JP4948826B2 - Non-human animals with age-related memory impairment suppressed - Google Patents

Description

  The present invention relates to a non-human animal in which a gene encoding a protein kinase A (PKA) catalytic subunit is mutated.

  Aged memory impair (AMI) is the most prominent phenotype of brain aging, and it also affects older people who do not suffer from age-related neurological disorders such as Alzheimer's disease (AD). It is a disorder that occurs (Reference 1). Age-related memory impairment (AMI) is a common phenomenon in many animals, from Drosophila to humans. Anatomical and physiological changes in AMI are disclosed in the literature (Foster, TC Brain Res Brain Res Rev 30, 236-249, 1999; Neurobiol Aging 20, 125-136, 1999). The genetic pathways involved in individual aging have been extensively investigated by examining longevity mutants. On the other hand, there are still many unclear points about the gene pathways related to AMI, and the correlation between individual aging and AMI is also unclear (Reference 2). Senile memory impairment such as senile dementia is expected to be a major cause of hindering social participation of elderly people in an aging society. However, since an effective model animal has not yet been established, elucidation of the process leading to expression at the molecular level and development of preventive / therapeutic drugs are greatly delayed.

  The inventor has previously characterized genetics of AMI using Drosophila with a short life span and excellent genetic analysis, and in the wild type, amn gene-dependent medium-term memory (MTM) It was found to specifically decrease (Reference 3). In other words, in contrast to other memory mutants, the amn gene mutant did not decrease memory by aging. The neuropeptide, the product of the amn gene, is expressed in DPM cells (dorsal paired medial cells) that project to mushroom bodies necessary for memory and learning, and is an important step in the formation of metaphase memory that requires the amn gene It has been suggested that cAMP signal is regulated (Reference 4). That is, AMI may be caused by the activity of the amn gene that decreases with aging. However, the expression level of the amn gene did not decrease with age, and AMI was not improved even if the amn gene was overexpressed in DPM cells (Reference 3).

  As a study on PKA memory using Drosophila, it has been reported that memory impairment occurs when a PKA inhibitor is expressed (Non-patent Document 1). In this report, inhibitory peptides are expressed in large quantities by heat shock to suppress PKA activity.

  It has also been reported that memory is improved a little when cAMP inhibitor analogue is expressed in the prefrontal cortex (Non-patent Document 2).

For the purpose of elucidating the molecular mechanism of AMI expression, many analyzes have been conducted in mammalian models, particularly humans, regarding what gene expression changes in the brain with aging (AMI) (Jiang et al. al, PNAS 98, 1930-1934, 2001; Lu et al, Nature 429, 883-891, 2004). However, despite attempts to classify by various algorithms and assign meanings to AMI, none of them are out of speculation and have not been directly verified. There are two reasons (failures) that are not directly verified. One is a large number of genes that show changes in expression with age, which includes many AMI-independent expression changes (many false positives), and the other is mammals. This is because the model has a long life span of years, and molecular genetic analysis (analysis by creating a mutant and aging) in an aging body is difficult.
Special Table 2000-515734 JP 2005-160361 A Drain, P. et al., Neuron 6, 71-82, 1991 Ramos, BP et al., Neuron 40, 835-845, 2003

  An object of the present invention is to provide a method for screening a non-human animal in which a gene encoding a PKA catalytic subunit is mutated and a substance that is a target of a substance that suppresses or ameliorates age-related memory impairment.

  In order to solve the above-mentioned problem, the present inventor first hypothesized that a signal pathway in the mushroom body downstream of the amn gene is inhibited by aging, resulting in memory impairment (Reference Document 5). ). In other words, we conducted intensive research in anticipation that an important step in the development of AMI occurs in mushroom bodies, and that genes that regulate AMI in the mushroom bodies will be found (Reference 6). As a result, non-human animals that have genetically suppressed the activity of the PKA catalytic subunit (DC0-PKAc) encoded by the DC0 gene by 40-50% can specifically suppress AMI without affecting lifespan. As a result, the present invention has been completed.

That is, the present invention is as follows.
(1) A non-human animal in which a gene encoding a protein kinase A catalytic subunit is mutated.

  The gene encoding the protein kinase A catalytic subunit is preferably DC0 in Drosophila and its homologous gene in mammals. In the non-human animal, age-related memory impairment may be suppressed or improved. Further, the non-human animal may be one in which the activity of protein kinase A is reduced to 50 to 60% of the wild-type activity due to gene mutation. Examples of non-human animals include Drosophila. Further, non-human animal dnc in which a gene involved in PKA activity regulation described in Example 4 is mutated is also included.

(2) A method for identifying a target substance of a substance that suppresses or improves age-related memory impairment, the following steps:
(A) measuring the expression pattern of the gene or protein derived from the non-human animal and the wild-type non-human animal according to (1) above,
(B) identifying the substance which becomes the target of the substance which suppresses or improves age-related memory impairment by comparing the obtained expression pattern.

(3) Using the non-human animal-derived protein and the wild-type non-human animal-derived protein described in (1) above, the presence or absence or level of phosphorylation was measured and compared, and phosphorylation was suppressed from the measurement results obtained. A method for identifying a substance that is a target of a substance that suppresses or ameliorates age-related memory impairment, characterized by identifying a protein.

  According to the present invention, a non-human animal in which a gene encoding a PKA catalytic subunit is mutated is provided. Among the non-human animals of the present invention, a fly model such as Drosophila can effectively perform genetic analysis of senile memory impairment in a short period of time. Therefore, the non-human animal of the present invention is expected to greatly contribute to identification and development of prophylactic / therapeutic targets by elucidating the genetic function of senile memory impairment.

  The present invention relates to a mutant non-human animal in which a gene encoding a PKA catalytic subunit is mutated, and preferably relates to a Drosophila that is a heterozygote of the gene.

1. SUMMARY OF THE INVENTION The present inventor considers that mushroom bodies that receive neuropeptides of the amn gene product have the cause of AMI, and attempted to search for strains that are abnormal in the expression of AMI from mutants of genes that are expressed in mushroom bodies. It was found that heterozygous mutant DC0 / + of gene DC0 encoding the catalytic subunit of kinase A (PKA) has strong AMI expression suppression. The present inventors have shown that AMI can be suppressed in non-human animals that have genetically suppressed PKA activity by 50-60%. The present invention provides two obstacles described in the background art which have been conventionally considered by providing genetic conditions for suppressing AMI, and by using Drosophila which has a shorter lifespan than mice and the like. To overcome.

  In the present invention, a mutant in which AMI is suppressed is produced in a model animal having a short lifespan (heading), and the gene and / or protein expression variation with aging in this mutant is compared with the wild type. That is, changes in gene and / or protein expression with aging under conditions where AMI does not occur (mutants) and gene and / or protein expression with aging under conditions in which AMI occurs (wild type) By comparing the fluctuations and examining the differences, false positives showing expression fluctuations unrelated to AMI can be eliminated. In addition, which gene animals and / or proteins are important for AMI expression in model animals that have a shorter lifespan than mice, etc., molecular genetic analysis in aging bodies is easier than in mammalian models Can be analyzed. If a gene and / or protein important for AMI expression can be found and identified in a short-lived model animal, then the function of the gene and / or protein against AMI can be verified by molecular genetic analysis in a mammalian model. it can. Thus, genes and / or proteins important for AMI expression that have been verified in mammalian models are potential targets for substances to suppress or improve AMI.

  The present invention solves the conventional biggest problem that AMI-suppressed mutants have not existed so far, and provides a AMI-suppressed mutant.

  The present inventor has found that the memory ability of amnesiac (amn), a Drosophila memory mutant, no longer decreases with age. This suggests that it is the substance of AMI that the memory involved in the amn gene is specifically impaired. Here, the amn gene is assumed to encode a neuropeptide that regulates the activity of adenyl cyclase (AC) in the mushroom body of the memory center, which is the nerve center of olfactory learning and olfactory memory (Reference 4).

  Therefore, in order to identify genes involved in AMI, the inventor searches for mutations in genes that have mushroom bodies as the main expression region, such as abnormalities such as AMI enhancement / suppression. went. As a result, it was found that the heterozygous mutant (DC0 / +) of the gene DC0 encoding the catalytic subunit of PKA was greatly suppressed without affecting the life span. DC0 / + mutants show normal high memory at an early age, and this memory is retained by aging.

  The present inventor uses either method when overexpressing the DC0 gene using the GAL4-UAS system or increasing the PKA activity by using a heterozygote of dnc (cAMP-degrading enzyme gene). I found that AMI would occur early, even if I had. Furthermore, the inventor has also found that AMI occurs normally in methuselah (mth) mutants that live longer as opposed to DC0 / +.

  These findings indicate that PKA (DC0-PKA), which has a catalytic subunit encoded by the DC0 gene, specifically controls AMI without affecting lifespan, and that extending lifespan suppresses AMI. It is shown that it is neither necessary nor sufficient condition.

  Drain et al. Have reported that memory impairment occurs when a PKA inhibitor is expressed in Drosophila (Neuron vol. 6, pp.71-82, 1991, Reference 12). However, their method expresses a large amount of inhibitory peptide by heat shock and suppresses all PKA activity, whereas the DC0 mutant used in the present invention has a mutation in the PKA catalytic subunit. The PKA activity is retained by 50 to 60% compared to the PKA activity of normal animals (Reference Document 11). Therefore, the method of inhibiting PKA using the DC0 mutant of the present invention is different from the method of Drain et al.

  In addition, Ramos et al. Have reported that when cAMP inhibitor analogue is expressed in the prefrontal cortex, the memory ability deteriorated is slightly improved (Reference Document 19). It is different in that it includes maintaining normal memory when young.

2. Heteromorphic animals of PKA catalytic subunit (1) PKA catalytic subunit
PKA is an enzyme that transfers the γ-phosphate group of ATP to a specific serine or threonine hydrosyl group of proteins, and is widely distributed in eukaryotic cells. This enzyme consists of a regulatory subunit with a molecular weight of about 40,000 to which cAMP binds and a catalytic subunit with a molecular weight of about 50,000. When two molecules of cAMP bind to one regulatory subunit, the catalytic subunit and the regulatory subunit are combined. The unit dissociates and becomes active. The present invention has been found that such active PKA causes memory impairment (AMI) due to aging. Therefore, in the present invention, mutations are introduced into the catalytic subunit that plays an important role in the cAMP signaling pathway involved in the memory process, and at the same time, the PKA activity is mildly reduced without suppressing all of the early learning. To provide animals that suppress AMI without affecting memory or memory and without affecting life span.

  Preferred catalytic subunits of PKA of the present invention include, for example, DC0 which is a catalytic subunit of PKA expressed in the fly brain, and Cβ and Cα which are mouse homologues of DC0 (Huang et al, J. Biol Chem vol 277, pp19889-19896, 2002), and the like. In particular, the catalytic subunit DC0 of PKA expressed in the fly brain is preferable. Hereinafter, DC0 will be described.

  The DC0 gene encodes the catalytic subunit of PKA expressed in the fly brain, and its homo-deletion mutant is embryonic lethal (Reference 11). On the other hand, when the activity of DC0-PKA is suppressed to about 10% of the wild type in adults, the formation of memory is inhibited. Of particular interest is the temperature-sensitive mutant of DC0, which, like amn, causes defects in metaphase memory (Ref. 13). DC0-PKA is prominently expressed in mushroom lobes and calyces (Reference 7).

  The DC0 gene includes genomic DNA encoding DC0, as well as its mRNA and cDNA. The base sequence of the DC0 gene is known, and the sequence can be easily obtained through a public database such as GenBank (GenBank Accession No. X16969). The nucleotide sequence of the gene for DC0 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. For animals whose sequences are not registered in public databases, clone the PKA catalytic subunit gene from homology with known PKA catalytic subunit genes and determine the sequence by conventional methods. Can do. For example, a genomic DNA library of the animal is prepared, the library is screened using a known PKA catalytic subunit gene derived from the genetically closest species, or a part thereof, as a probe, and the target PKA Catalytic subunit genes can be identified. For the genetic engineering method, for example, Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)) can be referred to.

(2) Hetero mutant
Since the homozygous mutant of the catalytic subunit of PKA is embryonic lethal, the animal of the present invention is preferably a heterozygous mutant. In addition, this mutant is a low-morphic mutant that partially lost its function as a PKA catalytic subunit due to base substitution in the PKA catalytic subunit gene on the chromosome (hypomorph mutant). The PKA activity is mildly suppressed and retains 50-60% of the wild-type PKA activity (that is, the activity is reduced by 40-50% compared to the wild-type). ). PKA activity is reduced to 50-60% milder than wild type, allowing mutant animals to suppress AMI without affecting early learning and memory. Yes. However, if PKA activity increases or decreases excessively, learning memory is impaired. In addition, such a function that the PKA activity decreases mildly was acquired in an allele-specific manner.

  Here, the “mutation” includes a mutation caused by deletion of a part or all of a gene and no longer functioning, and a mutation caused by base substitution or transposon factor insertion. Such mutation can be introduced by gene targeting method, site-directed mutagenesis method, X-ray irradiation, mutagenesis by carcinogen or enhancer trap method.

(3) Production Method The heterozygous mutant animal of the gene encoding the PKA catalytic subunit of the present invention includes, but is not limited to, non-mammals such as Drosophila and mammals other than humans such as rats and mice. It is not a thing. In particular, Drosophila melanogaster, a fruit fly, has a short life span and is optimal for behavioral genetic analysis of old individuals (Exp Gerontol 37, 1347-1357, 2002), and is preferred as the animal of the present invention.

  In the present invention, in order to produce a heterozygous mutant animal of a gene encoding a PKA catalytic subunit in a fly such as Drosophila, the PKA catalytic subunit gene on the chromosome is mutated using a known genetic manipulation means. Can be done. For example, when a strain having a transposon factor inserted in the vicinity of the DC0 gene and a strain having an enzyme transposase for transferring the transposon factor are crossed, retransfer of the transposon factor occurs in the next-generation (F1) germ cells. At this time, the transposon factor may be transferred while holding the surrounding genes, but if it is transferred again with some or all of the DC0 gene, the function of the PKA catalytic subunit encoded by the DC0 gene will be partly or entirely It will be lost. It is also possible to create so-called gene targeting by homologous recombination based on the DC0 gene sequence and creation of a reduced function mutant by RNA interference. PKA catalytic subunit gene mutation can be performed by mutating a part of the PKA catalytic subunit gene by deletion, substitution, or addition by genetic manipulation including the above. As described above, the site where deletion, substitution, or addition is performed, or the base that is substituted or inserted, as long as the PKA activity of the obtained mutant is maintained at 50 to 60% compared to the PKA activity of normal animals. It is not particularly limited.

  Introduction of a vector into a fly such as Drosophila can be performed using a known method. For example, it can be achieved by introducing the constructed targeting vector into, for example, an embryo. As the introduction method, a known introduction method such as microinjection can be used.

  In addition, in order to produce a heterozygous mutant animal of the PKA catalytic subunit of the present invention in a non-human mammal such as a mouse, known genetic engineering techniques such as gene targeting, Cre-loxP system, and somatic cell clone are used. Can be used. For example, a modification of part or all of the base sequence of the gene encoding the PKA catalytic subunit is introduced into a totipotent cell, and a cell into which the modified gene has been introduced is selected. Next, a chimeric individual can be produced by introducing the selected genetic modification (deletion, destruction, mutation, etc.) into a fertilized egg.

  Specifically, all or part of a gene fragment having homology to the DC0 gene obtained from a gene library constructed from an animal brain by a method such as PCR, lac-Z gene, neomycin resistance gene, etc. If necessary, introduce a marker gene such as the diphtheria toxin A fragment (DT-A) gene or the herpes simplex virus thymidine kinase (HSV-tk) gene into the 5 'end as necessary for targeting. Create a vector. This targeting vector is linearized and introduced into embryonic stem cells (ES cells) by electroporation (electroporation) or the like, and homologous recombination is performed. From the homologous recombinants, select ES cells that are resistant to antibiotics such as G418 or ganciclovir (GANC) by staining with X-gal, and the above functions of genes having homology to the DC0 gene are selected. The expressed target embryonic stem cells are microinjected into the blastocyst of the animal. Thereafter, when the blastocyst is returned to the foster parent, a chimeric mouse is produced, and this chimeric animal is mated with a wild type animal, a heterozygous mutant animal can be obtained.

3. Method for identifying screening target (target substance) of substance that suppresses or improves age-related memory impairment
In order to develop preventive / ameliorating drugs for AMI, it is important to clarify the molecular mechanism of AMI expression and to find in vivo molecules / genes that are targets of AMI preventive / ameliorating drugs.

  Age-related memory impairment (AMI) is suppressed in non-human animals in which the gene encoding the PKA catalytic subunit is mutated, particularly in the heterozygous mutant Drosophila DC0 / + of DC0. By utilizing this, in the present invention, it becomes possible to identify a molecule or gene in a living body targeted by a prophylactic / ameliorating agent for AMI. For example, if a protein that disappears when the brain ages due to aging does not disappear in the same-age mutant with suppressed AMI or has a low level of disappearance, the protein is a memory disorder caused by aging. It can be said that it is a marker protein that acts in a direction that does not cause odor. Therefore, a compound that expresses such a marker protein in an aging state is useful as an agent for preventing or treating AMI. The present invention is a method for identifying a marker protein or gene that acts in a direction that does not cause memory impairment caused by aging, and a marker protein / gene that causes memory impairment caused by aging. Hereinafter, DC0 / + will be described as an example of non-human animals in which the gene encoding the PKA catalytic subunit is mutated.

  DC0 / +, a heterozygous mutant of the gene DC0 that encodes the catalytic subunit of PKA, is an AMI-suppressing mutant that was first discovered by the present inventor, with PKA activity suppressed to 50-60%. Yes. From this, DC0 / + can be used to identify molecules and / or genes that are key to suppression of AMI expression. For example, two specific methods are illustrated below.

  One is the expression dynamics of DC0 / + (under AMI suppression conditions) and wild-type non-human animals (hereinafter referred to as “wild-type”) (under AMI expression conditions) with aging by DNA chip or two-dimensional electrophoresis. Is a method for comparatively identifying different genes or proteins. The other is to isolate DC0 / + (under AMI suppression conditions) and wild type (under AMI expression conditions) by isolating PKA substrates or phosphorylated proteins involved in AMI expression with a phosphorylated protein recognition column. ) Is a method for comparatively identifying kinetic changes associated with aging. Genes and / or proteins identified by such methods are potential target molecules for the prevention and / or improvement of AMI and thus therapeutic drug development. Each method will be described below.

(1) Identification method of target substance of AMI preventive / improving drug by comparison of kinetics of gene or protein with aging in DC0 / + (under AMI suppression condition) and wild type (under AMI expression condition) The method comprises the following steps:
(A) measuring the expression pattern of the gene or protein derived from the non-human animal of the present invention and the gene or protein derived from the wild-type non-human animal, respectively;
(B) by identifying the target substance of the substance that suppresses or ameliorates age-related memory impairment by comparing the obtained expression patterns.

  “Target substances” are substances that increase or decrease in relation to the expression of AMI. Substances that serve as markers (indicators) when screening for substances that can suppress or improve AMI (candidate substances for AMI therapeutics) Say. Substances that can suppress or improve AMI act on this marker. For example, if they are markers that show an increase associated with AMI expression, they can suppress the expression, and markers that show a decrease associated with AMI expression. If present, it is a substance that can promote its expression, and is preferable as a candidate substance for a prophylactic / ameliorating agent for AMI.

  Specifically, the expression pattern (dynamics) due to aging of the protein expressed in the wild type is analyzed. For example, a protein expressed in a wild-type young body (7 days old) is labeled with green fluorescence, and a protein expressed in an old body (20 days old) is labeled with red fluorescence on the same gel. Develop with 2D electrophoresis. The expression pattern thus developed is referred to as panel A. Then, in the protein that is expressed in the young body and disappears by aging, a green spot that is fluorescence of the young body appears. In a protein expressed in both the young body and the old body (a protein that does not change in expression with aging), a green fluorescent spot appears reflecting the green spot of the young body and the red spot of the old body. A protein that does not express in the young body but increases in expression with aging appears as a red fluorescent spot, which is the fluorescence of the old body. By measuring the relative intensity (quantity ratio) of these fluorescent spots with an imaging analyzer, proteins at each age can be quantified. In the analysis results at this stage, it can be considered that proteins whose expression level fluctuates with aging include those that increase or decrease in association with AMI, but many of them are false positives.

  On the other hand, the expression patterns of proteins expressed in wild-type and DC0 / + aging bodies are also analyzed. For example, proteins expressed in DC0 / + (under AMI suppression) aging bodies are labeled with green fluorescence, and wild type (under AMI conditions) aging bodies are labeled with red fluorescence, two-dimensionally on the same gel. Develop by electrophoresis and measure protein expression pattern. The expression pattern thus developed is referred to as panel B. Then, proteins that are expressed more in DC0 / + (under AMI suppression) aging than wild-type (under AMI) aging will reflect the fluorescent label of the DC0 / + aging, green spots Produce. Proteins that are expressed higher in wild-type (AMI conditions) aging bodies than in DC0 / + aging bodies (under AMI suppression conditions) will produce a red fluorescent spot reflecting the fluorescent label of wild-type aging bodies . Proteins that are expressed in both wild-type (under AMI) and DC0 / + (under AMI-suppressed) aging bodies and whose expression levels do not change are the wild-type aging body fluorescent label and DC0 / + Both aged fluorescent labels are reflected to produce a yellow spot.

  Here, by comparing the expression patterns of the same spot in the above two systems (comparison between panel A and panel B), it is possible to find changes in the expression of proteins involved in the expression of AMI excluding false positives. For example, the spots with strong green fluorescence in both systems are proteins whose protein expression decreases with age in the wild type (read from panel A), and expressed with age in DC0 / + (under AMI suppression conditions) Means that the protein does not decrease (can be read from panel B). Therefore, a compound capable of maintaining the expression of this protein can be a candidate substance for an agent for suppressing or improving AMI. A compound that induces the expression of a protein that is not expressed in the wild type but that is first observed in an aging body in DC0 / + (under AMI suppression conditions) can also be a candidate substance for an AMI suppression or improvement agent.

  On the other hand, in the wild type, it is a protein whose expression level increases with aging (red spot in panel A), and in DC0 / + (under AMI suppression conditions), the protein whose expression level decreases (in panel B) Red spots) are proteins that promote AMI. Substances that suppress the expression of such proteins can also be employed as candidate substances for suppressing or improving AMI in the present invention.

  The gene expression kinetics can also be analyzed by comparing expression patterns by the same analysis using a DNA chip.

(2) Identification method of AMI prophylactic / ameliorating drug target by comparing kinetic changes of phosphorylated PKA substrate protein between DC0 / + (under AMI suppression conditions) and wild type (under AMI expression conditions) Measure and compare the presence or level of phosphorylated protein using non-human animal-derived protein and wild-type non-human animal-derived protein in which the gene encoding the unit is mutated. Provided is a method for identifying a substance that is a target of a substance that suppresses or ameliorates age-related memory impairment, characterized by identifying a protein in which phosphorylation is suppressed in a non-human animal in which an encoding gene is mutated.

  In DC0 / +, the PKA activity is suppressed to 50-60%, so the amount of phosphorylated PKA substrate that ultimately causes neurological dysfunction is reduced compared to the wild type, thereby suppressing the expression of AMI. It is thought that. That is, it is considered that neuronal dysfunction, that is, AMI occurs when the amount of phosphorylated PKA substrate that ultimately causes neurological dysfunction increases with aging. The term “phosphorylated substrate” as used herein includes all proteins that are phosphorylated by PKA, such as ion channels, transcription, translation factors, and other kinases.

  Therefore, if a DC0-PKA substrate protein whose phosphorylation is suppressed by DC0 / + can be identified, a prophylactic / ameliorating drug for AMI, and a therapeutic drug can be obtained by searching for compounds that inhibit the phosphorylation or accumulation of the protein. Can be expected to develop.

  Phosphorylated DC0-PKA substrate protein is isolated by a phosphorylated protein recognition column (phosphorylated column). Since the activity of PKA is reduced in DC0 / +, the amount of phosphorylated protein that binds to the phosphorylated column is reduced compared to the wild type. Therefore, the phosphorylated protein bound to the phosphorylated column is recovered. For example, the phosphorylated protein derived from DC0 / + is labeled with green fluorescence, and the phosphorylated protein derived from wild type is labeled with red fluorescence on the same gel. In two-dimensional electrophoresis development, the phosphorylated protein spot with more wild than DC0 / + becomes a spot with strong red fluorescence. It can be said that the phosphorylated protein which becomes such a spot is a phosphorylated substrate of DC0-PKA.

  Whether the phosphorylated protein isolated from the phosphorylated column is actually a DC0-PKA substrate can be easily verified in vitro by phosphorylation with recombinant DC0-PKA.

  In addition, because PKA regulates the activity of transcription factors such as CREB by phosphorylation, DC0 / + suppresses the activity of PKA, resulting in the expression of genes encoding proteins that cause neuronal dysfunction. It may be suppressed. The identification of the target substance focusing on this point is as described in (1) above.

  The present invention can also screen for a substance capable of suppressing or improving AMI by contacting the test substance with the non-human animal of the present invention and analyzing the expression pattern of the target substance identified by the above method. . Substances screened in this way can be applied to prevent and improve AMI.

  In the present invention, the “non-human animal” includes both the whole body of a non-human animal body and a limited tissue or organ. In the case of limited tissues or organs, it includes those extracted from animals.

  A method for measuring or evaluating age-related memory impairment biochemically against the background that PKA activity causes age-related memory impairment includes, for example, a method for measuring PKA activity. PKA activity is measured by measuring PKA activity in the Drosophila head using a standard ELISA method for analyzing phosphorylation of substrate peptides by PKA. The analysis can be performed by preparing a calibration curve using recombinant PKAc in the presence of cAMP.

  Another method is to measure the expression level of the transcript of the DC0 gene. The expression level of the DC0 gene transcript can be quantified by, for example, real time PCR using total RNA derived from the head of Drosophila.

  Furthermore, as a method for evaluating memory impairment behaviorally, there is an analysis of changes in learning and memory behavior by a typical odor conditioning experiment. A typical odor conditioning experiment is a standard single cycle training modified from the methods described in the literature (Tully and Quinn, 1985, reference 10). For example, as a conditional stimulus for a predetermined number of Drosophila, an aversive odor (for example, 3-octanol [OCT]) is given together with an electric shock, and after a break, another aversive odor (for example, 4-methylcyclohexanol [MCH]) is given. ) By giving without electric shock.

  As a method for evaluating whether the learning memory behavior disorder is caused by a learning memory disorder, a memory experiment for evaluating responsiveness to an olfactory sense or an electric shock can be given. These can be assessed by having unconditioned flies choose between OCT and air, MCH and air, electric shock and no shock. If an unconditioned fly chooses air for OCT or MCH and chooses no electric shock for electric shock, then the failure of learning and memory behavior exhibited by this fly is odor or electric shock susceptibility It can be evaluated based on learning and memory impairment, not on the other.

  As a search for seeds for prophylactic / improving drugs for age-related memory impairment using PKA activity as an index, search for compounds that moderately inhibit the activity of recombinant PKA from a compound library using recombinants of DC0-PKA and DC0 mouse homolog Cβ Can also be done.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

  In addition, the experiment method of the experiment used in the Example is as follows.

Flies used W (CS10) used as a wild-type fly is a white-eye Canton-S obtained by crossing w ¹¹¹⁸ to Canton-S 10 times as already reported. All mutants except dnc ¹ used in this study were used in crosses with w (CS10) at least 6 times. For dnc ¹ whose gene mutation location was unknown, heterozygous individuals born by mating with w (CS10) were used. Since the dnc gene is on the X chromosome, all hetero individuals of the dnc gene are female. For control of the dnc ¹ / + experiment, female w (CS10), male w (CS10), and male born by mating male dnc ¹ and female w (CS10) were used. As a result, there was no difference in learning, memory, and odor reactivity among all controls. This showed that the genetic background did not affect the dnc ¹ / + results.

  Unless otherwise indicated, all flies were kept under 25 ± 2 ° C., 60 ± 10% humidity, and 12 hours light-dark period. About 50 flies for behavioral experiments and about 20 flies for life tests are kept in vials containing food, and transferred to vials containing new food every 2-3 days. Changed.

  In the mating of mutants without the mini-white marker, it was confirmed by RT-PCR that the mutant gene was retained for each generation.

Analysis of memory Typical odor conditioning (Ref. 10) uses two types of odors that flies dislike (3-octanol [OCT] and 4-methylcyclohexanol [MCH]). Approximately 100 flies were given the first scent (CS +) with an electric shock for 60 seconds (electric shock was applied for 1.5 seconds, 60V DC electric shock at 3 second intervals). After the first odor presentation, after a 45-second break, the next odor (CS-) was given for 60 seconds, this time without electric shock. In the memory test, trained flies were allowed to choose CS + and CS-. The performance index (PI) was 0 when the flies were split 50:50, and 100 when the flies were separated 0: 100 away from the smell of CS +. The responsiveness to olfaction and electric shock in the memory experiment was examined by selecting non-trained flies to select OCT and air, MCH and air, with or without electric shock. In the mutants and aging bodies used in the present invention, no remarkable abnormality was observed in the odor and the response to shock.

Quantification of PKA activity PKA activity in the Drosophila head was examined by a standard ELISA method (MESACUP Protein Kinase Assay Kit, MBL, Nagoya, Japan) for analyzing phosphorylation of substrate peptides by PKA.
The head extract was obtained by the following procedure. First, Drosophila was frozen with dry ice and the head was separated. Next, about 100 heads were ground with a pestle using 200 μL of sample preparation buffer (MESACUP Protein Kinase Assay Kit). Thereafter, the impurities were removed by centrifugation to obtain a head extract. Analysis was performed in the presence of 2 μM cAMP concentration, and a calibration curve was prepared using recombinant PKAc (New England Biolabs). Each data was obtained from the average of the results of a minimum of 4 independent experiments.

Expression of DC0 gene transcript
Comparison of DC0 transcript expression levels was quantified using real time PCR (Applied Biosystems, model 7500). Drosophila heads were isolated using dry ice, and total RNA was isolated using TRIzol reagent (Invitrogen). cDNA was synthesized using High Capacity cDNA Archive Kit (Applied Biosystems), and quantitative PCR was performed using DC0 and GAPDH2-specific primers and SYBR-Green. The primers used are as follows.
DC0-fwd: CAAGGAGGAGTTCGAGGACA (SEQ ID NO: 3)
DC0-rev: GTGCTGGACGATCATGACAC (SEQ ID NO: 4)
GAPDH2-fwd: GCGGTAGAATGGGGTGAGAC (SEQ ID NO: 5)
dGAPDH2-rev: TGAAGAGCGAAAACAGTAGC (SEQ ID NO: 6)
PCR was performed under normal conditions. DC0 expression was obtained by normalizing GAPDH2 expression. Each data consists of an average of at least 4 independent experiments.

Immunohistochemistry The head of adult Drosophila 3 to 5 days after removal of the snout was fixed with 4% formaldehyde for 4 hours. Then, it was washed with ethanol for 30 minutes × 2 times and further with toluene for 10 minutes × 3 times. The head was immersed in 100% paraffin four times for 1 hour each and then embedded in paraffin. 5 micron slices were collected and rehydrated with xylene for 5 min x 3 times and reduced EtOH for 5 min x 3 times. DC0 antibody was obtained from Daniel Kalderon (Columbia University, New York). The antibody was used at a dilution of 1: 400 with PBHT (0.02M NaPO ₄ , 0.5M NaCl, 0.2% Triton X-100 (pH 7.4)) and 5% goat serum. The FITC-conjugated anti-rabbit secondary antibody was diluted 1: 2000 before use.

Screening for single gene variants affecting AMI and identification of DC0 mutant Drosophila
To identify single-gene mutations that affect AMI, DC0 (references 7, 11, 13), volado (reference 8), fasciclin 2 (reference 9), dunce (reference 10), rutabaga (reference) In addition to the known mutants, the GAL4 transposon factor line that shows a reporter signal in the mushroom body, etc., was provided with mutants of about 100 genes mainly expressed in the mushroom body. We tested memory for 1 hour in young and aged animals, and searched for inhibitory mutants of AMI using the decrease in memory for 1 hour as an index.

  Known mutants such as DC0, volado (vol), fasciclin 2 (Fas II), dunce (dnc), rutabaga (rut) induce gene mutation by ingesting wild type with X-ray irradiation or carcinogens It was produced. On the other hand, the GAL4 transposon factor strain is a strain in which a gene expression disorder is caused by the insertion of a transposon factor including the yeast transcription factor GAL4 around the enhancer region. GAL4 recognizes the UAS sequence of the gene and induces gene expression downstream of the UAS sequence. Therefore, when a GAL4 transposon factor strain and a reporter strain having a reporter gene (eg, a gene encoding a fluorescent protein such as GFP) downstream of the UAS sequence were crossed, the expression was suppressed by the insertion of the GAL4 transposon factor in the next generation (F1). Instead of a gene, expression of a reporter protein is observed in the expression region (also called an enhancer trap line). The GAL4 transposon factor strain used in this study is a strain in which the reporter is expressed in the F1 mushroom body by crossing with the reporter strain, and the expression of the gene expressed in the mushroom body is expected to be suppressed. is there.

AMI is characterized by significant memory one hour after odor conditioning on the 20th day after emergence, so the 1 hour memory is compared between the young mutant (Day 1) and the old mutant (Day 20). (FIG. 1a). FIG. 1 a shows a typical example of 1-hour and 20-day 1-hour memory of a gene variant expressed in mushroom bodies. Line 1 is the wild type of control, 2-10 is enhancer GAL4, 11 is fasII ^rd2 , 12 is DC0 ^B3 / +, 13 is vol ¹ , 14 is dnc ¹ , 15 is rut ²⁰⁸⁰ (N number is 4-6 in all groups).

  In the wild type, taking into account that the memory score (Performance index) decreases by 20-30 at 20 days of age, two strains (No. 8 and 12) suppress the decrease in memory due to aging, and three strains (2 , 3, 6), it was found that the decrease in memory became more prominent (FIG. 1b). FIG. 1b shows the difference between the 1 hour memory on day 1 and the 1 hour memory on day 20 of the gene variant expressed in the mushroom body. “+” Indicates that the memory has greatly declined. On the other hand, “*” indicates that the memory loss is smaller than the wild type of the control. In addition, with the dnc mutant homozygote 14 and the homozygous rut mutant 15, the one-hour memory of young flies was already extremely low, so accurate AMI could not be measured in these strains.

Among these, line 12 is a heterozygous DC0 (DC0 ^B3 / +) of the hypomorphic mutation of the DC0 gene. The young DC0 ^B3 / + had normal memory, but the memory was retained even when it became an aging body. DC0 ^B3 mutants were crossed with wild-type control strains for 10 generations, and even after equilibrating the genetic background, AMI was significantly suppressed in DC0 ^B3 / +. Therefore, it was clarified that the suppression of AMI is due to the DC0 gene mutation and not the genetic background effect (FIG. 1d). FIG. 1d shows a 1 hour memory score. It was shown that AMI was suppressed in flies of DC0 ^B3 / + mated with 10 generations of w (CS10) and DC0 ^H2 / + mated with 5 generations to eliminate genetic background effects (all genes Type and N number is 8-12).

  The DC0 gene encodes the catalytic subunit of PKA expressed in the fly brain, and the homozygous mutant is embryonic lethal. DC0 plays an important role in the cAMP signaling pathway involved in memory processes. Overexpression of PKA regulatory subunits that do not respond to cAMP and PKA inhibitory genes (PKI) suppresses DC0-PKA activity, thereby inhibiting memory formation (Reference 12). Of particular interest is that temperature-sensitive mutants of DC0 have been reported to have defects in metaphase memory similar to amn (Reference 13). As in previous reports, it was confirmed that DC0-PKA was notably expressed in mushroom lobes and calyces, but not in kenyon cells (FIG. 1c) (reference 7). FIG. 1c is a DC0-PKA immunofluorescence photograph using an anti-DC0-PKA antibody, showing the localization of the DC0-PKA protein in the mushroom body.

The PKA activity of DC0 ^B3 / + was reduced to about 60% of the wild type PKA activity as a control (FIG. 1e). FIG. 1e compares the PKA activity of the DC0 ^B3 / + and DC0 ^H2 / + flies. The PKA activity of DC0 ^B3 / + and DC0 ^H2 / + flies is 65.6 ± 4.8% and 57.2 ± 2.1%, respectively, compared to the wild type (+ / +) (N = 4). In addition, all the data in this specification are shown by the average +/- standard error.

To elucidate whether AMI suppression of DC0 ^B3 / + is allele-specific, we compared memory between young and old flies in DC0 ^H2 / +. DC0 ^H2 is the second strongest hypomorphic mutant of DC0 with activity close to the PKA activity of DC0 ^B3 / + (FIG. 1e). DC0 ^H2 / +, like DC0 ^B3 / +, showed normal memory at a young age and AMI was significantly suppressed (FIG. 1d). This indicates that AMI is suppressed when PKA activity decreases universally. Moreover, although learning memory is impaired by excessive increase or decrease of PKA activity, a more subtle decrease in activity of about 40% does not affect learning and memory of young flies, and prevents memory decrease due to aging. It has been suggested.

Relationship between lifetime of DC0 mutant and AMI suppression
Since AMI is generally considered to result from individual aging, DC0 / + mutants may suppress AMI by delaying individual aging. In order to investigate the possibility, the lifetime of DC0 ^B3 / + was examined. Surprisingly, the life span of DC0 ^B3 / + flies was no different from wild type. In males, wild type 41.6 days (N = 238) versus DC0 / + is 39.2 days (N = 239), in females wild type 36.3 days (N = 236), DC0 / + is 37.0 days (N = 227) (FIG. 2a).

In wild-type flies, AMI appeared 15 days after emergence. On the other hand, in DC0 ^B3 / +, no remarkable AMI was observed until 30 days after emergence (FIG. 2b). FIG. 2b shows that AMI expression is delayed in DC0 ^B3 / +. A decrease in 1-hour memory appears on day 15 in the wild type control, whereas it appears only on day 30 in DC0 ^B3 / + flies (P <0.05 by day 0 DC0 ^B3 / The PI values at + and 30 days are 79.53 ± 3.0 (N = 6) and 65.0 ± 5.6 (N = 6), respectively). In other words, these results show that when PKA activity decreases by about 40%, the occurrence of AMI is delayed more than twice without affecting the lifespan, and it can be considered as a separate phenomenon from individual aging and AMI. Yes.

In order to evaluate in more detail the suppression of AMI by DC0 mutations, memory retention curves were examined and compared with wild type, DC0 ^B3 / + and DC0 ^H2 / + flies at 1 and 20 days of age. As a result, in the aged body (after 20-day-old) wild type, the 1-hour memory was remarkably reduced due to the specific decrease in the medium-term memory, and only slight changes were observed in the 0-hour memory and the 7-hour memory. On the other hand, neither DC0 ^B3 / + nor DC0 ^H2 / + showed any change in the memory retention curve due to aging (FIGS. 2c and d). Figures 2c and d show wild-type memory retention curves of DC0 / + mutant and control on day 1 and day 20. There is no difference in memory retention between flies of DC0 ^B3 / + (c) and DC0 ^H2 / + (d). Note that all data in FIGS. 2b-d are N = 6-12.

As a result, there was generally no change in memory retention curves with aging at DC0 / +, indicating that AMI is suppressed when PKA activity decreases.

Relationship between extended life and AMI suppression
The AMI suppression model system suggests a relationship between AMI and individual aging. However, it is unclear whether only one major in vivo pathway regulates the phenotype most relevant to aging, or whether many in vivo pathways are involved. Aging of cardiac function is regulated by the insulin / insulin-like growth factor pathway (IIS) as well as individual aging (14, 15). Therefore, aging of heart function is considered to be controlled by the same mechanism as individual aging. Similarly, reducing metabolic activity in mammals suppresses AMI in the same way as extending lifespan. On the other hand, a decrease in metabolic activity due to excessive caloric restriction has been reported to prolong life but not inhibit AMI (Reference 16). In addition, compared with other animals, the period from the start of menopause to the death due to longevity is relatively long in humans. These indicate that these two processes can be considered separately. In addition, as described above, there is no extension of life in DC0 / + in which AMI is suppressed. In other words, AMI shows that it can be suppressed without affecting the life span, but this means that AMI and individual aging can be considered separately.

  To further investigate the correlation between individual aging and AMI, flies were reared under two conditions that could extend their lifespan. One is to keep under calorie restriction, and the other is to keep under low temperature. Similar to the suppression of IIS, these also extend their lifespan through an overlapping mechanism of metabolic suppression. As expected, wild-type flies bred under calorie restriction have increased lifespan by approximately 30%, with average life spans of 37.6 to 48.4 days for males and 27.4 to 37.8 days for females (FIG. 3a). Wild-type flies reared at low temperatures are about twice as long, and males have lifespans from 52.3 to 95.2 days and females from 40.5 to 82.6 days (Fig. 3c).

  As seen in mammals, 20-day-old flies reared under caloric restriction had a higher memory score than controls of the same day-age (FIG. 3b). FIG. 3b shows that AMI was suppressed by calorie restriction. The PI value of 1-hour memory on the first day after emergence (1 day of age) is 69.4 ± 2.9 (N = 10). Calorie-limited 20-day-old 1-hour memory (53.1 ± 2.9, N = 10) is significantly higher than normally-bred 20-day-old 1-hour memory (42.0 ± 2.3, N = 10) (P <0.01, T test). On the other hand, even with calorie restriction, significant memory decline from day 1 is observed (P <0.001, T test).

  Similar to the calorie restricted flies, the 20-day-old flies kept at low temperature also showed higher memory scores than the controls. This shows that both conditions improve the AMI as well as prolong the lifetime (FIG. 3d). FIG. 3d shows that AMI was suppressed by low temperature breeding. PI value is 72.8 ± 3.4 (N = 12) at 1 day of age, 52.8 ± 3.1 (N = 8) at 20 days of chilled breeding, and 38.0 ± 1.7 (N = 8) at 20 days of normal breeding (P <0.005 T test: 20-day-old rearing at 18 ° C. compared with rearing at 24 ° C.). However, even when kept at low temperature, significant memory loss was observed at 20 days of age compared to 1 day of age (P <0.001 T test: comparison between 20 days of age reared at 18 ° C and 1 day of age). In particular, flies reared at low temperatures live twice as long as control flies. Therefore, if aging and AMI are equally suppressed by low temperature feeding, the memory score of low temperature fed 20 day flies should not differ from the memory score of young flies.

  These results indicate that even if there is a relationship between AMI and life, the conditions for maximizing the life are not necessary and sufficient to suppress AMI.

In order to further verify whether extension of individual aging is sufficient for suppression of AMI, AMI was examined using methuselah (mth), a long-lived mutant (Reference Document 17). Under our experimental conditions, mth ¹ lives 30% longer than the control wild type (FIG. 3e). FIG. 3e shows the extension of the lifetime of the mth ¹ mutant. The average life span of male mth ¹ and wild-type flies was 48.1 days (N = 198) and 35.3 days (N = 202), respectively. The life expectancy of female mth ¹ and wild type was 43.2 days (N = 194) and 33.7 days (N = 196), respectively.

Nevertheless, similar to wild-type flies, significant AMI expression occurred 15 days after emergence (FIG. 3f). In wild type, significant AMI was observed from 15 days of age [P <0.001 T test: wild type 1 day and 15 days of age; 74.8 ± 1.3 (N = 8) and 52.8 ± 3.1 (N = 8)]. mth ¹ also showed a remarkable AMI at 15 days of age, similar to the wild type. [P <0.001 T test: mth ¹ 1 and 15 days: 61.5 ± 1.6 (N = 8) and 39.4 ± 3.7 (N = 8)]
In other words, memory loss at 15 days of age is indistinguishable between mth ¹ and wild type. This result is similar to the previous result that motor activity declines normally even in mth (Reference 18). These facts suggest that although individual aging and AMI are in partial agreement, prolonged individual aging is neither a necessary nor a sufficient condition for AMI suppression.

Relationship between DC0-PKA activity and AMI
Since the decrease in DC0-PKA activity strongly suppresses AMI, there is a possibility that the decrease in memory ability is reduced by increasing the expression of DC0 due to aging. Overexpression of DC0 in the mushroom body results in significant memory impairment (FIG. 4a). FIG. 4a shows that when DC0-PKA is overexpressed in mushroom bodies, memory decreases for the same time as aging bodies. In the wild type (+ / +) and the strain (c747 / PKAc +) in which the DC0 gene was forcibly expressed in the mushroom body, 1-hour and 20-day-old memories were examined respectively. c747 is an enhancer trap line (GAL4 transposon factor line) that specifically expresses GAL4 in a mushroom body, and PKAc + is a line having a DC0 gene downstream of the UAS sequence. That is, c747 / PKAc + expresses a catalytic subunit having constant activity in the mushroom body under GAL4 / UAS control. All data are N = 6.

  However, neither PKA activity nor DC0-PKA protein levels changed with aging (FIG. 4b). FIG. 4b shows that DC0 expression and PKA protein levels do not change as flies age. Total RNA was isolated from day 1 and day 20 wild-type flies. The level of DC0 transcript was quantified using quantitative PCR. Furthermore, it was normalized at the level of GAPDH2 transcript. The activity of PKA was measured with a 1-day-old wild type and 20-day wild-type head protein extract under a cAMP concentration of 2 μM. Samples were standardized with a calibration curve made from purified recombinant PKAc. When the average expression / activity of 1-day-old flies was defined as 100%, the expression / activity of 20-day-old flies was almost the same as that of 1-day-old. Each data is shown as an average from 4 independent experiments.

Although there was no increase in DC0-PKA expression due to aging, AMI may still be enhanced by increasing DC0-PKA activity. Therefore, in order to investigate whether AMI is generated earlier when DC0-PKA activity is increased by increasing cAMP level, AMI was examined with a mutant of gene dunce (dnc) encoding cAMP-specific phosphodiesterase. Compared to homo (see FIG. 1a), heterozygous mutant females (dnc ¹ / +; dnc is present on the X chromosome) of dnc ^1, a large memory impairment is not indicated when young. Surprisingly, the control wild type female showed AMI from day 15 after emergence (see Fig. 2b), whereas the dnc ¹ / + female became adult and already had AMI on the fifth day. Woke up (Figure 5). That is, when comparing 1 hour memory between wild type females and dnc1 / + females for 1 hour, 5 days old and 10 days old, AMI has not yet occurred at 10 days old in wild type, but dnc1 / + Has already seen significant AMI at 5 days of age (P <0.02). The respective data are shown as mean ± standard error, N = 8. Male flies with the same genetic background as dnc1 / + females (F1 males obtained by mating dnc1 males with wild-type females, have no dnc1 mutation, but other gene backgrounds are dnc1 / + females Shows the memory and AMI indistinguishable from the wild male and female.

  This indicates that the enhanced AMI produced by dnc1 / + is not due to genetic background effects. The 10-day-old dnc1 / + female showed normal avoidance against OCT and MCH used in the memory test (Fig. 5), indicating that the increase in AMI is not due to olfactory disturbance. .

In addition, protein phosphatase 1 (Pp1-87B) mutants cause AMI early. In addition, the present inventors have found that AMI is suppressed in flies in which a PKA regulatory subunit that suppresses DC0-PKA is overexpressed in mushroom bodies. From these results, it was shown that when the activity of DC0-PKA is lowered, the onset of AMI is delayed, but when the activity is raised, AMI occurs earlier.

  In this example, proteins obtained from wild-type and DC0 / + mutants were developed by two-dimensional electrophoresis, and the kinetics of proteins expressed in the wild-type due to aging (FIG. 6A), wild-type and DC0 / The expression levels of proteins expressed in / + aging bodies were compared (FIG. 6B).

  Fig. 6A shows that proteins expressed in wild-type young bodies (7 days old) are labeled with green fluorescence and proteins expressed in old bodies (20 days old) are labeled with red fluorescence. It is a figure which shows the result developed by dimensional electrophoresis. If it is a protein that does not change in expression with age, it is a yellow fluorescent spot.If it is a protein whose expression decreases with age, it is a spot with strong green fluorescence.If it is a protein that increases expression with age. The relative intensity (quantity ratio) was quantified with an imaging analyzer as a spot with strong red fluorescence.

  Figure 6B shows the same gel with DC0 / + (AMI-suppressed) aged bodies labeled with green fluorescence and wild-type (AMI-occurring) aged bodies labeled with red fluorescence. It is a figure which shows the result developed by two-dimensional electrophoresis above. Thus, for example, a spot with strong green fluorescence is a protein that is expressed in DC0 / + (AMI-suppressed) aging bodies higher than in wild-type (AMI-occurring) aging bodies.

  Next, the expression of the same spot was compared between FIG. 6A and FIG. 6B, and the expression change of the protein involved in the expression of AMI was examined (FIG. 6C). For example, in both FIGS. 6A and 6B, in the case of a spot with strong green fluorescence, the expression decreases with aging in the wild type (under the condition where AMI occurs), but with aging in DC0 / + (under the condition where AMI is suppressed) Therefore, it can be said that maintaining the expression of this protein is important for the suppression of AMI. In FIG. 6C, it can be said that proteins showing spots 3 and 6 are useful for AMI suppression. Whether or not this protein expression maintenance is important for AMI suppression is simply determined by whether or not AMI is suppressed in the same way as DC0 / + when the decrease in expression of this protein is suppressed by genetic manipulation in the wild type. Can be verified.

References
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It is a figure which shows that AMI is suppressed by DC0 / + mutation. FIG. 3 shows that the DC0 mutant suppresses AMI without acting on the life span. It is a figure which shows that extending a lifetime is not a necessary and sufficient condition for suppression of AMI. It is a figure which shows that PKA activity changes in relation to aging. It is a figure which shows that AMI promotes when PKA activity increases. It is the photograph which compared the dynamics (A) by the aging of the protein expressed with a wild type, and the expression (B) of the protein expressed with the aging body of a wild type and DC0 / +.

Sequence number 3: Primer sequence number 4: Primer sequence number 5: Primer sequence number 6: Primer

Claims (3)

A non-human animal in which the gene encoding the protein kinase A catalytic subunit is mutated and the activity of the protein kinase A in the brain is reduced to 50-60% of the wild-type activity is treated as a model for suppressing or improving age-related memory impairment. a method of use as Rudo thereof,
It said non-human animal is Drosophila, the protein kinase A catalytic subunit is DC 0,
Said method . A method for identifying a target substance of a substance that suppresses or improves age-related memory impairment ,
(A) a gene encoding a protein kinase A catalytic subunit is mutated, non-human animal-derived gene or protein activity of the protein kinase A is reduced to 50% to 60% of the wild-type activity in the brain, and the wild-type Measure the expression patterns of genes or proteins derived from non-human animals,
By comparing (b) obtained expression pattern, seen including identifying a substance to be a target of substances which inhibit or ameliorate age-related memory disorders,
The non-human animal is Drosophila and the protein kinase A catalytic subunit is DC0;
Said method. A protein derived from a non-human animal in which the gene encoding the protein kinase A catalytic subunit has been mutated to reduce the activity of the protein kinase A in the brain to 50-60% of the wild-type activity , and from a wild-type non-human animal protein was measured and compared the presence or level of phosphorylated with, from the measurement results obtained, you identify proteins that activity of the protein kinase a was phosphorylated suppressed in non-human animals with a reduced, age- A method of identifying a substance that is a target of a substance that suppresses or improves memory impairment ,
The non-human animal is Drosophila and the protein kinase A catalytic subunit is DC0;
Said method .