemic and hemorrhagic strokes [37, 38]. In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has at least one risk factor for stroke. In some embodiments the subject has two risk factors for . In some embodiments the subject has three risk factors for stroke. In some embodiments the subject has four risk factors for stroke. In some embodiments the subject has more than four risk factors for stroke. In some embodiments the subject has high blood pressure. In some embodiments the subject has high blood cholesterol. In some embodiments the subject has a familial history of stroke. In some embodiments the subject has heart disease. In some embodiments the subject has diabetes. In some embodiments the subject has a brain aneurysm. In some embodiments the subject has arteriovenous malformations. In some embodiments the subject has vasculitis. In some embodiments the subject has sickle cell e. In some embodiments the t has a bleeding disorder. In some embodiments the subject has a history of use of roidal anti-inflammatory drugs (NSAIDs). In some ments the subject smokes tobacco. In some ments the subject drinks large amounts of alcohol. In some embodiments the subject uses illegal drugs. In some embodiments the subject is obese. In some embodiments the subject is overweight. In some embodiments the subject has a lack of physical activity. In some embodiments the subject has an unhealthy diet.

The examples indicate that the compositions of the invention may be useful for treating brain injury and aiding recovery when administered before the injury event occurs. Therefore, the compositions of the invention may be particularly useful for treating brain injury when administered to subjects at risk of brain injury, such as stroke.

In n embodiments, the compositions of the ion are for use in reducing the damage caused by a potential brain injury, ably a stroke. The itions may reduce the damage caused when they are administered before the potential brain injury occurs, in particular when administered to a patient identified as at risk of a brain injury.

The examples indicate that the compositions of the invention may be useful for treating brain injury and aiding recovery when administered after the injury event occurs. Therefore, the compositions of the invention may be particularly useful for treating brain injury when administered to subjects following a brain injury, such as .

In some embodiments, the compositions of the invention treat brain injury by reducing c damage. In some embodiments, the compositions of the invention treat brain injury by improving motor function. In some embodiments, the compositions of the invention treat brain injury by improving muscle strength. In some embodiments, the compositions of the invention treat brain injury by improving . In some embodiments, the itions of the invention treat brain injury by improving social recognition. In some embodiments, the compositions of the invention treat brain injury bmproving ogical function.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Treatment of brain injury may refer to, for example, an alleviation of the severity of ms.

Treatment of brain injury may also refer to reducing the neurological impairments following stroke.

Compositions of the invention for use in treating stroke may be provided to the subject in advance of the onset of stroke, for example in a patient identified as being at risk of . itions of the invention for use in treating stroke may be ed after a stroke has occurred, for example, during recovery. Compositions of the invention for use in treating stroke may be ed during the acute phase of recovery (i.e. up to one week after stroke). Compositions of the invention for use in treating stroke may be provided during the subacute phase of recovery (i.e. from one week up to three months after stroke). Compositions of the invention for use in treating stroke may be ed during the chronic phase of recovery (from three months after ).

In certain embodiments, the compositions of the invention are for use in combination with a secondary active agent. In certain embodiments, the compositions of the invention are for use in combination with aspirin or tissue plasminogen activator (tPA). Other secondary agents include other antiplatelets (such as ogrel), anticoagulants (such as heparins, warfarin, apixaban, dabigatran, edoxaban or rivaroxaban), antihypertensives (such as diuretics, ACE inhibitors, calcium channel blockers, beta- blockers or alpha-blockers) or statins. The itions of the invention may improve the patient’s response to the secondary active agent.

In certain embodiments, the itions of the invention reduce the effect of ischemia on tissues. In n embodiments, the compositions of the ion reduce the amount of damage to tissues caused by ischemia. In certain embodiments, the tissues damaged by ischemia are the cerebral tissues. In certain embodiments, the compositions of the invention reduce necrosis or the number of necrotic cells.

In certain embodiments, the compositions of the invention reduce apoptosis or the number of tic cells. In certain embodiments, the compositions of the invention reduce the number of necrotic and apoptotic cells. In certain embodiments, the compositions of the invention prevent cell death by necrosis and/or apoptosis. In n embodiments, the compositions of the invention prevent cell death by necrosis and/or sis caused by ischemia. In certain embodiments, the compositions of the ion improve the recovery of the tissue damaged by ia. In certain embodiments, the compositions of the ion improve the speed of clearance of necrotic cells and/or apoptotic cells.

In certain embodiments, the compositions of the invention improve the efficacy of the clearance of necrotic cells and/or apoptotic cells. In n embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues. In certain embodiments, the compositions of the invention improve the replacement and/or ration of cells within tissues damaged by ischemia. In certain embodiments, the compositions of the invention improve the overall histology of the tissue (for example upon a biopsy).

Modes ofadministration Prefera, the compositions of the invention are to be administered to the gastrointestinal tract in order to enable delivery to and/ or partial or total colonisation of the intestine with the bacterial strain of the [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm invention. Generally, the compositions of the invention are administered orally, but they may be administered ly, intranasally, or via buccal or sublingual .

In certain embodiments, the compositions of the invention may be administered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may be administered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), tic hard fat (e.g. ire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.

In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), aneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other le access ports.

The compositions of the ion may be administered once, or they may be administered sequentially as part of a treatment n. In certain embodiments, the compositions of the invention are to be administered daily.

In certain embodiments of the ion, treatment according to the invention is accompanied by assessment of the patient’s gut microbiota. Treatment may be repeated if delivery of and / or partial or total colonisation with the strain of the invention is not ed such that efficacy is not observed, or treatment may be ceased if delivery and/ or partial or total sation is sful and efficacy is observed.

In certain embodiments, the composition of the invention may be stered to a pregnant , for example a mammal such as a human in order to prevent an inflammatory or autoimmune disease developing in her child in utero and / or after it is born.

The itions of the invention may be administered to a patient that has been diagnosed with a neurodegenerative disease, or that has been identified as being at risk of a neurodegenerative disease.

The compositions may also be administered as a prophylactic measure to prevent the development of egenerative disease in a healthy patient.

The compositions of the invention may be administered to a patient that has been identified as having an abnormal gut microbiota. For example, the patient may have reduced or absent colonisation by Megasphaera, and in particular Megasphaera massiliensis.

The compositions of the invention may be administered as a food product, such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment of humans, although they may be used tnat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used.

Compositions Generally, the ition of the invention comprises bacteria. In preferred embodiments of the ion, the composition is ated in freeze-dried form. For example, the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a ial strain of the invention. ably, the composition of the invention comprises lyophilised bacteria. lisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [39], [], [4 l H.

Alternatively, the composition of the invention may comprise a live, active bacterial culture.

In some embodiments, the bacterial strain in the composition of the invention has not been inactivated, for example, has not been heat-inactivated. In some embodiments, the bacterial strain in the composition of the invention has not been killed, for example, has not been heat-killed. In some embodiments, the bacterial strain in the composition of the invention has not been attenuated, for example, has not been ttenuated. For example, in some embodiments, the bacterial strain in the composition of the invention has not been killed, inactivated and/or ated. For example, in some embodiments, the bacterial strain in the composition of the invention is live. For example, in some embodiments, the bacterial strain in the composition of the ion is Viable. For example, in some embodiments, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine. For example, in some embodiments, the bacterial strain in the composition of the invention is viable and capable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of live bacterial strains and bacterial strains that have been killed.

In preferred embodiments, the ition of the ion is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from degradation until delivery at the target on through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any riate encapsulation method may be used. Exemplary ulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier es, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. ce on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [42] and [43].

The chsition may be stered orally and may be in the form of a tablet, capsule or powder.

Encapsulated products are red because Megasphaera are anaerobes. Other ingredients (such as [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm vitamin C, for example), may be included as oxygen scavengers and prebiotic ates to improve the delivery and / or partial or total colonisation and survival in viva. Alternatively, the probiotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.

The ition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effective amount of a bacterial strain of the invention. A therapeutically effective amount of a bacterial strain is sufficient to exert a beneficial effect upon a patient. A therapeutically effective amount of a bacterial strain may be sufficient to result in delivery to and / or partial or total colonisation of the patient’s intestine.

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 103 to about 1 x 1011 colony g units (CPU); for example, from about 1 x 107 to about 1 x 1010 CPU; in another example from about 1 x 106 to about 1 x 1010 CFU.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 106 to about 1 x 1011 CFU/g, respect to the weight of the ition; for example, from about 1 x 108 to about 1 x 1010 CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.

Typically, a tic, such as the composition of the invention, is optionally ed with at least one suitable prebiotic compound. A prebiotic compound is y a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and transgalacto- oligosaccharides.

In certain embodiments, the probiotic composition of the present invention includes a prebiotic compound in an amount of from about 1 to about 30% by , respect to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: - oligosaccharides (or FOS), short-chain -oligosaccharides, inulin, t- oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan—oligosaccharides (or COS), beta— glucans, arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for city shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the sion of the beet sugar and including a saccharose molecule to which three glucose les are bonded.

In certain embodiments, the compositions of the invention are used in combination with another therapeutic compound for treating or preventing the neurodegenerative disorder. In some embodiments, the compositions of the invention are stered with nutritional supplements that modulnieuroprotection or neuroproliferation. In preferred embodiments, the nutritional supplements comprise or consist of ional Vitamins. In n embodiments, the Vitamins are vitamin B6, [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm magnesium, dimethylglycine (vitamin B16) and vitamin C. In certain embodiments, the compositions of the invention are administered in combination with another probiotic.

In n embodiments, the compositions of the invention are for use in enhancing the effect of a second agent on a neurodegenerative e. The immune modulatory effects of the compositions of the invention may make the brain more susceptible to conventional therapies such as Levodopa, dopamine agonists, MAO-B tors, COMT inhibitors, Glutamate antagonists, or anticholinergics, which are exemplary secondary agents to be administered in combination (sequentially or contemporaneously) with the compositions of the invention.

The compositions of the invention may comprise pharmaceutically able excipients or carriers.

Examples of such suitable excipients may be found in the reference [44]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are bed, for example, in reference [45]. Examples of suitable carriers e lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical itions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, n, natural sugars such as glucose, anhydrous lactose, free—flow lactose, beta-lactose, corn ners, natural and synthetic gums, such as , tragacanth or sodium te, ymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

The compositions of the invention may be formulated as a food product. For example, a food t may provide nutritional benefit in addition to the eutic effect of the invention, such as in a nutritional supplement. Similarly, a food product may be ated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a ceutical composition. In certain ments, the composition of the invention is formulated as a milk-based product. The term "milk-based product" means any liquid or semi-solid milk— or whey— based t having a varying fat content. The milk- based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products.

Another important group includes milk beverages, such as whey beverages, fermented milks, u milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.

[Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm In some embodiments, the compositions of the invention comprise one or more bacterial s of the genus Megasphaera and do not contain bacteria from any other genera, or which comprise only de s or biologically irrelevant amounts of bacteria from another genera. Thus, in some embodiments, the invention provides a composition comprising one or more bacterial strains of the genus Megasphaera, which does not contain bacteria from any other genera or which comprises only de minimis or biologically irrelevant amounts of bacteria from another genera, for use in therapy.

In some embodiments, the compositions of the invention comprise one or more ial strains of the species Megasphaera massilz'ensis and do not contain bacteria from any other species, or which se only de minimis or ically irrelevant amounts of bacteria from another species. Thus, in some embodiments, the invention es a composition sing one or more bacterial strains of the s Megasphaera massiliensis, which does not contain bacteria from any other species or which comprises only de minimis or biologically irrelevant amounts of bacteria from r species, for use in therapy.

In some embodiments, the compositions of the invention comprise one or more ial strains of the species Megasphaera massiliensis and do not contain bacteria from any other Megasphaera species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from r Megasphaera s. Thus, in some ments, the invention provides a composition comprising one or more bacterial strains of the species Megasphaera massilz'ensz's, which does not contain ia from any other Megasphaera species or which comprises only de minimis or biologically irrelevant amounts of bacteria from another Megasphaera species, for use in therapy.

In certain embodiments, the compositions of the invention contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism.

In some embodiments, the invention provides a ition comprising a single bacterial strain of the genus Megasphaera, which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy.

In some embodiments, the invention provides a composition comprising a single bacterial strain of the s Megasphaera massilz’ensis, which does not contain bacteria from any other s or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy.

In some embodiments, the compositions of the ion comprise more than one bacterial strain. For example, in some embodiments, the compositions of the invention comprise more than one strain from within and, (finally,same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains),do not contain bacteria from any other species. In some embodiments, the itions of the invention comprise less than 50 strains from within the same species (e.g. less than [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1-20,1-19,l-18, 1-15, 1-10,l-9,l-8,1-7,1-6,1-5,1-4,1-3,1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains from within the same species and, ally, do not contain bacteria from any other species. The invention comprises any combination of the foregoing.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises the Megasphaera ial strain as part of a microbial consortium. For example, in some ments, the haera bacterial strain is present in ation with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from other genera with which it can live symbiotically in vivo in the intestine. For example, in some embodiments, the composition comprises a bacterial strain of Megasphaera in combination with a bacterial strain from a different genus. In some embodiments, the microbial consortium comprises two or more bacterial strains obtained from a faeces sample of a single organism, e.g. a human. In some embodiments, the ial consortium is not found together in nature. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from faeces s of at least two different organisms. In some embodiments, the two different organisms are from the same species, e.g. two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal.

In some embodiments, the composition of the invention additionally comprises a bacterial strain that has the same safety and therapeutic y characteristics as strain MRx0029, but which is not MRx0029, or which is not a Megaspkaera massiliensz's.

In some ments in which the composition of the invention comprises more than one bacterial strain, species or genus, the individual bacterial strains, species or genera may be for separate, simultaneous or sequential administration. For example, the composition may comprise all of the more than one bacterial strain, species or genera, or the ial strains, s or genera may be stored separately and be administered separately, simultaneously or sequentially. In some embodiments, the more than one bacterial strains, species or genera are stored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the ion is obtained from human adult faeces.

In some embodiments in which the composition of the invention comprises more than one ial strain, all of the bacterial s are obtained from human adult faeces or if other bacterial strains are present they are present only in de minimis amounts. The ia may have been cultured subsequent to being obtained from the human adult faeces and being used in a composition of the invention.

As meDned above, in some embodiments, the one or more Megasphaera bacterial strains is/are the only therapeutically active agent(s) in a composition of the invention. In some embodiments, the [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm bacterial strain(s) in the composition is/are the only therapeutically active agent(s) in a composition of the invention.

The compositions for use in accordance with the invention may or may not require marketing al.

In certain embodiments, the ion provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides the above pharmaceutical composition, n said bacterial strain is spray dried. In certain embodiments, the invention provides the above pharmaceutical ition, wherein the ial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the invention provides the above ceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is capable of partially or totally colonising the intestine. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable and capable of partially or totally sing the intestine.

In some cases, the lyophilised bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

The itions of the invention can comprise pharmaceutically acceptable ents, diluents or carriers.

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a neurodegenerative disorder when administered to a subject in need thereof.

In certain embodiments, the invention provides ceutical composition comprising: a bacterial strain of the invention; and a ceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a neurodegenerative er.

In certain embodiments, the invention provides the above pharmaceutical ition, wherein the amount of the bacterial strain is from about 1 X 103 to about l X 1011 colony forming units per gram with respect to a weight of the composition.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is stered at a dose of l g, 3 g, 5 g or 10 g.

In n embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutnus, nasal, , and sublingual.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm ed set by kjm In certain embodiments, the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.

In certain embodiments, the invention provides the above pharmaceutical ition, comprising a diluent selected from the group consisting of l, glycerol and water.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium , sodium te, magnesium stearate, sodium benzoate, sodium e and sodium chloride.

In certain embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a vative, an antioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid.

In certain embodiments, the invention provides the above ceutical composition, wherein said bacterial strain is lyophilised.

In certain ments, the invention provides the above pharmaceutical composition, n when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, s after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed ner is a sachet or bottle. In some embodiments, the composition ofthe invention is provided in a syringe comprising a ition as bed herein.

The composition of the present invention may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a tablet or capsule. In some embodiments, the capsule is a gelatine capsule cap”).

In some embodiments, the compositions of the invention are administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual stration by which the compound enters the blood stream directly from the mouth.

Pharmnltical formulations suitable for oral stration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm tablets; soft or hard capsules containing multi- or nano-particulates, s (e.g. s solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an enteric formulation, i.e. a gastro-resistant formulation (for example, resistant to gastric pH) that is suitable for delivery of the composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to degradation under gastric conditions.

In some embodiments, the enteric formulation comprises an c coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric- coated tablet or an enteric-coated capsule, or the like. The enteric g may be a conventional enteric coating, for example, a conventional g for a tablet, capsule, or the like for oral delivery. The formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the c formulation is intrinsically enteric, for example, gastro-resistant without the need for an c coating. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling al. In some embodiments, the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule ses a shell that does not contain any film forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [46 D. In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from el).

In some embodiments, the formulation is a soft capsule. Soft capsules are capsules which may, owing to additions of ers, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the e shell, have a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gelatine or starch. ne-based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as, for e, orally or rectally, soft capsules can have various , they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional processes, such as, for e, by the Scherer process, the Accogel process or the droplet or blowing process.

Culturing methods The bacterial strains for use in the present invention can be cultured using rd iology techniD as detailed in, for example, references [47], [] and [49].

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm The solid or liquid medium used for e may be YCFA agar or YCFA medium. YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO3 (0.4 g), cysteine (0.1 g), K2HPO4 (0.045 g), KHZPO4 (0.045 g), NaCl (0.09 g), $O4 (0.09 g), MgSO4 ' 7HZO (0.009 g), CaClz (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 ug), cobalamin (1 ug), p-aminobenzoic acid (3 ug), folic acid (5 ug), and pyridoxamine (15 ug). ial strainsfor use in vaccine compositions The inventors have identified that the ial strains of the invention are useful for ng or preventing neurodegenerative disorders. This is likely to be a result of the effect that the bacterial strains of the invention have on the host immune system. Therefore, the compositions of the invention may also be useful for preventing neurodegenerative ers, when administered as e compositions. In certain such embodiments, the bacterial strains of the invention may be killed, vated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration Via injection, such as via subcutaneous ion.

General The ce of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular y, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references [50] and [51,57], etc.

The term “comprising” encompasses ding” as well as “consisting” e.g. a composition “comprising” X may consist exclusively ofX or may include something additional e.g. X + Y.

The term “about” in relation to a numerical value x is optional and means, for example, x:10%.

The word “substantially” does not exclude “completely” eg. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences. This ent and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [58]. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open y of 12 and a gap extension y of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [59].

Unless cally stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional ening steps. Also, steps may bnnbined, omitted or performed in an alternative order, if appropriate.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm ed set by kjm Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combined with other ed features, to provide r ments. In particular, embodiments highlighted herein as being suitable, typical or red may be combined with each other (except when they are mutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1 — Efi‘icacy ofbacterial inocula to act as a rotectant Summary Neuroblastoma cells were treated with compositions comprising bacterial s according to the invention. The SH—SYSY neuroblastoma cells used are dopamine producing and stablished as an in vitro model for studying neurodegenerative diseases. The ability of the bacterial strains to increase roliferation was observed. The neuroblastoma cells were also treated with dopaminergic neurotoxin 1-methylphenylpyridinium (MPP), which induces permanent symptoms of son’s disease in neuroblastoma cells. The ability of the bacterial strains to act as a neuroprotectant against MPP was igated.

Material and Methods ial strain Megasphaera massiliensis MRx0029; Parabacteroz'des distasonis MRX0005 Cell line SH-SYSY neuroblastoma cells were purchased from ECCACC (Cat. no: 94030304) and were grown in MEM (Sigma Aldrich, cat n. M2279) supplemented with Nutrient Mixture F-12 Ham (Sigma Aldrich, cat 11. N4888).

Method Once grown the SH—SYSY neuroblastoma cells were plated on 96-well plate at 11,000 cells/well and incubated for 2 days. The cells were then transferred to differentiation medium (which contains PBS at 1%) and 10 uM retinoic acid (Sigma Aldrich, cat. n. R2625-100MG). Differentiation medium was replaced every other day and cells were harvested at 7 day of differentiation. Cells were pre-treated with or without MPP (Sigma Aldrich, cat. 11. D048—1G) for 8 hours. Subsequently, cells were treated with 10% bacterial supernatant and incubated overnight. Cell viability was measured by using CCK-8 reagent (Sigma h, Cell Counting Kit — 8, cat. n. 96992-3000TESTS-F) and read at 450nm wavelength.

Results The was of these experiments are shown in Figure 1. Treatment of neuroblastoma cells with MRxO or MRX0005 led to an increase in the proliferation of neurons. Neuroblastoma cells that were treated with MPP together with the bacterial strain had increased cell viability compared to the [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm cells treated with MPP alone (which had decreased viability).These data show that the bacterial strain can act as a neuroprotectant. The protective effect was greater for MRX0029-treated cells, which rescued viability more than the positive control cells treated with Quercetin. These data show that the bacterial strains can act as a neuroprotectant e 2 — Efficacy ofbacterial inocula to reduce IL-6 secretion.

Sum—mark Activation of proinflammatory cytokines has been associated with neuron damage in neurodegenerative disease. Lipopolysaccharide (LPS) is a known ator of the proinflammatory cytokine IL-6. Human glioblastoma astrocytoma cells were treated with compositions comprising ial strains according to the invention in ation with LPS to observe their ability to modulate the levels of IL-6.

Material and Methods Bacterial strain Megasphaera massiliensis MRX0029 Cell line MG U373 is a human glioblastoma astrocytoma derived from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4mM L-Glut, 1X MEM Non essential Amino Acid solution and 1X Sodium Piruvate.

Method Once grown the MG U373 cells were plated on 24-well plate at 100,000 cells/well. The cells were treated with LPS (lug/mL) alone or with 10% of bacteria supernatant from MRX0029 for 24h. A control was also performed where the cells were ted in untreated media. Afterwards the cell free supernatants were collected, centrifuged at 10,000g for 3min at 4°C. IL-6 was measured using the Human lL-6 ELISA Kit from Peprotech (cat n.#900-K16) according to manufacturer ctions.

Results The results of these experiments are shown in Figure 2. Treatment of neuroblastoma cells with LPS and the bacteria strain led to a se in the level of lL-6 secreted.

Example 2b — Efficacy ofbacterial inocula to modulate IL-8 secretion.

Summary As inflammation plays a pivotal role in neurodegenerative diseases and lL-8 has been shown to have ray-positive s, the effect of compositions comprising bacterial strains of the invention and LPS on the activation of lL-8 were assessed. Human glioblastoma astrocytoma cells were treated [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm with compositions comprising bacterial strains according to the invention in ation with LPS to observe their ability to modulate the levels of IL-8.

Material and Methods Bacterial strains Megasphaera massiliensis MRX0029; Parabacteroides distasonis 5 Cell line MG U373 is a human glioblastoma astrocytoma derived from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10% PBS, 1% Pen Strep, 4mM L-Glut, 1X MEM Non essential Amino Acid solution and 1X Sodium Piruvate.

Method Once grown the MG U373 cells were plated on 24-well plate at 100,000 cells/well. The cells were treated with LPS (lug/mL) alone or with 10% of bacteria supernatant from MRX0029 for 24h.

Afterwards the cell free supernatants were collected, centrifuged at 10,000g for 3min at 4°C. IL-8 was measured using Human IL-8 ELISA Kit from Peprotech (cat n.#900-K18) ing to manufacturer instruction.

Results The results of these experiments are shown in Figure 3. Treatment of neuroblastoma cells with the bacteria strains lead to an increase in IL-8 secretion ndently of the presence of LPS.

Example 2C — Efficacy ofbacterial inocula to reduce clein-induced inflammation.

Summary Neuroinflammation plays a pivotal role in Parkinson’s e and a-synuclein has been shown to induce neuroinflammation in viva. Therefore, the ability of the bacteria strains of the invention to inhibit a-synuclein-induced nflammation was assessed. A co-culture of human glioblastoma astrocytoma cells and neuroblastoma cells were d to wild-type a-synuclein and the mutant isoforms E46K and A53T and treated with itions comprising ial strains according to the invention. The ability of the bacteria strains to t a-synuclein—induced ion of IL-6 was then tested.

Material and Methods Bacterial strains MegasDera massiliensis MRX0029; Parabacteroides distasonis MRX0005 [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Cell Zine MG U373 is a human glioblastoma astrocytoma derived from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat n. M-2279) mented with 10% FBS, 1% Pen Strep, 4mM L-Glut, 1X MEM Non-essential Amino Acid solution and 1X Sodium Piruvate.

SH—SYSY is a human astoma cell line derived from a malignant neuroblastoma and can be purchased from Aldrich (cat 11. 94030304-1VL). The cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2mM amine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 ug/ml streptomycin. Cells on growth medium were plated on 96-well plate at 11,000 cells/well and placed in the incubator. After 2 days, media were replaced with differentiation medium (growth medium containing 1% FBS) and 10 uM ic acid. Differentiation medium was replaced every other day and cells were used after 7 days of differentiation.

Method SHSY5Y cells were plated on 12 well plates at a density of 50,000 cells/well. The cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 ug/ml streptomycin. Cells on growth medium were plated on 96-well plate at 11,000 cells/well and placed in the incubator. After 2 days, media were replaced with differentiation medium (growth medium containing 1% FBS) and 10 uM retinoic acid.

Differentiation medium was replaced every other day and cells were used after 7 days of differentiation. U373 were plated on 12 transwell plates (0.4um polyester ne, Costar) at a density of 50,000cells/well for 72 hrs. Cells were co—cultured together for 24hrs before treatment in differentiation medium (growth medium containing 1% FBS without retinoic acid).

Thereafter cells were treated with l oc-synuclein (Wt, A53T, E46K) in the presence or absence of 10% ia supernatant for 48 hrs. Cell free Supernatants were collected, won at 10000g for 3 min at 4°C, aliquoted and stored at -80 0C. Human IL-6 and IL-8 were ed as described above.

The results of these experiments are shown in Figure 4. Treatment of cells with wild-type (x-synuclein and the mutant isoforms E46K and A53T induced moderate secretion of IL-6 (Figure 4A). The 0t-syn— induced secretion of IL-6 was inhibited in cells treated with the bacteria strains (Figure 4A). The reduction in IL-6 secretion was greatest on administration of MRX0029.

[Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Example 3 — Efi‘icacy erial inocula to reduce NFK’B activation Summary Activation of the NFKB promoter leads to the production of proinflammatory cytokines including IL- IB, IL-lOL, IL-18, TNFoc and IL-6. The NFKB promoter can be activated by OL—synuclein and LPS by stimulating the TLR4 ligand. ons in oc-synuclein, such as oc-synuclein A53T, are implicated in familial Parkinson’s. ent of neuronal cells with LPS simulates Parkinson’s caused by environmental factors. The ability of itions comprising bacterial strains according to the invention to inhibit the activation of the NFKB promoter was investigated.

Material and Methods Bacterial strain Megasphaera massiliensis MRX0029 Cell line Human Hek blue TLR4 were purchased from Gen (cat n. hkb-htlr4). Human Hek blue TLR4 were grown in DMEM high glucose (Sigma Aldrich, cat n. D-6171) supplemented with 10% PBS, 1% Pen Strep, 4mM L-Glut, Normocin and 1X HEK Blue selection solution.

Method Once grown the Human Hek blue cells were plated in 96 well plates at 25,000 cells/well in 4 replicates.

One set of cells were treated with oc-synuclein A53T (lug/mL) alone or with 10% of bacteria supernatant from 9 for 22h. The second set of cells were treated with LPS (10 ng/mL, from Salmonella enterica serotype urium, Sigma Aldrich, cat 11. L6143) alone or with 10% of ia atant from MR029 for 22h. The cells were subsequently spun down and 20ul of the supernatant was mixed with 200ul of Quanti Blue reagent (InvivoGen, cat 11. rep-qb2), incubated for 2 h and ance read at 655nm.

Results The results of these experiments are shown in Figure 5 and 6. Figure 5 shows that the activation of the NFKB promoter by 0L-synuclein is inhibited by MRx0029. Figure 6 shows that the activation of the NFKB promoter by LPS is inhibited by MRX0029.

Example 4 — Efficacy ofbacterial inocula to alter antioxidant capacity The y of compositions comprising bacterial strains according to the invention to alter the antioxidant capacity. The antioxidant capacity of the bacterial strain was established using the well- knoerTS (2,2'-azino-bis(3-ethylbenzothiazolinesulphonic acid)) assay.

[Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm Bacterial strain Megasphaera massiliensis MRx0029 Method Bacterial cells (106 or greater) were collected and centrifuged. They were resuspended in assay buffer (using three times the pellet ). The suspension was sonicated on ice for 5 minutes and then spun down at 12,000 x g for 10 minutes. The supernatant was removed and measured using the ABTS assay kit produced by Sigma Aldrich (code ), in accordance with manufacturer’s instructions.

Results The results of these experiments are shown in Figure 7. Figure 7 shows that 9 has an antioxidant capacity of approximately 2mM ed to Trolox.

Example 5 — Efi‘icacy erial inocula to alter lipidperoxidation levels Summary The ability of compositions comprising bacterial strains according to the invention to alter lipid peroxidation levels was investigated. The thiobarbituric reactive substances assay (TBARs) was used to measure the by-products of lipid peroxidation.

Material and Methods Bacterial strain haera massiliensis MRx0029 Method Bacterial cells (106 or greater) were collected and centrifuged, a wash step was performed with isotonic saline before the pellet was re-suspensed in potassium chloride assay buffer. The suspension was sonicated on ice for 10 minutes and then spun down at 10,000 x g for 10 minutes. The supernatant was removed and the level of lipid peroxidation evaluated using the thiobarbituric ve substances assay.

Results The results of the experiments are shown in Figure 8. Figure 8 shows that MRX029 is able to inhibit lipid peroxidation by approximately 20 %, which is a higher antioxidant capacity than the positive control, butylated hydroxytoluene (1% w/v).

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Example 6 — Efi‘icacy ofbacterial inocula on histone ylase ty Summary The ability of compositions comprising bacterial strains according to the invention to alter histone deacetylase activity was investigated. Dysregulation of histone deacetylase has been implicated in the pathogenesis associated with age-associated neurodegenerative diseases.

Material and Methods Bacterial strain Megasphaera massiliensis MRx0029 Cell line The cell line HT-29 was used because e deacetylase is present.

Method Cell free supernatants ofstationary phase bacterial cultures were isolated by centrifugation and filtering in a 0.22 uM filter. HT-29 cells were used 3 days’ post nce and stepped down in 1 mL DTS 24 hours prior to commencement of the experiment. The HT-29 cells were challenged with 10 % cell free supernatant diluted in DTS and was is left to incubate for 48 hours. Nuclease proteins were then extracted using the Sigma Aldrich Nuclease extraction kit and samples were snap frozen prior to HDAC activity measurement. HDAC activity was assessed etrically using the Sigma Aldrich (UK) kit.

Results The results of the experiments are shown in Figure 9. Figure 9 shows that MRx0029 is able reduce the levels of histone deacetylase activity.

Example 7 — Level 0findoleproduction in ia Summary The y of the bacteria of the invention to produce indole was investigated. Indole has been implicated in attenuating inflammation and oxidative stress.

Material and Methods ial strain Megasphaera massiliensis MRx0029 ATCC 11775 is a bacterial reference strain that is known to e indole.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm Method Intact bacterial cells in stationary phase were incubated with 6mM Tryptophan for 48 hours. Bacterial species which possess the enzyme tryptophanase will utilise tryptophan as a ate to produce indole. Following the 48 hour incubation period, the supernatant was removed and added to Kovac's reagent for quantification of indole. Standards, stock solutions and reagents were prepared using standardised methods validated se.

Results The results of the ments are shown in Figure 10. Figure 10 shows that MRx0029 has the capacity to produce indole from tryptophan, at concentrations of approximately 0.2mM.

Example 8 — Level 0fkynurenineproduction in bacteria Summary The ability of the bacteria of the invention to produce kynurenine was investigated. Dysregulation of the kynurenine pathway can lead to activation of the immune system and the accumulation of potentially neurotoxic nds. Alterations in the kynurenine metabolism may be involved in the development of son’s es.

Bacterial strain Megasplzaera massiliensis MRx0029 DSM 17136 is a strain ofBacteroides copricola that is known to produce kynurenine.

Method Cell free supernatants ofstationary phase bacterial cultures were isolated by centrifugation and filtering in a 0.22 uM filter and frozen until use. Kynurenine standards, stock solutions and reagents were prepared using standardised methods validated in—house. Sample were d with trichloroacetic acid and centrifuged at 10,000xg for 10 minutes at 4°C. The supernatant was collected and dispensed into a 96 well plate. Ehrlich’s reagent was used for kynurenine detection and added at a ratio of 1:1.

Results The results of the experiments are shown in Figure 11. Figure 11 shows that MRx0029 has the capacity to produce kynurenine at a tration of imately 40 uM.

Example 9 — Levels ofDopamine, DOPAC andHVA in striatum in bacteria-treatedMPTP mice Parkinson's disease is a common neurodegenerative er whose cardinal clinical features include , slowness of movement, stiffness, and postural instability. These ms are primarily utable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the consent loss of their ting nerve fibers in the striatum [60]. Mice treated with MPTP (1- methylphenyl-1,2,3,6-tetrahydropyridine) selectively lose significant numbers of nigrostriatal [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm dopaminergic neurons [61]. MPTP induced loss of dopaminergic cells in ntia nigra mimics the clinical condition in Parkinson’s disease and is therefore a usefiil model to test arkinsonian drugs.

The aim of this study was to evaluate the s ofMRX0029 anaerobic ia using MPTP lesioned mice. 48 male mice were allocated to 4 different treatment groups (groups A, B, E and I, with n=12 animals in each group). The ent groups are shown in Table 1 below and the project time course is outlined below.

Table 1: Treatment groups Treatment Lesion Group n Sa e y evef t | 1 Substance Dose Route Schedule Substance Dose Route Schedule A 12 Vehicle (PBS) p.o. 18 days: day(- Vehicle (0.9% i.p. dayO 14) - day3 saline) B 12 Vehicle (PBS) p.o. 18 days: day(- MPTP 4x 20 i.p. dayO 14) - dav3 mG/kq MR 0029 E 12 Mega§phaera S1/S2 2 x10"8 p.o. 18 days: day(- MPTP 4x 20 i.p. dayO bacteria 14) - day3 mg/kg (chi) Vehicle (PBS) - - p.o. 18 days: day(- | 12 14) _ days MPTP 4x 20 i.p. dayO mg/kg oindazole 50 mg/kg i.p. day0 (2x i.p.) Groups A, B, E and I were treated daily for 18 days via oral gavage with either bacteria (MRx0029 — group E), or vehicle (PBS). Oral treatment d 14 days before MPTP lesion. Group 1 animals received a daily vehicle (PBS) p.o. (per oral) treatment and were injected i.p. (intraperitoneal) with the reference drug 30 min before and 90 min after first MPTP on day 0. The application volume for p.o. and vehicle treatment was 200 pl per mouse. ia strain of group B was from glycerol stocks (gly).

For oral treatment, s for applications were stored in vial containing 70% Ethanol and were flushed before and after each use with distilled water. Every treatment group had its own gavage and ethanol vial and distilled water vial. The tubes and gavages were not changed between the groups.

Directly before treatment each syringe was flushed with N2.

On day 0 MPTP (20 mg/kg bodyweight (b.w.) 4 times, 2h inter-treatment interval) was injected i.p. in animals of groups B, E and 1. One group of animals (A) was sham lesioned by i.p. administration of the MPTP vehicle (0.9% saline). The application volume was 10 ul per g body weight. Weighing of the animals was performed before the MPTP treatment to dose the animals ing to their actual body weight. Afterwards animals received the daily p.o. treatment.

Formulation ofpreparationsfor dosing andpreparation ofglycerol stocks for dosing D [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm MRX0029 Megasphaera sp.

Name of the Bacteria strain: Storage condition/stability: -80 °C Vehicle: 1x PBS Treatment dosages: 2 x 10"8 bacteria Administration: 200m Lot N umber: n/a For ent group E (MRX0029) l.) 1 glycerol stock was taken from the -80 °C freezer and placed under anaerobic conditions (anaerobic jar with sachet) at 37 °C in order to thaw (this took 30—40 mins). 2.) The completely thawed glycerol stock was centrifuged at 6000 x g for 10 min at room temperature. 3.) The supernatant was ded without disturbing the pellet (e.g. using a pipette). 4.) 4.22 mL of e pre-warmed (37 °C) 1 x PBS was added and gently mixed using a e.

.) The mice were dosed with 200 ”L of the bacterial solution. The animals were dosed within 15 mins after resuspension of the pellet with PBS.

Reference drug groug formulation Name of the Reference item: 7-Nitroindazole Storage condition/stability: -20°C Vehicle: Peanut 0” Treatment dosages: 50 mg/kg Administration: i.p. (30 min before and 90 min after 1StMPTP treatment) Batch Number: MKB86671V The appropriate amount of 7-Nitroindazole was dissolved in peanut oil to reach the final concentration of 50 mg/kg.

[Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Materials and s Animals Mouse line: C57BLT<A6J (IJAXTM Mice Strain) Provider: Charles River Laboratories Age at start: "10 weeks Sex: Male Number of animals: 48 Specific handling ofAnimals and Randomization Gloves were d between each treatment group and sprayed with 70% l solution between each cage of the same group to minimize the risk of contamination whenever animals were handled (e. g.: ent, behavioural testing, cleaning and tissue sampling).

The treatment was at random and ated daily so as to prevent the same groups being treated at the same time each day. Animals were randomized per cage at the tissue sampling.

Tissue sampling andprocessing On day 4 animals of all groups were sacrificed and brains were collected. Therefore, mice were deeply anesthetized by Pentobarbital injection (600mg/kg).

Blood (approximately 500 pl) was collected by heart re. Mice were then ardially perfused with 0.9% saline and brains were removed and hemisected. The left hemisphere was subdivided into striatal tissue (for HPLC), substantia nigra tissue as well as residual brain, weighed and immediately frozen and stored at -80°C. Instruments and surfaces which were in contact with the animals had to be cleaned with 70% l before the next animal was dissected.

Biochemical Analysis ofDopamine, DOPAC andHVA levels with HPLC in striatum The striatal samples (n=6 from each treatment group; total 24 samples) were mixed at a ratio of 1:10 (w/v) with 0.2 M perchloric acid including 100 uM EDTA-2Na and nized at 0 °C in a glass- pestlemicro-homogenizer. Following standing for 30 min on ice, the homogenates were fuged at ,000 RPM for 10 minutes in a refrigerated centrifuge Biofuge Fresco (Heraeus Instruments, Germany). The supernatants were carefully aspirated and mixed with 0.4 M Na—acetate buffer, pH 3 at a ratio 1:2 (v/V) and filtered through a 0.22 um centrifugal filter (Merck Millipore, Germany) for 4 min at 14 000 g at 4 °C. The filtrates were stored at -80 °C before HPLC is.

HPLC analysis Concenions of DA, DOPAC and HVA in the striatal samples were determined by column liquid chromatography with electrochemical detection [62;63]. The HPLC system (HTEC-500, Eicom Corp, [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Kyoto, Japan) including a pulse-free microflow pump, a degasser and an amperometric detector equipped with a glassy-carbon electrode operating at +0.45 V vs. an Ag/AgCl ref. electrode was used.

Samples were injected by use of a CMA/200 Refrigerated Microsampler (CMA/Microdialysis, Stockholm, Sweden). The chromatograms were recorded and ated by use of a computerized data acquisition system (DataApex, Prague, Czech Republic). DA, DOPAC and HVA were separated on a 150 x 2.1 i.d. mm column (CA5-ODS, Eicom Corp., Kyoto, Japan). The mobile phase ted of 0.1 M phosphate buffer at pH 6.0, 0.13 mM EDTA, 2.3 mM sodium-l-octanesulfonate and 20 % (v/v) methanol. The ion limit (signal-to-noise ratio = 3) for DA was estimated to 0.5 fmol in 15 ul (0.03 nM) injected onto the column.

Results Administration of bacteria strains was well tolerated by the animals. On the MPTP lesion day and if necessary on the day afterwards a red light was used to warm the s. If animals were in bad conditions (felt cold, dehydrated, abnormal behaviour), they were supplied with wet food and subcutaneous saline treatment if necessary.

For analysis of Dopamine, DOPAC and HVA , striatal tissue of 6 animals per ent group were used. Data were analyzed by using Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test or One-way analysis of variance followed by Bonferroni post hoc test (A vs. all(*), B vs. all, 1 vs. all (#)). */# = p<0.05; ** = p<0.01; *** = p<0.001.

The healthy animals in group A had high levels of Dopamine, DOPAC and HVA whereas MPTP treatment in group B reduced this and the positive control (group I) recovered the production to some degree (Figure 12). Animals p I tended to have higher Dopamine levels than the bacteria treated group and group B. DOPAC (a Dopamine metabolite) levels in general were significantly lower in animals of group B compared to DOPAC levels of unlesioned animals of group A (Figure 12B). cantly, treatment with 9 (group E) was found to recover tion of Dopamine and DOPAC (Figures 12A and 12B, respectively). Treatment with MRX0029 may therefore be useful for treating or preventing egenerative disorders.

Example 10 — Efficacy ofbacteria to alter neurite outgrowth Neurite wth is an ant process for the development of connections between neurons. The ability of bacterial strains and c acids to induce neurite outgrowth was therefore tested by measuring transcriptional levels of microtubule associated protein MAP2, a specific neuronal differentiation marker.

Bactegfi'train Megasphaera massiliensis MRX0029.

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm Method SHSY5Y were plated in 10cm petri dishes a density of 2x106 cells. After 24h cells were treated in differentiation medium (growth medium containing 1% FBS without RA) with 10% bacteria supernatants or YCFA+, 10uM RA, 200uM hexanoic acid or 200uM valproic acid, for 17 hrs. There after entative images were taken using phase st EVOS XL core microscope at 40X/0.65 magnification. Cells were collected, and total RNA was isolated according to RNeasy mini kit protocol (Qiagen). cDNAs were made using the high capacity cDNA reverse transcription kit (Applied Biosystems). Gene sion was measured using qPCR. GAPDH was used as internal control. Fold change was calculated according to the 2M“) method.

Immunofluorescence and Confocal copy Cells were seeded onto 8 well chamber slides (Marienfeld Laboratory Glassware) at 5x104 cells/well overnight and were treated with 10% bacterial supernatant for 24 hrs. For differentiation, cells were treated with 10nM Retinoic acid for 5 days before treating with ial supernatant. Cells were then fixed with 4% paraformaldehyde in PBS for 20 s at room temperature (RT). Fixed cells were washed with PBS, and permeabilized with 1% Triton X-100 in PBS for 10 minutes. After washing with PBS, the slides were incubated with blocking buffer (4% BSA/PBS) for 1hr at RT before adding anti-MAP2 antibody (sc-74421, Santa Cruz Biotechnology Inc) diluted in 1% BSA/PBS for 12hr at 4°C. They were then washed twice with PBS, ed by tion with Alexa Flour 488 conjugated anti-mouse (Molecular Probes Inc) and Alexa Flour 594 conjugated Phalloidin 757, Abcam) for 1hr at RT. After washing 3X with PBS, the slides were mounted with Vectorshield containing DAPI (Sigma, Aldrich). Slides were viewed using a Zeiss Axioscope microscope equipped with a 63x/1.2 W Korr objective and filter sets suitable for detection of the fluorochromes used. Manual exposure times for the digital acquisition of images immuno-labelled with MAP-2 were kept constant ng comparison between different wells and treatments. Phalloidin (F-actin) and DAPI exposure times varied to suit the field of view. ised fields of view were acquired using a QImaging camera controlled by Image Pro Plus software. Images were saved as TIFs and opened in Adobe Photoshop CC 20151.2 and overlays of the MAP-2, DAPI and Phalloidion images overlaid and merged.

Representative images were selected to illustrate the differences in abundance and location of the proteins examined s The results are shown in Figure 13. Figure 13A shows representative microscopy images of undifferentiated SHSY-5Y cells incubated with each of the acids and bacteria supernatants. Treatment of cells with MRX0029 d a neuron-like phenotype, showing similar features to cells treated with retinoic acid (which is used for terminal differentiation of neuroblastoma cells), where cell bodies are bigger? pyramidal-shaped, with neurites and processed branching out to network with neighbour1cells. gure 13B shows that 9 significantly upregulates MAP2 in undifferentiated [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm neuroblastoma cells. Phalloidin (an actin cytoskeleton-binding agent) ng further proved a different arrangement of cytoskeletal structure in cells treated with MRx0029, further supporting the neuronal differentiation hypothesis for MRx0029 (Fig. 13B).

Example 11 — Efficacy ofbacterial inocula to reduce oxidative levels in cells Background The tion of reactive oxygen species contributes to the pathology of neurodegenerative diseases.

The ability of bacterial strains to protect entiated SHSY-5Y and U3 73 cells from reactive oxygen species (ROS) generated by treatment with Tert-Butyl Hydrogen Peroxide (TBHP) was investigated.

Material and Methods Bacterial strain haera massiliensis MRX0029 Method SHSY-5Y cells were plated in black flat bottom 96 well plate at density of 5000 cells/well and placed in the C02 incubator. After 24 h, media were replaced with differentiation medium (growth medium containing 1% FB S) and 10 uM retinoic acid. Differentiation medium was replaced every other day.

On Day 10 the differentiation medium was removed and cells were washed with pre-warmed PBS and stained with 10uM DCFDA molecular probe for 20 mins in growth medium containing 1% FBS. Then cells were washed with pre-warmed PBS again and treated with 100uM TBHP in the presence or absence of 10% bacteria supernatant for 2h. Fluorescence intensity was measured using TECAN plate reader at Ex/Em 485/530 nm.

Results The s of the experiments are shown in Figure 14. Figure 14b shows that 9 is able to inhibit ROS production in differentiated SHSY-5Y neuroblastoma cells. 9 did not have an effect on the generation of ROS in U373 astroglioblastoma cells (Figure 14a). This shows that this aspect of the antioxidant effect is neuron-specific.

Example 12 — neuroprotection RA-differentiated SHSY—5Y cells were treated with MPP+, the active metabolite of MPTP, a chemical widely used to mimic in vitro and in vivo some of the features of PD pathology. Cell viability was ed as the rate of mitochondria respiration (Figure 15). Both MRx0005 and MRx0029 showed significant effects and promote per se an increase of the mitochondria metabolic activity in SHSY-5Y cells. MRX0029 showed complete protection from MPP+, restoring cell viability nearly to the same level of ted cells and higher than quercetin positive control. 5 protection was about 20% comtho YCFA-MPP+ treated sample, about the same ed for the quercetin positive control(Fig. l .

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm ed set by kjm Example 13 — Further analysis ofthe mechanism ofhistone deacetylation inhibition Introduction The gut microbiota, with its immense diversity and metabolic capacity, represents a huge metabolic reservoir for production of a vast variety of molecules with ial to influence HDAC activity. Few studies have assessed the HDAC tory activity of microbially-derived metabolites other than te, which has been shown to t HDAC and is associated with improvement of motor function in Huntington’s disease [64]. The inventors therefore sought to determine which lites are responsible for HDAC inhibition and further elucidate the isms by which inhibition is Material and Methods Bacterial culture and cell-free supernatant collection Pure cultures of bacteria were grown anaerobically in YCFA broth until they reached their nary growth phase. Cultures were centrifuged at 5,000 x g for 5 minutes and the cell-free supernatant (CFS) was filtered using a 0.2 uM filter (Millipore, UK). 1 mL aliquots ofthe CFS were stored at -80 °C until use. Sodium butyrate, hexanoic and valeric acid were obtained from Sigma Aldrich (UK) and suspensions were prepared in YCFA broth.

SCFA and MCFA fication ofbacterial supernatants Short chain fatty acids (SCFAs) and medium chain fatty acids (MCFAs) from bacterial supernatants were analysed and quantified by MS Omics APS as follows. Samples were acidified using hydrochloride acid, and deuterium ed internal standards where added. All samples were analyzed in a randomized order. Analysis was performed using a high polarity column (ZebronTM ZB-FFAP, GC Cap. Column 30 m x 0.25 mm x 0.25 mm) installed in a GC (7890B, Agilent) coupled with a quadropole or (59977B, Agilent). The system was controlled by ChemStation (Agilent). Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in [65].

Specific HDAC activity analysis Specific HDAC inhibition activity was analysed for HDAC], 2, 3, 4, 5, 6, 9 using fluorogenic assay kits for each type of HDAC (BPS Bioscience, CA). Assays were conducted according to manufacturer’s instructions and each sample were performed in ates. Cell free supernatants were diluted 1 in 10 and exposed to specific HDAC proteins provided in the kit to maintain consistency n methods.

Results HistorDacetylase—inhibiting gut commensal microbial metabolites are butyrate and valeric acid [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm MRx0029, whose supernatant showed strong HDAC inhibition in both HT29 whole cells and HT29 cell lysates, produced valeric acid and ic acid at mean concentrations of 5.08 mM and 1.60 mM, respectively (Figure 16A and C).

To investigate which metabolites were responsible for the strain-induced HDAC inhibition, different concentrations of hexanoic acid, valeric acid and sodium butyrate were measured for their HDAC inhibition on whole HT-29 cells and on HT-29 cell lysate. The s in Fig. 16B show significant (P<0.05) inhibition of HDAC activity by sodium butyrate on whole cells as well as on the cell lysate, while hexanoic acid did not show significant inhibitory activity. c acid inhibited total HDAC ty (* (p<0.05), ** (p<0.005), *** (P<0.001), **** (p<0.0001)).

Potent total HDAC inhibitors investigated target class IHDA Cs.

The specific HDAC inhibition profile of the test bacteria strain was investigated. Specific HDAC inhibition assays (BPS ence, CA) were carried out for Class 1 and Class II HDACs. The ability of the bacterial strain to inhibit HDAC s was compared to butyrate, hexanoic and valeric acid.

Our results demonstrate that MRX0029, is a very potent inhibitor of Class 1 HDAC enzymes (HDACl, 2 and 3). Inhibition of class II HDACs was not as significant (data not shown).

Discussion The strain with HDAC inhibitory activity produced significant amounts of valeric acid and hexanoic acid as well as significant amounts of sodium butyrate (Figure 16C). When tested as pure substances, valeric acid and sodium butyrate resulted in significant HDAC inhibition (p<0.0001).

Interestingly, the results for specific HDAC activity show that the tested strain is a potent tor of Class I HDACs, and particularly HDAC2 e 17 and 18). Class I HDACs (HDACl, 2, 3 and 8) reside in the nucleus and are tously expressed in several human cell types. HDACs 1—3 share more than 50% homology, but have distinct ures and cellular functions [66]. They are primarily involved in cell survival, eration and differentiation, and thus their inhibition may be useful is wide array ofdiseases [67]; [68]; [69]; [70]; [71].

Example 14 — Level ofBDNF secretion in SHSY-5Y cells ound Brain-derived neurotrophic factor (BDNF) is a ubiquitous molecule in the brain associated with neural development, neuro—protection and neuro-regeneration. BDNF not only protects against neurodegeneration but also mental disorders like depression and anxiety, which are quite common amongst patients diagnosed with PD or AD.

[Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm Methods SH-SY5-SY were plated in 24 wells plate at density of 60,000 cells/well and placed in the incubator.

After 24 h, media were replaced with differentiation medium (growth medium containing 1% FBS) and 10 uM retinoic acid. Differentiation medium was ed every other day and cells were used on day 10 of differentiation. For the treatment differentiation medium was removed and replaced with 450ul of full growth media and 50 ul of bacteria SN was added to the treated wells or YCFA+ was added as negative Control.

Results The results are shown in Figure 19, which shows that administration ofMRX0029 in combination with retinoic acid increases the secretion of BDNF from differentiated neuroblasoma cells. Compositions sing commensal bacteria and organic acids may therefore be useful in therapy.

Example 15 — Metaboliteproduction — metabolites in the brain ound Metabolites present in bacteria atants can directly influence the host response to oxidative stress, o-cell communication and neuroprotection. Metabolites that play a key role in neurological processes were ed during the ex vivo screening in brain tissue ofmice fed with MRXOOOS and MRX0029.

Methods Animals BALBc (Envigo, UK) adult male mice were group housed under a 12 h light-dark cycle; rd rodent chow and water were available ad libitum. All experiments were performed in accordance with European guidelines ing approval by University e Cork Animal Ethics Experimentation Committee. Animals were 8 weeks old at the start of the experiment.

Study Design Animals were allowed to habituate to their holding room for one week after arrival into the animal unit. They receive oral gavage (200uL dose) of live biotherapeutics at a dose of 1 X 109 CFU for 6 consecutive days between 15:00 and 17:00. On day 7, the animals are decapitated, and tissues are harvested for experimentation.

Tissue Collection Animals were sacrificed in a random fashion regarding treatment and testing condition; sampling occurred between 9.00 am. and 1:00 pm. Trunk blood was collected in potassium EDTA (Ethylene Diamia‘etra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at —80 0C for further analysis. The brain was quickly excised, dissected and each brain region was snap- [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm frozen on dry ice and stored at -80 0C for further analysis. Spleen was removed and processed immediately after culls for ex—vivo immune stimulation. inal tissue (2 cm segments of ileum and colon closest to the caecum were excised, and the furthest lcm of tissue from the caecum were used) were mounted into the Using chambers for inal permeability assay. The caecum was removed, weighted and stored at -80 °C for SCFAs is.

Monoamine Analysis Neurotransmitter concentration was analysed by HPLC on samples from the brainstem. Briefly, brainstem tissue was sonicated in 500 pl of chilled mobile phase spiked with 4 ng/40 ul of N-Methyl -HT (Sigma Chemical Co., UK) as internal standard. The mobile phase contained 0.1 M citric acid, 5.6 mM octane-l-sulphonic acid (Sigma), 0.1 M sodium ogen phosphate, 0.01 mM EDTA (Alkem/Reagecon, Cork) and 9% (v/v) methanol (Alkem/Reagecon) and was adjusted to pH 2.8 using 4 N sodium ide /Reagecon). Homogenates were then centrifuged for 15 min at 22,000 X g at 4 °C and 40 ul of the atant injected onto the HPLC system which consisted of a SCL 10- Avp system controller, LECD 6A electrochemical or (Shimadzu), a LC-10AS pump, a CTO- 10A oven, a A autoinjector (with sample cooler maintained at 40 C) and an online Gastorr er (ISS, UK). A reverse-phase column (Kinetex 2.6 11 C18 100 X 4.6 mm, Phenomenex) maintained at 30 °C was employed in the separation (Flow rate 0.9 ml/min). The glassy carbon working electrode combined with an Ag/AgCl reference electrode azu) operated a +0.8 V and the chromatograms generated were analyzed using Class-VP 5 software dzu). The neurotransmitters were identified by their characteristic retention times as determined by standard injections, which run at regular intervals during the sample analysis. The ratios of peak heights of analyte versus internal standard were measured and compared with standard injection. Results were expressed as ng of neurotransmitter per g fresh weight of tissue.

Metabolite analysis For GC-metabolite analysis, samples of bacterial supernatants were derivatized with methyl chloroformate using a slightly modified version of the protocol described in [72]. All samples were analyzed in a randomized order. Analysis was performed using GC (7890B, Agilent) coupled with a quadropole detector B, Agilent). The system was controlled by ChemStation (Agilent). Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in [65].

For fatty acid analysis samples were acidified using hydrochloride acid, and deuterium labelled internal standards where added. All samples were analyzed in a randomized order. Analysis was med using a high polarity column (ZebronTM ZB-FFAP, GC Cap. Column 30 m x 0.25 mm x 0.25 mm) installed in a GC (7890B, Agilent) coupled with a quadropole detector (59977B, Agilent). The system was cDolled by ChemStation (Agilent). Raw data was converted to netCDF format using [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in [65]. s — neurotransmitter production The results are shown in Figure 20, which shows that in brains of mice fed with MRx0029, noradrenaline levels are increased (p=0.0507), accompanied with a slight increase of serotonin and 5- HLAA. These data support the metabolite analysis set out below, suggesting that MRx00029 is a major producer of 4-hydroxyphenylacetic acid, a known antioxidant [73]. More importantly, 4- hydroxyphenylacetic acid is a synthetic intermediate ofdopamine and norepinephrine and an important bio-active molecule [74]. In fact, in PD, degenerative changes extend beyond the nergic system, affecting equally the serotonergic and energic systems, which in turn leads to decreased levels of serotonin (5-hydroxytryptamine, 5-HT) and enaline (norepinephrine) in both striatal and extra-striatal structures [75]. L-DOPA targets mainly the dopamine-related es of PD, however it does not address the decreases in both 5-HT and noradrenaline. Adding to this is that the longer is the duration ofL-DOPA treatment, the more Visible are a range of motor and nonmotor complications (e.g. dyskinesia, psychiatric symptoms) [76]. Therefore, these data trate that ia that produce organic acids, such as 4-hydroxyphenylacetic acid, may be useful in therapy, in particular in the treatment of neurodegenerative diseases.

Results — metabolite production Metabolites present in bacteria supernatants can directly influence the host response to oxidative stress, cell-to-cell communication and neuroprotection in the specific. lites in the atant of cultures of MRX0029 and MRX0005 were analysed and the results are shown in Figure 21.

A few metabolites showed a striking difference between the two strains analysed. The concentration of succinic acid was particularly elevated in MRX0005. Interestingly, the ratio sample/media for 4- hydroxyphenylacetic acid was significantly higher in MRx0029 (Fig. 21A).

Fatty acid is in the supernatants revealed an interesting dichotomy in the two strains: 5 produced mainly acetic and propanoic acid, while MRx0029 produced butanoic, pentanoic and hexanoic acid, both in the linear and branched forms (Fig. 21B). The two s looked very different and in particular, the tion of succinic acid and 4-hydroxyphenylacetic acid by MRx0005 and MRx0029 tively was notable (Figure 21A). Furthermore, 5 seems to produce more C2 and C3 short chain fatty acids, while MRx00029 produced more C4 (butyrate) and both linear and branched medium chain fatty acids, including hexanoic acid.

Succinic acid is a Krebs cycle metabolite ed in oxidative phosphorylation. Oxidative phosphorylation complex is akey step for synaptic trafficking of proteins and vesicles to proximal and distal Dons [77]. Its dysfunction has been reported in neurodegenerative disorders including Alzheimer’s disease, Parkinson’s e and Spinocerebellar ataxia type 1 [78]. These findings are [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm particularly interesting as succinic acid can augment mitochondrial activity and support able neurons in neurodegenerative disease d to misfolded proteins including PD [79]. BDNF and succinic acid have both a similar tive activity not only in neuro-degeneration but also in mental disorders like depression and anxiety, which are quite common amongst ts diagnosed with PD or AD.

Figure 21B also demonstrates that MRX0029 is a butyrate oic acid) producer. This may be significant because butyrate has a known role is reducing impermeability of the blood brain barrier, which has a neuroprotective effect [80]. This property of 9 (and other neuroprotective bacteria) may contribute to its efficacy.

Example I6 - tion ofthe mRNA expression oftightjunction proteins by MRx0029 Since recent evidence suggests that intestinal dysfunction and inflammation is a non-motor symptom associated with PD, the ability of the bacterial strains of the invention to cause any intestinal barrier dysfunction was investigated. HT29-mtx epithelial, mucin-producing cell monolayers [81] were used as an in vitro model to evaluate gut barrier disruption and immune stimulation following treatment with MRx0005 and MRx0029. Differentiated HT29-mtx cells exposed to phorbol 12-myristate acetate (PMA) secreted a cant amount of IL-8; in contrast treatment for 24h with MRx005 and MRx0029 bacterial supernatants, induced an even lower secretion of IL-8 compared than both untreated and YCFA-treated cells (Fig. 22A).

The ability of MRx0005 and MRx0029 to regulate epithelial permeability by modifying intracellular signal transduction involved in the expression and localization of proteins involved in the gut barrier formation was then investigated.

RNA was ed and tative RT-PCR (qRT-PCR) analysis was performed to terize the changes in gene expression of tight junction proteins during incubation with MRx0005 and MRx0029.

The administration of MRx0029 enhanced Occludin, Vlillin, Tight Junction Protein 1 and 2 (respectively TJPl and TJP2) mRNA sion after 2h incubation (Fig. 22B). In contrast, exposure to MRx0005 did not alter the gene expression of tight junction proteins indicating that the two strains act differentially on the intestinal barrier.

The in vitro s were compared with data from the ex vivo parallel analysis on the gut of mice fed with MRx0005 and 9. Gene expression of TJP2 and in was quantified in the colon and ileum. The ex vivo data perfectly mirror the in vitro data as MRx0029 was able to significantly up- regulate TJPl and Occludin (p=0.073) in the colon region of the murine intestine (Fig. 22C+22D).

MRXOQ was also able to decrease the permeability function in the colon of the same mice (Fig.22E+2 .

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Materials and methods - RNA extraction and qPCR analysis Total RNA was extracted using the RNeasy mini kit n, Manchester, JUK) according to the manufacturer‘s instructions, and the RNA concentration determined by absorbance at 260/280nm using a ophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington, DE). For mRNA expression is, cDNA was prepared from total RNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, UK) according to the cturer's instructions. The reverse transcription reactions were performed in a Thermo cycler (Biometra, y) at 25°C for 10min, 37°C for 120min, and 85°C for 5 min, hold on at 4°C. Resulting cDNA was amplified in duplicates by the SYBR-Green PCR assay, and products were detected on QuantStudio 6 flex real-time PCR machine ed Biosystems, UK) using a standardised profile (initial denaturation of 95°C for 10 minutes, followed by 40 cycles of 15 seconds of denaturation at 95°C and 60 seconds of annealing/extension at 60/65°C, depending on the primers. A dissociation stage was added after the 40 cycles to generate a melting curve. Analysis was performed using the Applied Biosystems QuantStudio Real-Time PCR re V1.2. The primer sequences for Actin, Villin, Occludin TJPl and TJP2 are provided in the sequence listing.

Example 16 — Stability testing A ition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25°C or 4°C and the container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%, ?5%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3 , 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols.

Example 1 7 Methods Animals The animals and study design used were the same as for Example 15. ial strains 0 755: Parabacteroides distasonis (MRX005) 0 Megasphaera massiliensis (MRX0029) Tissue Collection Animals were sacrificed in a random fashion ing treatment and testing condition; sampling Diamioccurrfletween 9.00 am. and 2:30 pm. Trunk blood was collected in potassium EDTA (Ethyleneetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm -80 °C for further is. The brain was y excised, dissected and each brain region was snap- frozen on dry ice and stored at —80 °C for further analysis. Spleen was removed, collected in 5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758 Sigma + 10 % FBS (F7524, Sigma) + 1% Pen/Strep (P4333, Sigma)) and processed immediately after culls for o immune stimulation. Intestinal tissue (2 3cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm 2cm oftissue from the caecum were used) were mounted into the Ussing chambers for intestinal permeability assay. The caecum was removed, weighted and stored at -80 °C for SCFAs analysis.

Monoamine Analysis The neurotransmitter tration was ed as described in Example 10 Spleen Cytokine Assay Spleens were collected immediately in 5mL RPMI media following sacrifice and cultured immediately. Spleen cells were first homogenised in this RPMI media, followed by 5 mins incubation with lml of RBC lysis buffer 389001 ROCHE, Sigma). A further 10 ml of RPMI media was added, followed by 200G centrifugation for 5 mins. The supernatant was then d through 40um strainer. Cells were counted and seeded (4,000,000/mL media). After 2.5 h of adaptation, cells were stimulated with lipopolysaccharide (LPS-2 rig/ml) or concanavalin A (ConA-2.5 ug/ml) for 24 h. ing stimulation, the supernatants were harvested to assess the cytokine e using lammatory Panel 1 (mouse) V-PLEX Kit (Meso Scale Discovery, Maryland, USA) for TNFOL, IL-10, IL-lB, Interferon y, CXCL2 and IL6. The analyses were performed using MESO QuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, SECTOR S 600.

Gene Expression Analysis Total RNA was ted using the mirVanaTM miRNA Isolation kit (Ambion/Llife logies, Paisley, UK) and DNase treated (Turbo DNA-free, Ambion/life technologies) according to the manufacturers recommendations. RNA was quantified using NanoDropTM spectrophotometer o Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer's instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent, Stockport, UK) according to the cturer's procedure and an RNA integrity number (RIN) was calculated. RNA with RIN value >7 was used for subsequent experiments. RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA kit (Applied Biosystems, Warrington, UK) according to manufacturer's ctions. Briefly, Multiscribe Reverse Transcriptase (50 U/uL) (1)(2)(1)(10) was added as part of RT master mix, incubated for 25°C for 10 min, 37°C for 2 h, 85°C for 5 min and stored at 4°C. Quantitative PCR was d out using probes (6 carboxy cein - FAM) designed by Ap ' Biosystems to mouse specific targeted genes, while using B—actin as an endogenous control.

Amplifiion reactions contained 1 ul cDNA, 5 ul of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to a total of 10 ul by the addition of RNase-free water. All reactions [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm were performed in triplicate using 96-well plates on the LightCycler®480 System. Thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for amplicon contamination, each run ned no template controls in triplicate for each probe used. Cycle threshold (Ct) values were recorded. Data was normalized using B-actin and ormed using the 2-AACT method and presented as a fold change vs. control group.

Short Chain Fatty Acids Analysis in the Caecal Content Caecum t was mixed and vortexed with MilliQ water and incubated at room temperature for 10 min. Supernatants were obtained by centrifugation (10000 g, 5 min, 4 °C) to pellet ia and other solids and filtration by 0.2um. It was transferred to a clear GC vial and 2-Ethy1butyric acid (Sigma) was used as the internal standard. The concentration of SCFA was analyzed using a Varian 3500 GC flame-ionization system, fitted with a with a ZB-FFAP column (30 m x 0.32 mm x 0.25 mm; Phenomenex). A standard curve was built with different concentrations of a standard mix containing acetate, nate, iso-butyrate, n—butyrate, isovalerate and valerate (Sigma). Peaks were ated by using the Varian Star Chromatography Workstation version 6.0 software. All SCFA data are expressed as . tical Analysis Normally distributed data are presented as mean i SEM; Non-parametric datasets are presented as median with quartile range. Unpaired two-tailed t-test were applied to analyse parametric data and Mann-Whitney test was used for non-parametric. Spearman's rank ation coefficient was ed for the correlation analysis in the pooled datasets. A p value < 0.05 was deemed significant in all cases.

Results — Neurotransmitter production The results in Figure 23 show the effect ofMRx005 treatment on the concentration of neurotransmitters in the brain of mice. Most notably, treatment with MRx005 leads to a decrease in dopamine.

Results — Gene expression Expression of genes for neurotransmitter receptors [serotonin receptor la(5-HTR1a), dopamine D1 receptor, GABA receptor subunit B1, GABAA receptor, NMDA2A (Grin2A) and NMDA2B (Grin2b) receptor], inflammatory markers [IL-1E3, 1L6, CDllb, TNFor and TLR4], and endocrine markers [corticosterone releasing factor (CRF), corticosterone releasing factor receptors 1 and 2 (CRFRl, CRFR2), brain-derived neurotrophin factor (BDNF), vasopressin receptor, oxytocin or, glucocorticoid receptor and mineralocorticoid receptor] were analysed in brain tissue from the ampus, amygdala and prefrontal cortex. hippo Figurefij-38 show the changes in gene expression after MRX005 or MRX0029 treatment in the al, amygdala and prefrontal . Treatment with MRx0029 led to an increase in [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm glucocorticoid receptor sion in the amygdala (Figure 31C). Figure 32A shows that MRX005 significantly increased the expression of BDNF in the amygdala, while ent with MRx0029 significantly increased the expression of TLR4 in the amygdala (Figure 32).

Both MRx005 and MRx0029 can se expression of CD1 lb in the amygdala (Figure 33A), while the expression of IL-6, Grin2a and Grin2b is reduced after MRx005 treatment es 33B-D). In addition, MRx005 and MRx0029 significantly increased the expression of GABRA2 and sed the expression of GABBRl in the amygdala. ent with MRx005 led to a significant increase in the expression ofBDNF in the prefrontal cortex (Figure 35B).

Discussion MRx005 and MRx0029 administration caused changes in gene expression, especially in the amygdala.

Results — Efi”ect on Tpk] and [DO-I expression Figure 39 shows that MRx0029 can significantly increase the expression phan hydroxylase- 1 (Tphl) in the colon and that MRX005 treatment can increase lDO-l expression in the colon. Treatment with MRX005 increased the expression of Tphl and IDOl in the ileum (Figure 40).

Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-l) the first and imiting enzyme in the tryptophan/kynurenine pathway while phan hydroxylase l (Tphl), an m of the enzyme tryptophan hydroxylase, responsible for the synthesis of serotonin. These data suggest that MRx0029 and MRx005 may affect serotonin levels and the tryptophan/kynurenine pathway.

Results — Effect on tryptophan metabolite levels Figure 41 shows the effect of treatment with MRx005 on the levels of circulating kynurenine and tryptophan.

Results — Effect on cvtokine expressionflom splenocytes The ex-vivo splenocyte assay involves challenging the splenocytes (cells isolated from the spleen - a main organ involved in immune defence), with a bacterio- or viral-mimetic challenge.

MRX005 significantly reduced the levels of interferon-y in splenocytes following a challenge with LPS (Figure 42). In addition, MRX005 reduced the levels of eukin-6 and tumour is factor after a challenge with LPS (Figures 44 and 45, respectively). Treatment with MRx0029 led to a reduction in interferon-y, interleukin-18 and interleukin-6 following a challenge with LPS (Figures 42, 43 and 44, respectively).

Treatment with MRx005 and MRx0029 led to an increase in the levels of the chemoattractant CXCLl (Figuru).

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Results — Effect on Caecal Snort Chain Fatty Acid Levels Short chain fatty acids (SCFAs) are produced when non-digestible fibres from the diet are fermented by bacteria in the gut. The effects of MRXOOS administration are shown in Figure 48.

Example 18— Further analysis ofMRX029 and MRX005-ina‘uced changes in gene expression levels Methods Cell line SH—SY5Y cells Bacterial strains 0 755: cteroia’es distasonis (MRX005) 0 Megasplzaera massiliensis 29) qPCR SHSY5Y were plated in 10cm petri dishes a density of 2x106 cells. After 24h cells were treated in differentiation medium (growth medium containing 1% FBS without RA) with 10% ia supernatants or YCFA+, lOuM RA, 200uM hexanoic acid or 200uM ic acid, for 17 hrs. There after representative images were taken using phase contrast EVOS XL core microscope at 40X/0.65 magnification. Cells were collected, and total RNA was isolated according to RNeasy mini kit protocol (Qiagen). cDNAs were made using the high capacity cDNA reverse transcription kit (Applied Biosystems). Gene expression was measured using qPCR. GAPDH was used as internal control. Fold change was calculated according to the 20AM) method. The primer sequences for MAP2, DRD2, GABRB3, SYP, PINKl, PARK7 and NSE are provided in the sequence listing. -labelling and cell imaging Cells were seeded onto 8-well chamber slides (Marienfeld Laboratory are) at 5x104 well overnight and were treated with 10% bacterial supernatant for 24 h. For differentiation, cells were d with 10 nM RA for 5 days before treating with cell-free bacterial supernatant for 24 h.

Afterwards, the cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room ature (RT). Fixed cells were washed with PBS, and permeabilized with 1% Triton X-100 in PBS for 10 minutes. After washing with PBS, the slides were incubated with blocking buffer (4% BSA/PBS) for 1 h at RT before adding anti-MAP2 antibody or B3-tubulin (sc-74421 and sc-80005 respectively, Santa Cruz Biotechnology Inc) diluted in 1% BSA/PBS for 12 h at 4°C. They were then washed twice with PBS, ed by incubation with Alexa Flour 488 ated anti-mouse (Molecular Probes Inc) and Alexa Flour 594 conjugated Phalloidin (abl76757, Abcam) for l h at RT. After washing 3X with PBS, the slinere staining with DAPI and d with Vectashield® (Vector Laboratories). Slides were viewe ng a Axioskop 50 microscope (Zeiss) equipped with a 63x/ 1 .2 W Korr objective and filter [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm sets suitable for detection of the fluorochromes used. Manual exposure times for the digital acquisition of images immuno-labelled with MAP-2 were kept constant allowing comparison n different wells and treatments. Phalloidin (F-actin) and DAPI exposure times varied to suit the field of view.

Randomised fields of view were acquired using a QImaging camera controlled by Image Pro Plus software. Images were saved as TIFF files and opened in Adobe Photoshop CC 2015.1.2. Images of the MAP-2, DAPI and Phalloidin images were then overlaid and merged. Representative images were selected to illustrate the differences in nce and location of the proteins examined.

Immunoblotting SH-SY5Y cells cultured under the indicated conditions described above, treated with MRx0005 and MRx0029 for 24h and then lysed in RIPA buffer containing cocktail of protease inhibitors (Roche Diagnostics, UK). Protein concentration was estimated using the BCA n assay kit (Pierce Biotechnology, Rockford, IL), separated by SDS-PAGE and transferred to a PVDF membrane.

Membranes were then blocked with 5% non-fat dry milk or 5% BSA and ted overnight at 4°C with the primary antibodies (respectively MAP2 and B3-tubulin). The blots were then ted with the riate horseradish peroxidase (HRP)-conjugated ary antibody, and proteins were detected by uminescence ion kit e Biotechnology, Rockford, IL). For both MAP2 and B3-tubulin, B-actin served as a l to monitor protein loading variability amongst samples.

Results and Discussion Gene expression Figures 13a (graph insert) and 49 show the MRx0029 and MRXOOS-induced changes in sion levels of Actin, Villin, Occludin TJPl, TJP2, MAP2, DRD2, GABRB3, SYP, PINKl, PARK7 and NSE.

Microscopy and Immunoblotting Figure 50 shows the change in the level of expression of MAP2 in SHSY5Y cells as determined by confocal microscopy. The expression levels of MAP2 and ulin were also quantified by immunoblot analysis. The results shown in Figure 50M and N indicate that MRX029 induces an increase in the level expression ofMAP2 Sequences SEQ ID N021 (Megasphaera massiliensis gene for 16S ribosomal RNA, partial sequence, strain: NP3 - JX424772.1) Q1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac l gagaagagat gagaagcttg cttcttatca attcgagtgg caaacgggtg agtaacgcgt 121 aagcaacctg cccttcagat ggggacaaca gctggaaacg gctgctaata ccgaatacgt ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm 18. tctttccgcc gcatgacggg aagaagaaag ggaggccttc gggctttcgc aggg 24; gtct gattagctag ttggaggggt aacggcccac caaggcgacg atcagtagcc ; ggtctgagag gatgaacggc cacattggga ctgagacacg gcccagactc ctacgggagg 36- cagcagtggg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgaacga 42; tgacggcctt cgggttgtaa gtta tatgggacga acagggcatc ggttaatacc 48; cggtgtcttt gacggtaccg taagagaaag ccacggctaa gcca gcagccgcgg 54; taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggcgc gcaggcggca 60; tcgcaagtcg gtcttaaaag tgcggggctt aaccccgtga ggggaccgaa actgtgaagc 66; tcgagtgtcg gagaggaaag cggaattcct agtgtagcgg tgaaatgcgt agatattagg 72; aggaacacca gtggcgaaag cggctttctg gacgacaact gacgctgagg cgcgaaagcc 78; aggggagcaa acgggattag ataccccggt ggcc gtaaacgatg gatactaggt _ gtaggaggta tcgactcctt ctgtgccgga gttaacgcaa taagtatccc gcctggggag cgca aaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagtat i gtggtttaat tcgacgcaac aacc agcc ttgacattga ttgctacgga Z aagagatttc cggttcttct tcggaagaca agaaaacagg tggtgcacgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctat cttctgttgc cagcacctcg gact cagaagagac agac aatgcggagg ggga L tgacgtcaag tcatcatgcc ccttatggct tgggctacac acgtactaca atggctctta Z atagagggac gcgaaggagc gatccggagc aaaccccaaa aacagagtcc cagttcggat tgcaggctgc aactcgcctg catgaagcag gaatcgctag taatcgcagg tact Z gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgaa tcac Z acccgaagcc ggtgaggcaa ccgcaaggaa ccagccgtcg aaggtggggg cgatgattgg 150; gtcg taacaaggt SEQ ID N022 (consensus 16S rRNA sequence for Megasphaera massiliensis strain MRX0029) TGAGAAGCTTGCTTCTTATCGATTCTAGTGGCAAACGGGTGAGTAACGCGTAAGCAACCTGCCCTTCAGATGGGGAC TGGAAACGGCTGCTAATACCGAATACGTTCTTTCCGCCGCATGACGGGAAGAAGAAAGGGAGGCCTTCGGG CTTTCGCTGGAGGAGGGGCTTGCGC1CTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGAT1CAGTAGCC GGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTT CCGCAATGGACGAAAGTCC1GACGGAGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCC‘GTTATAC‘G GGACGAACAGGACATCGGTTAATACCCGGTGC1CTTTGACGGTACCGTAAGAGAAAGCCACGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCATCGCAAGT CGGTCTTAAAAGTGCGGGGCC1TAACCCCGTGAGGGGACCGAAACTGTGAAGCTCGAGTGTCGGAGAGGAAAGCGGAA TTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACGACAACTGA CGCTGAGGCGCGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGC1CCTGGCCGTAAACGATGGATACTAGG TGTAGGAGGTATCGACTCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGGCTG AAACTCAAAGGAAC1TGACGGGGGCCCGCACAAGCGGTGGAGTAC‘GTGGTTTAATTCGACGCAACGCGAAGAACCTC1A CCAAGCCTTGACATTGATTGCTACGGAAAGAGATTTCCGGTTCTTCTTCGGAAGACAAGAAAACAGGTGGTGCACGG CTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCTGTTGCCAGCACC 40 TCGGGTGGGGACC1CAGAAGAGACTGCCGCAGACAATGCGGAGGAAGGCGGGGATGACGTCAAGTCATCAC‘GCCCCC1T ATGGCTTGGGCTACACACGTACTACAATGGCTCTTAATAGAGGGAAGCGAAGGAGCGATCCGGAGCAAACCCCAAAA ACAGAGTCCCAGC1TCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCAGGAATCGCTAGTAATCGCAGGC‘CAGCAC“A CTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCGGTGA GGCAACCGCAAG Primers used for qPCR (with SEQ ID NO in brackets) llheverse sequence GATCAAGATCATTGCTCCTC (3) TTGTCAAGAAAGGGTGTAAC (4) IGAPDH GTGGAAGGACTCATG (5) ATGCCAGTGAGCTTCCCGTTC (6) l MAP�-2���-;; CTCAGCACCGCTAACAGAGG (7) CATTGGCGCTTCTCTCCTC (8) lo_c_c_l_u_d_i_n�-;; AAGAGGAATTTTGACACTGG (9) GCCATGTACTCTTCACTTTC (10) ITJl CACTGGTGAAATCC (11) CTCTTGCTGCCAAACTATCT (12) ITJP2 CCCTCCCCTGGATCAGGAT (13) GCCATCAAACTCGTCCATCA (14) lvillin CATTACCTGCTCTACGTTTG (15) AGATGGACATAAGATGAGGTG (16) SEQ ID NO: 17 (consensus 16S rRNA sequence for cteroides distasonis strain MRX0005) AMCCGGGTGGCGACCGGCGCACGGGTGAGTAACGCGTATGCAACTTGCCTATCAGAGGGGGATAACCCGGCGAAAGT CGGACTAATACCGCATGAAGCAGGGATCCCGCATGGGAATATTTGCTAAAGATTCATCGCTGATAGATAGGCATGCG TTCCATTAGGCAGTTGGCGGGGTAACGGCCCACCAAACCGACGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACA TTGGTACTGAGACACGGACCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCTGAACC AGCCAAGTCGCGTGAGGGATGAAGGTTCTATGGATCGTAAACCTCTTTTATAAGGGAATAAAGTGCGGGACGTGTCC CGTTTTGTATGTACCTTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGT TATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTTTTAAGTCAGCGGTGAAAGTCTGTGGCTCAACCATAG AATTGCCGTTGAAACTGGGAGGCTTGAGTATGTTTGAGGCAGGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGAT ATCACGCAGAACCCCGATTGCGAAGGCAGCCTGCCAAGCCATTACTGACGCTGATGCACGAAAGCGTGGGGATCAAA CAGGATTAGATACCCTGGTAGTCCACGCAGTAAACGATGATCACTAGCTGTTTGCGATACACTGTAAGCGGCACAGC GAAAGCGTTAAGTGATCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG CGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGTTTGAACGCATTCGGACMGAKGTGGAA ACACATTTTCTAGCAATAGCCATTTGCGAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAG TGCCATAACGAGCGCAACCCTTGCCACTAGTTACTAACAGGTAAAGCTGAGGACTCTGGTGGGACTGCCAGCGTAAG CTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTGTTACAATGGCGTGG ACAAAGGGAAGCCACCTGGCGACAGGGAGCGAATCCCCAAACCACGTCTCAGTTCGGATCGGAGTCTGCAACCCGAC TCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCAAGCCATGGGAGCCGGGGGTACCTGAAGTCCGTAACCGCGAGGATCGGCCTAGGGTAAAACTGGTGACTGG GGCTAAGTCGTACGGGG Primers and probes used for ex vivo qPCR (with SEQ ID NO in brackets) Ex vivo Name Forward Sequence e Sequence Probe ACTB GAT TAC TGC TCT GGC TCC TAG GAC TCA TCG TAC TCC TGC TIG(19) M/CTG GCC TCA /ZEN/CTGTCC ACC (18) TIC C/31ABkFQ/(20) GAPDH AAT GGT GAA GGT CGG TGT G GTG GAG TCA TAC TGG AAC ATG TAG /56-FAM/TGC AAA TGG /ZEN/CAG CCC TGG (21) (22) TG/31ABkFQ/(23) BDNF GCT GCC TIG ATG TTI ACT TIG GCA ACC GAA GTA TGA AAT AAC CA /56-FAM/ACC AGG TGA /ZEN/GAA GAG AC(24) (25) TGA TGA CCA TCC /31ABkFQ/ {26) IL6 AGC CAG AGT CCT TCA GAG A TCC TIA GCC ACT CCT TCT GT(28) /56-FAM/CCT ACC CCA /ZEN/ATI TCC AAT (27) GCT CTC CT/31ABkFQ/(29) Additional primers used in qPCR (with SEQ ID NO in brackets) Gene ID Forward sequence Reverse sequence ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm NSE CCCTGTATCGTAAGAACGGT (30) GCCACCATTGATCACGTTGA (31) PINK1 CAACTAGCCCCTC (32) GGCAGCACATCAGGGTAGTC (33) PARK? GTGATGTGGTCATTT (34) CTGTGCGCCCAGATTACCT (35) svp GGCTTCATGGCATCAACTTCA CTCGGCTTTGTGAAGGTGCT (36) (37) REFERENCES Spor et al. (2011) Nat Rev Microbial. 9(4):279-90.

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Kehr J., and ake T. (2006) Monitoring brain chemical signals by microdialysis. In: Encyclopedia of Sensors, Vol. 6. (Eds. C.A. Grimes, E.C. Dickey and M.V. Pishko) American Scientific Publishers, USA. 287-312. el and Zukin (2008) Curr Opin Pharmacol, 2008. 8(1): 57-64 Johnsen et al (2017) Journal ofChromatography A. 1503: 57-64 West and Johnstone (2014) J Clin Invest. 124, 30-39 Glauben et al. (2006) JImmunol, 176: 5015-5022 Angiolilli et al. (2017) Ann Rheum Dis, 76: 277-285 Gonneaud et al. (2014) JInflamm, 11: 43 Alenghat et al. (2013) Nature, 504: 153-157 Felice et al. (2015) Ailment Pharmacol Ther, 41: 26-38 Smart et al (2010) Nature Protocols. 10: 1709-29 Weon et al. (2016) Huot et al., (2015) Parkinson 's Disease Scatton et al. (1983) Brain Res, 275(2): 321-8 Hely eta. (2005) Mov Disord. 20(2): 190-9 Budd and Nicholls (1998) Essays Biochem. 33:43-52 Ebadi et al (2001) Biol Signals Recept. 10(3 -4):224-253 Ferro et al (2017) PLoS One 2017, 12(12):e0188425 Michel and Prat (2016) Ann Transl Med. 4(1): 15.

Gagnon et al (2013) JMicrobiological Methods. 94: 274-279 [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Print Out nal in Electronic Form) 0-1 Form PCT/R0/134 Indications Relating to Deposited Microorganism(s) or Other Biological Material (PCT Rule 13bis) 01 PmmmdUWW PCT Online Filing Version 3.5.000.256e MT/FOP 20141031/0.20.5.2O International Application No.

Applicant's or agent's file reference P0 7 0 7 7 2W0 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: 1-1 Page 11 1-2 'me 16—20 1-3 Identification of deposit 11 Name Ofdeposnaryin'StitUtion NCIMB National Collections of Industrial, Food and Marine Bacteria (NCIMB) 12 NMm$onmmmmwmwmmn NCIMB Ltd, on Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom 166 u 12 March 2015 (12.03.2015) 134 Accession Number NCIMB 4 23 8 2 16 Designated States for Which All ations Indications are Made The indications made below relate to the deposited rganism(s) or other biological material referred to in the description on: 2-1 page 8 2-2 “"6 12-15 2-3 Identification of deposit 2-3—1 Name of depositary institution NCIMB National Collections of Industrial, Food and Marine ia 2-3—2 NMm$onmmmmwmwmmn NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom 266 Dmequmw 13 July 2017 (13.07.2017) 264 Accession Number NCIMB 42787 Designated States for Which All designations Indications are Made FOR RECEIVING OFFICE USE ONLY This form was received with the international application: (yes or no) 041 Authorized officer SUBSHTUTESHEET(RULE26) ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Print Out (Original in Electronic Form) FOR INTERNATIONAL BUREAU USE ONLY 0-5 This form was ed by the international Bureau on: 01 Authorized officer SUBSTITUTE SHEET (RULE 26)

Claims (21)

1. Use of a ition comprising a bacterial strain of the genus Megasphaera, wherein the bacterial strain has a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2, in the manufacture of a medicament for ng or preventing a neurodegenerative disorder in a 5 patient.

2. The use of claim 1, wherein the medicament is for treating or preventing a e or condition selected from the group consisting of Parkinson’s disease, including ssive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski me, 10 normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor e disease; prion e; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild 15 cognitive impairment; HIV-related ive impairment and corticobasal degeneration.

3. The use of claim 2, n the medicament is for treating or preventing Parkinson’s disease. 20

4. The use of any one of the preceding claims, wherein the medicament is for treating or preventing early-onset egenerative disease.

5. The use of any one of the preceding claims, wherein the medicament is for preventing or ng onset or progression of a neurodegenerative disorder.

6. Use of a composition comprising a bacterial strain of the genus Megasphaera, wherein the bacterial strain has a 16s rRNA ce that is at least 95%, identical to SEQ ID NO:2, in the manufacture of a medicament for treating a brain injury in a patient. 30

7. The use of claim 6, wherein the brain injury is stroke, such as cerebral ischemia, focal cerebral ischemia, ischemic stroke or hemorrhagic stroke.

8. The use of any one of the preceding claims, wherein the bacterial strain is of Megasphaera massiliensis.

9. The use of any one of the preceding claims, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2, or wherein the bacterial strain has the 16s rRNA sequence represented by SEQ ID NO:2.

10. The use of any one of claims 1-3, wherein the medicament ses a bacterial strain of the species Megasphaera massiliensis.

11. The use of claim 6, wherein the brain injury is brain injury resulting from a stroke.

12. The use of any one of the preceding claims, wherein the ment is formulated for oral administration.

13. The use of any one of the preceding , wherein the ition comprises one or 15 more pharmaceutically acceptable ents or carriers.

14. The use of any one of the preceding claims, wherein the bacterial strain is lyophilised.

15. The use of any one of the preceding claims, n the composition is a food 20 product.

16. The use of any one of the preceding claims, wherein the bacterial strain is a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787.

17. A cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787.

18. A composition comprising the cell of claim 17.

19. The composition of claim 18, comprising a pharmaceutically acceptable r or excipient.

20. A biologically pure culture of the Megasphaera massiliensis strain deposited under 35 accession number NCIMB 42787.

21. Use of a cell of the haera massiliensis strain deposited under accession number NCIMB 42787, in the manufacture of a medicament.