WO2023029210A1 - Pp2a agonist, and compound for purging and/or dissolving aging cells and/or inhibiting cell aging for use in treatment of mental disorder - Google Patents

Abstract

An application of a PP2A agonist or a compound for purging and/or dissolving aging cells and/or inhibiting cell aging during the preparation of a drug for preventing and treating mental disorders caused by normal aging. The mental disorder is selected from among cognitive impairment, affective disorders, and/or behavioral disorders, and the PP2A agonist is MPH, DT-061 or FTY720. The compound for purging and/or dissolving aging cells and/or inhibiting cell aging is selected from among ABT263, compounds that induce PP2A activity and/or expression increase, compounds that induce p53 activity and/or expression decrease, and compounds that induce p21 activity and/or expression decrease.

Description

PP2A agonists, compounds that remove and/or dissolve senescent cells and/or inhibit cellular senescence for the treatment of mental disorders technical field

The invention relates to the technical field of biomedicine, in particular to a PP2A agonist, a compound for clearing and/or dissolving senescent cells and/or inhibiting cell senescence for treating mental disorders.

Background technique

Mental disorders are syndromes characterized by disturbances in individual cognition, emotion, or volitional behavior. According to the presence or absence of organic factors, it is mainly divided into: organic mental disorders and functional mental disorders. Organic mental disorders are disorders caused by central nervous system diseases, metabolic disorders, alcohol substance abuse, poisoning, brain damage, encephalitis and other factors. Symptoms such as behavioral changes, such as delirium, amnestic syndrome, and dementia. Functional mental disorders are symptoms such as hallucinations, delusions, and mood disorders, such as anxiety disorders and schizophrenia, caused by the long-term interaction of biological, psychological, and social environmental factors.

Protein phosphatase 2A (protein phosphatase 2A, PP2A) is a serine/threonine phosphatase with a wide range of substrates, most of which involve transcription factors and protein kinases, and participate in many cell biological functions, such as energy metabolism, DNA damage It plays an important role in repair, protein translation, cell cycle regulation and signal transduction. Previous studies have shown that PP2A has a tumor suppressive effect; participates in the regulation of sugar and lipid metabolism, and abnormal function of PP2A can lead to the occurrence of tumors, diabetes, obesity, etc. At the same time, coronary atherosclerosis is also related to the abnormal variation of PP2A; it is involved in retinopathy And secondary pathological processes such as cardiomyopathy. In addition, the patent document CN101478977A discloses that the abnormal hyperphosphorylation of tau protein in the brain of Alzheimer's disease (AD) is partly due to the down-regulation of PP2A activity, so it can increase the activity of PP2A, especially the activity of the ABC isoform of PP2A The formulation of the drug will have clinical use to treat or prevent the development of neurodegenerative diseases. Patent document CN103893182A discloses that small molecule inhibitors of SET protein can increase the activity of PP2A in cells by inhibiting the interaction between SET protein and PP2A, reduce the phosphorylation level of protein kinases interacting with PP2A, and promote cell apoptosis. Change the cell cycle to play the corresponding biological role, and treat inflammation, neurodegenerative diseases, malignant tumors and autoimmune diseases with abnormal expression of SET protein. The master's thesis of Jilin University, "Experimental Study of Src Promoting Outgrowth of Injured Neurons and Growth Cone Formation Through PP2A", discloses that inhibiting PP2A activity promotes the growth of neurites and growth cone formation in injured neurons. Journal paper "Lu Wenhui, Qu Qiumin, Cao Hongmei. The effect of lithium on the expression of CDK5 and PP2A in the brain of rats exposed to chronic aluminum [J]. Journal of Xi'an Jiaotong University (Medical Sciences), 2010, 031(004): 463-466." It is disclosed that there is no significant difference in the PP2A content of rat cortex and hippocampus in the normal group, the chronic aluminum exposure lithium treatment group and the non-treatment group. Overall, the current relationship between PP2A and neurological and psychiatric disorders is unclear.

Both p53 and p21 are cyclin-dependent kinase inhibitors that play an important role in the initiation and maintenance of cellular senescence. ABT263 is a compound known to clear senescent cells.

In general, there are no reports of PP2A agonists in the prevention and treatment of mental disorders caused by normal aging or reduced PP2A activity or expression, nor the use of compounds that clear and/or dissolve senescent cells and/or inhibit cellular senescence , Research on the treatment of mental disorders mediated by accumulation of DNA damage and increased aging of nerve cells by clearing and/or dissolving senescent cells and/or inhibiting cell senescence.

Contents of the invention

The object of the present invention is to address the deficiencies in the prior art, and to provide the use of PP2A agonists, compounds for removing and/or dissolving senescent cells and/or inhibiting cell senescence for the treatment of mental disorders.

In the first aspect, the present invention provides the use of PP2A agonists in the preparation of drugs for preventing and treating mental disorders caused by normal aging.

As a preferred example, the mental disorder is selected from cognitive disorders, emotional disorders and behavioral disorders, and the phenotypes of the mental disorders are weakened ability to respond to the outside world, weakened ability to explore new things, irritability, anxiety, Depression, decreased social skills, decreased cognitive function, decreased ability to learn, or decreased ability to remember.

As another preferred example, the PP2A agonist is MPH, DT-061 or FTY720.

In the second aspect, the present invention provides the use of PP2A agonists in the preparation of drugs for the prevention and treatment of mental disorders caused by the reduction of PP2A activity and/or expression.

As a preferred example, the mental disorder is selected from cognitive disorders, emotional disorders and/or behavioral disorders.

More preferably, the phenotype of the mental disorder is weakened ability to respond to the outside world, weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, decreased cognitive function, decreased learning ability or memory ability decline.

As another preferred example, the mental disorder caused by the decreased activity and/or expression of PP2A is ADHD, bipolar disorder, schizophrenia or depression and anxiety.

As another preferred example, the PP2A agonist is MPH, DT-061 or FTY720.

In the third aspect, the present invention provides the application of PP2A agonists in the preparation of experimental reagents for reducing DNA damage, clearing senescent cells, dissolving senescent cells or inhibiting cell senescence.

In a fourth aspect, the present invention provides PP2A activity and/or expression as a biomarker for preparing a reagent or a kit for diagnosing age-related mental disorders.

As a preferred example, the age-related mental disorder is a mental disorder caused by normal aging.

More preferably, the mental disorder is a mental disorder closely related to age, selected from senile anxiety, senile depression, senile ADHD and senile bipolar disorder.

In a fifth aspect, the present invention provides a method for preventing and treating mental disorders in a subject in need due to normal aging or reduced PP2A activity and/or expression, comprising administering protein phosphatase 2A (PP2A) to the subject. ) agonist step.

In the sixth aspect, the present invention provides the use of compounds for eliminating senescent cells and/or dissolving senescent cells and/or inhibiting cell senescence in the preparation of drugs for preventing and treating mental disorders.

As a preferred example, the mental disorder is a mental disorder caused by the accumulation of DNA damage and/or the increase of aging nerve cells.

More preferably, said accumulation of DNA damage and/or increased senescence of nerve cells is caused by normal aging, or by decreased activity and/or expression of PP2A.

As another preferred example, the mental disorder is selected from cognitive disorders, emotional disorders and/or behavioral disorders.

More preferably, the phenotype of the mental disorder is weakened ability to respond to the outside world, weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, decreased cognitive function, decreased learning ability or memory ability decline.

As another preferred example, the compound for eliminating senescent cells and/or lysing senescent cells and/or inhibiting cell senescence is a small molecular compound or a biomacromolecule.

As another preferred example, the compound that eliminates senescent cells and/or dissolves senescent cells and/or inhibits cell senescence is selected from ABT263, compounds that cause increased activity and/or expression of PP2A, compounds that cause decreased activity and/or expression of p53 Compounds, and compounds that cause a decrease in p21 activity and/or expression.

In a seventh aspect, the present invention provides a method for preventing and treating mental disorders in a subject in need, comprising the step of administering to the subject a compound that eliminates senescent cells and/or dissolves senescent cells and/or inhibits cellular senescence .

As a preferred example, the mental disorder is a mental disorder mediated by accumulation of DNA damage and/or increased aging of nerve cells.

More preferably, said accumulation of DNA damage and/or increased senescence of nerve cells is caused by normal aging, or by decreased activity and/or expression of PP2A.

As another preferred example, the mental disorder is selected from cognitive disorders, emotional disorders and/or behavioral disorders.

More preferably, the phenotype of the mental disorder is weakened ability to respond to the outside world, weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, decreased cognitive function, decreased learning ability or memory ability decline.

As another preferred example, the compound for eliminating senescent cells and/or lysing senescent cells and/or inhibiting cell senescence is a small molecular compound or a biomacromolecule.

As another preferred example, the compound that eliminates senescent cells and/or dissolves senescent cells and/or inhibits cell senescence is selected from ABT263, compounds that cause increased activity and/or expression of PP2A, compounds that cause decreased activity and/or expression of p53 Compounds, and compounds that cause a decrease in p21 activity and/or expression.

In this paper, the "mental disorder" is a general term for different degrees of disorders in mental activities such as cognition, emotion, behavior and will. As known in the art, the "cognition" includes three processes: sensation and perception, memory and attention, and thinking process. The "sensation and perception" refers to using the eyes, ears, nose, tongue and skin to understand the surrounding things to understand the world. This process is the brain's perception of the existence of external objective things, that is, the result of reflecting the external things to the brain . The "memory" in the "memory and attention" refers to keeping the things perceived in the past in the brain, and the "attention" refers to the direction of human mental activities to certain things. The "thinking process" refers to the process in which the brain analyzes, synthesizes, and judges the information reflected in the brain by using past experience to draw conclusions. Said "emotion" refers to people's attitude and external expression towards any kind of thing. The above-mentioned "behavior and will" refers to the psychological process of people taking actions in order to achieve certain goals. Under normal circumstances, the three aspects of mental activity "cognition", "emotion", "behavior and will" are coordinated and coordinated with each other, and are consistent with the external environment. When there is a mental disorder, it shows the incoordination of "cognition", "emotion" and "behavior and will", or the "cognition", "emotion" or "behavior and will" are inconsistent with each other. The incompatibility of the external environment.

In this paper, the "mental disorders caused by the accumulation of DNA damage caused by normal aging and/or the increase of nerve cell aging" and "mental disorders caused by normal aging" both refer to mental symptoms that affect healthy aging. WHO believes that At the biological level, aging is the gradual accumulation of various molecular and cellular damages, leading to the gradual reduction of physiological reserves, the general decline of various abilities, and the increased risk of many diseases. But these changes are neither linear nor constant across populations; they are sporadicly correlated with age. For example, while some people over the age of 70 may enjoy life in good health, in good spirits, the vast majority are debilitated and require various types of support to meet their basic needs. Mental status changes (or disorders) proposed by WHO include: changes in coping ability; behavioral changes; memory loss; aggression; depression, anxiety and apathy; difficulty sleeping; delusions and hallucinations; repetitive behaviors; walking and getting lost; changes in judgment. In the elderly population, the appearance of one or more of the above symptoms indicates the decline of the mental or psychological status of the elderly. But this mental state change (or disorder) is distinct from Alzheimer's disease (AD) caused by β-amyloid (Aβ) deposits and tau tangles.

Herein, the "PP2A agonist" refers to a compound that directly or indirectly acts on and activates PP2A to produce a physiological response.

In this article, the full English name of MPH is Methlphenidate, also known as methylphenidate. DT-061 is a known PP2A agonist, also abbreviated as SMAP. FTY720 is a known PP2A agonist known as fingolimod.

The present invention has the advantage that:

1. The present invention finds that MPH has a therapeutic effect on the mental disorders of aged zebrafish, aged mice and ppp2r2c ^m/m zebrafish, and can eliminate its DNA damage (DDR) and aging nerve cells, so MPH can be used to prepare and prevent normal Drugs for mental disorders caused by aging or decreased PP2A activity.

2. The present invention finds that MPH increases the PP2A activity of aged zebrafish, aged mice and ppp2r2c ^m/m zebrafish, and is a PP2A agonist.

3. The present invention also finds that PP2A agonists can improve the mental disorders of ppp2r2c ^m/m zebrafish, aged zebrafish and aged mice, and eliminate their DNA damage (DDR) and aging nerve cells, so PP2A agonists can be used to prepare and prevent Drugs for mental disorders due to normal aging or decreased PP2A activity.

4. The present invention finds that aged zebrafish and mice and PPP2R2C ^m/m mutant zebrafish all have a certain degree of mental disorder, and all have severe DNA damage and cell senescence. Treatment with MPH or other PP2A agonists reversed psychotic symptoms, DNA damage, and senescent neurons in aged zebrafish and mice, as well as in PPP2R2C ^m/m mutant zebrafish. Further, in the PPP2R2C ^m/m mutant zebrafish that knocked out the aging-related gene p53 and the PPP2R2C ^m/m mutant zebrafish that knocked out the aging-related gene p21, it was observed that the symptoms of mental disorders in the mutant zebrafish were significantly improved, and PPP2R2C ^m The symptoms of mental disorders were also significantly improved after ABT263, which has the effect of clearing senescent cells, in ^/m mutant zebrafish. The above results fully demonstrate that removing senescent cells and/or dissolving senescent cells and/or inhibiting cell senescence is an effective way to treat mental disorders, and compounds that remove senescent cells and/or dissolve senescent cells and/or inhibit cell senescence can be used to prepare and prevent mental disorders Drug.

Description of drawings

Figure 1: Schematic showing the mutation of the zebrafish ppp2r2c gene using the CRISPR-Cas9 system. Yellow and red boxes show target exons and domains, respectively. The red dashed line shows the 4-nucleotide deletion in ppp2r2c. Black triangles show the positions of PCR primers used to amplify ppp2r2c mRNA.

Figure 2: RT-qPCR analysis of primers ppp2r2c-p1, ppp2r2c-p2 and ppp2r2c-p3 at different positions of the ppp2r2c gene (including before and after the ppp2r2c mutation region shown in (Figure 1), other regulatory subtypes of PP2A RT-qPCR analysis of the base genes (ppp2r2a, ppp2r2b, ppp2r2d) and ppp2r5c in adult wild-type and homozygous ppp2r2c mutant fish brains showed that the mRNA expression level of ppp2r2c was specifically decreased in this model, and the model construction Success (n=3 independent biological samples; unpaired two-sided t-test).

Figure 3: Experimental design. Behavioral testing of WT and ppp2r2c ^m/m (6 month old) fish with or without MPH treatment for 3 days.

Figure 4: Experimental scheme of photostimulation test and parameters for recording and analysis in photostimulation test. a, the experimental scheme of the photostimulation test; b, the representative figure of adult fish locomotor activity during the 30-second light period; c, the quantification of the burst state duration during the 30-second light period (n=9 for each group).

Figure 5: a, Representative locomotion trajectories (grey lines) in microscopy tests of WT and ppp2r2c ^m/m (6 months old) with and without MPH treatment for 3 days during the 5 min test period. b Quantification of the number of mirror attacks in 5 min intervals (WT-vehicle, WT-MPH and ppp2r2c ^m/m -vehicle, n = 9; ppp2r2c ^m/m -MPH, n = 8).

Figure 6: a. Representative locomotion trajectories (gray lines) of WT and ppp2r2c ^m/m (6 months old) with or without MPH treatment for 3 days during a 30-min trial of the open-field test, where the square indicates the central area . b is the cumulative time of adults staying in the central area (ppp2r2c ^m/m -vehicle, n=7; WT-vehicle and WT-MPH, n=8; ppp2r2 ^cm/m -MPH, n=9). Data are mean ± SEM, *P<0.05, **P<0.01, ns, not significant; two-way ANOVA.

Figure 7: Photostimulation, mirror image challenge, and open field tests with different psychotherapeutic drugs.

Figure 8: 3-day PP2A phosphatase assay in adult ppp2r2c ^m/m zebrafish brains (n = 3 independent biological samples, each containing two brains).

Figure 9: a is a representative graph of locomotor activity of WT and ppp2r2c ^m/m (6 months old) treated with or without DT-061, FTY720 and MPH+DT-061 during 30 seconds of light. b Quantification of burst state duration during 30 s illumination (WT, n=9; WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n=6, WT-FTY- 720 and ppp2r2c ^m/m -FTY-720, n=10; WT-MPH+DT-061 and ppp2r2c ^m/m- MPH+DT-061, n=11).

Figure 10: a is representative of the mirror challenge test of WT and ppp2r2c ^m/m (6 months old) with or without DT-061, FTY720 and MPH+DT-061 treatment for 3 days during the 5 min test period Sexual movement trajectory (grey line). b Quantification of the number of mirror attacks in a 5-min interval (WT-vehicle, n = 9; WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n = 6, WT-FTY- 720 and ppp2r2c ^m/m -FTY-720, n=10; ppp2r2c ^m/m- MPH+DT-061, n=11; WT-MPH+DT-061, n=12).

Figure 11: a is the representative movement of WT and ppp2r2c ^m/m (6 months old) with or without DT-061, FTY720 and MPH+DT-061 treatment for 3 days during the 30-minute open-field test trajectory (gray line). The box shows the central area. b is the cumulative time of adults staying in the central area (WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m- DT-061, n=9; WT-vehicle, n=10; WT-FTY -720 and ppp2r2c ^m/m -FTY-720, n=11, WT-MPH+DT-061 and ppp2r2c ^m/m- MPH+DT-061, n=6). c is the average speed of adult fish (WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n=9; WT-vehicle, n=10; WT-FTY-720 and ppp2r2c ^{m/m m} -FTY-720, n=11, WT-MPH+DT-061 and ppp2r2c ^m/m -MPH+DT-061, n=6). d is the total swimming distance of adult fish (WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n=9; WT-vehicle, n=10; WT-FTY-720 and ppp2r2c ^m/m -FTY-720, n=11, WT-MPH+DT-061 and ppp2r2c ^m/m -MPH+DT-061, n=6).

Figure 12: Immunofluorescence detection of NeuN (green) and γH2AX (red) in OT of WT and ppp2r2c ^m/m (6 months old) treated with or without DT-061, FTY720 and MPH+DT-061 for 3 days (Scale bar, 5 μm). Quantification of the percentage of γH2AX positivity in neurons (NeuN+) and non-neurons (NeuN) (positive values indicate the number of nuclei/total number of nuclei with at least 5 γH2AX foci in the nucleus, n=6/group; * indicates the statistical difference of the NeuN+ group , # indicates the statistical difference of the NeuN-group).

Figure 13: SA-β-gal and NeuN (green) immunofluorescence in OT of WT and ppp2r2c ^m/m (6 months old) treated with or without DT-061, FTY720 and MPH+DT-061 for 3 days Representative images of staining (scale bar, 15 μm). Arrows point to SA-β-gal+ neurons. Determination of neuronal SA-β-gal positive neuronal (NeuN+) and non-neuronal (NeuN) percentages (n=6/group; * indicates statistical difference of NeuN+ group, # indicates statistical difference of NeuN- group). Data are mean±SD, *P<0.05, **P<0.01, #P<0.05, ##P<0.01, two-way analysis of variance.

Figure 14: Experimental design for behavioral testing of 6-month-old (adult) and 22-month-old fish (old fish), with or without MPH or DT-061 treatment for 3 days.

Figure 15: Recording and analysis parameters during photostimulation experiments. a is a representative graph of locomotor activity during the 30-s turn-on period. b Quantification of burst state duration during a 30-s light-on period (6m, n = 8; 22m, n = 15; 22m MPH, n = 11; 22m-DT-061, n = 9; single factor variance analysis).

Figure 16: a is a representative locomotion trajectory (gray line) of an adult fish during a mirror attack test during a 5-minute trial. b Quantification of the number of mirror attacks in 5-min intervals (6m, n = 14; 22m, n = 15; 22m-DT-061, n = 12; one-way ANOVA).

Figure 17: a is a representative locomotion trace (gray line) during a 30 min open field test. The box shows the central area. b is the cumulative residence time of adult fish in the central area (3m, n=18, 22m, n=11; 22m MPH, n=10; 22m-DT-061, n=9; one-way analysis of variance).

Figure 18: a Representative confocal images of Neun (green) and γH2AX (red) co-staining in brains of 22-month-old fish treated with or without MPH or DT-061 for 3 days (scale bar, 5 μm). b, quantification of γH2AX-positive nuclei (positive values indicate at least 5 γH2AX foci) (WT-vehicle, n=4; WT-MPH, n=7; WT-DT-061, n=3; unpaired bilateral t test).

Figure 19: a is a representative image of SA-β-gal staining of 22-month-old WT fish treated with or without MPH or DT-061 for 3 days (scale bar, 15 μm). b is the quantification of the percentage of SA-β-gal positive cells (WT-vehicle and WT-MPH, n=6; WT-DT-061, n=4; one-way ANOVA).

Figure 20: PP2A phosphatase assay after treatment of aged fish with MPH or DT-061 (n = 3 independent biological samples, each group containing two brains; one-way ANOVA). The data are expressed as mean ± standard deviation, *P<0.05, **P<0.01.

Figure 21: Behavioral experimental design of 3-month-old and 14-month-old mice treated with MPH.

Figure 22: Number of transitions between light and dark chambers in light-dark switching experiments (n=5, 3m; n=8, 14m; n=8, 14m-MPH; one-way ANOVA).

Figure 23: Maximum latency in the Morris water maze test.

Figure 24: Frequency of platform crossing in the mouse Morris water maze experiment.

Figure 25: Time spent in the correct quadrant in the Morris water maze test (3m, n=4; 14m, n=4; 14m-MPH, n=5; one-way ANOVA).

Figure 26: a is the co-staining of Neun (green) and γH2AX (red) in the frontal and temporal lobes of 14-month-old mice compared with 3-month-old mice after treatment with MPH or vehicle Representative confocal images (scale bar, 25 μm). b Quantification of 3m, 14m and 14m-MPH frontal or temporal γH2AX-positive nuclei (3m, n = 6; 14m, n = 3; 14m-MPH, n = 3; unpaired two-sided t-test).

Figure 27: a is a representative image of SA-β-gal staining in frontal and temporal lobes of 14-month-old WT mice treated with or without MPH compared with 3-month-old mice (scale bar, 25 μm) . b is the quantification of the percentage of cells positive for SA-β-gal staining (n=3/group; unpaired two-sided t-test).

Figure 28: PP2A phosphatase assays in frontal and temporal lobes of mouse brain (3m, n=5; 14m, n=8; 14m-MPH, n=8; unpaired two-sided t-test). The data are expressed as mean ± standard deviation, *P<0.05, **P<0.01.

Figure 29: a is a representative result of locomotor activity during the 30-second light period of the photostimulation test. b, quantification of burst movement states during 30 s of light exposure (ppp2r2c ^m/m p53 ^-/- , n = 11; WT, ppp2r2c ^m/m and ppp2r2c ^m/m p21 ^-/- , n = 10; WT- ABT263, n=9; ppp2r2c ^m/m -ABT263, n=8; one-way ANOVA).

Figure 30: a is WT and ppp2r2c ^m/m treated with or without ABT263 for 3 days in the mirror attack experiment, and the representative trajectory of ppp2r2c ^m/m p53 ^-/- and ppp2r2c ^m/m p21 ^-/- (grey Wire). b is the quantification of the number of mirror attack times in 5-min intervals (ppp2r2c ^m/m p53 ^-/- , n = 11; WT, ppp2r2c ^m/m and ppp2r2c ^m/m p21 ^-/- groups, n = 10; WT-ABT263 and ppp2r2c ^m/m -ABT263, n = 9; one-way ANOVA).

Figure 31: a is the representative trajectory of ppp2r2c ^{m/m p53 -/- and ppp2r2c m/m p21 -/- in} WT and ppp2r2c ^m/m treated with or without ABT263 for 3 days in a 30-minute open field test (gray line). The box shows the central area. b Quantification of cumulative time spent staying in the central region (WT, ppp2r2c ^m/m and ppp2r2c ^m/m p21 ^-/- groups, n = 10; WT-ABT263, ppp2r2c ^m/m -ABT263 and ppp2r2c ^m/m p53 ^-/- , n=9; one-way ANOVA).

Detailed ways

The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1

1. Experimental method

1. All drug solutions were freshly prepared in PBS (MPH 1.08mg/kg, sodium valproate 300mg/kg, aripiprazole 0.6 mg/kg, ziprasidone 1.6mg/kg, DT061 and FTY720 5mg/kg) . Zebrafish were given food soaked in the drug solution for 3 days and behavioral assays were performed on day 4. Both 14- and 3-month-old C57B6/J mice were produced and maintained in the animal facility of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University. The protocol was approved by the Animal Experimentation Ethics Committee. 14-month-old and 3-month-old C57B6/J mice were given MPH (12.3mg/kg/d) by intragastric administration for 14 days.

2. Generation of CRISPR-Cas9 mutant zebrafish. The ppp2r2c mutant zebrafish was generated using the CRISPR-Cas9 system, and the ZiFiT Targeter software (http://zifit.partners.org/ZiFiT) was used to design the ppp2r2c exon 9 (sgRNA: 5′-GGGCAGAGATACC-3′( SEQ ID NO:1)) and cdkn1a/p21 exon 2 (sgRNA: 5′-GGTAATGGGCCGACTAGG-3′(SEQ ID NO:2)) guide RNA. The F0 generation was bred to 3 months of age and outcrossed with wild zebrafish for potential F1indel mutations. PCR amplification and sequencing of genomic DNA isolated from F1 zebrafish tails to identify ppp2r2c mutants (primers used for genotyping: fwd 5′-CaggcAgtgttGaagat-3′ (SEQ ID NO:3); rev 5′ -GTGCTGAGAGGCCACTAA-3' (SEQ ID NO:4)) and p21 mutants (primers for genotyping: fwd 5'-TCTGTGATTGTGTG-3' (SEQ ID NO:5); rev 5'-GAGTGCACATCGTTC-3' (SEQ ID NO: 6)).

3. Immunofluorescence imaging of brain slices. Zebrafish tissues were dissociated at 4°C and fixed overnight with 4% PFA, followed by dehydration with 30% sucrose overnight. Tissues were embedded in OCT and sectioned at 5 min. Cryosections and slides were fixed in 4% PFA for 30 minutes, then permeabilized and blocked with 0.5% Triton X-100, 2% FBS in 1×PBS for 1 hour at room temperature. Hybridization with primary antibodies was performed overnight at 4°C, followed by three washes with 0.1% Tween-20 in PBS. Hybridization with corresponding secondary antibodies (Invitrogen) was performed at 37°C for at least 2 hours. Finally, sections were incubated with 1x DAPI for 5 min at room temperature. Photographs were taken using a confocal laser scanning microscope (SP8; Leica).

4. Determination of PP2A phosphatase. Homogenates prepared from zebrafish brain, mouse frontal and temporal lobes were placed in 20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0 and 10 μg/mL each of aprotinin, leupitin and pepsin inhibitor, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride, and dissolved by adding 1% NP-40. Analysis was then performed using the PP2A Immunoprecipitation Phosphatase Assay Kit (Microwell) according to the manufacturer's instructions. Absorbance at a wavelength of 650 nm (A650) was measured in a microtiter plate reader (BioTAK).

5. Zebrafish behavior analysis.

Place an adult fish in a standard mating tank (21 x 10 x 7.5 cm) containing system water to a depth of 6 cm and allow it to acclimate for 15 min before transferring to an automated observation and video tracking system (ZebraLab; Viewpoint Life Sciences).

Photostimulation test: An adult fish is placed in the dark and allowed to acclimatize for 15 minutes. The lights were then turned on for 30 s, and the activity was recorded using the ZebraLab quantification software module. The quantitative test focuses on zebrafish activity, which represents the total amount and frequency of zebrafish movement in the tank. The software automatically records the zebrafish's position and compares the previous position with the new position. Changed pixels are considered active. We obtained two thresholds (burst threshold of 100 and freeze of 20) using wild-type fish. The burst threshold indicates that 95% of the moving pixels change below this value. A frozen value means that the pixel changes below this value when the fish stops moving. If the value of the moving surface is above the burst threshold, the activity will be recorded as burst activity and the software will automatically record the duration of the activity.

Mirror Attack Test: A mirror was placed outside the end of the tank and the fish's aggressive behavior was monitored for 5 consecutive minutes using the ZebraLab tracking software module.

Open field test: Zebrafish activity was monitored continuously for 30 min, using video tracking software (to record and analyze the time spent in the central area (30% of the total area).

6. SA-β-gal staining. Senescence β-galactosidase staining kit (Beyotime) was used. Follow the manufacturer's instructions for analysis. Images were taken with a Zeiss A2 microscope.

7. Mouse behavior measurement.

Morris water maze experiment: The Morris water maze experiment was carried out on blue circular pools and platforms with diameters of 120cm and 10cm respectively. The circular area is divided into 4 quadrants and a platform area. There are 4 different shaped papers hanging around the pool. The experiment took a total of 6 days. For the first 5 days, fix the platform in a fixed position, then place the mice in the pool with their heads facing the wall from each quadrant. The incubation period and distance of the mice entering the platform were recorded by the software of Shanghai Xinruan Information Technology Co., Ltd., as the judgment of the learning ability of the mice. On day 6, the procedure was repeated after removal of the platform. Record the frequency with which the mouse crosses the platform area, the time in the correct quadrant, and the average distance from the platform location as a judgment of its memory.

Light/dark transition experiments: We used an apparatus consisting of a light-emitting, open-topped, opaque plexiglass box (25 × 25 × 30 cm) and a black, top-closed, opaque plexiglass box (20× 20×30 cm) connected together. Animals were placed in a light box and moved freely from light to dark for 5 min through a connecting door (12 x 5 cm). The light box is illuminated by a desk lamp. A mouse was considered to be in the light box if all four paws were in the light box. The number of transitions between light and dark chambers is collected.

2. Experimental results

1. Construction of zebrafish model of abnormal mental behavior (Figure 1, Figure 2).

Figure 1: Schematic showing the mutation of the zebrafish ppp2r2c gene using the CRISPR-Cas9 system. Yellow and red boxes show target exons and domains, respectively. The red dashed line shows the 4-nucleotide deletion in ppp2r2c. Black triangles show the positions of PCR primers used to amplify ppp2r2c mRNA.

Figure 2: RT-qPCR analysis of primers ppp2r2c-p1, ppp2r2c-p2 and ppp2r2c-p3 at different positions of the ppp2r2c gene (including before and after the ppp2r2c mutation region shown in (Figure 1), other regulatory subtypes of PP2A RT-qPCR analysis of the base genes (ppp2r2a, ppp2r2b, ppp2r2d) and ppp2r5c in adult wild-type and homozygous ppp2r2c mutant fish brains showed that the mRNA expression level of ppp2r2c was specifically decreased in this model, and the model construction Success (n=3 independent biological samples; unpaired two-sided t-test).

2. MPH can reverse the abnormal mental behavior exhibited by the ppp2r2c ^m/m model (Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7).

Figure 3: Experimental design. Behavioral testing of WT and ppp2r2c ^m/m (6 month old) fish with or without MPH treatment for 3 days.

Figure 4: Experimental scheme of photostimulation test and parameters for recording and analysis in photostimulation test. a, the experimental scheme of the photostimulation test; b, the representative figure of adult fish locomotor activity during the 30-second light period; c, the quantification of the burst state duration during the 30-second light period (n=9 for each group).

Figure 5: a, Representative locomotion trajectories (gray lines) in microscopy tests of WT and ppp2r2c ^m/m (6 months old) with and without MPH treatment for 3 days during the 5 min test period. b Quantification of the number of mirror attacks in 5 min intervals (WT-vehicle, WT-MPH and ppp2r2c ^m/m -vehicle, n = 9; ppp2r2c ^m/m -MPH, n = 8).

Figure 6: a. Representative locomotion trajectories (gray lines) of WT and ppp2r2c ^m/m (6 months old) with or without MPH treatment for 3 days during a 30-min trial of the open-field test, where the square indicates the central area . b is the cumulative time of adults staying in the central area (ppp2r2c ^m/m -vehicle, n=7; WT-vehicle and WT-MPH, n=8; ppp2r2 ^cm/m -MPH, n=9). Data are mean ± SEM, *P<0.05, **P<0.01, ns, not significant; two-way ANOVA.

Figure 7: Photostimulation, mirror image challenge, and open field tests with different psychotherapeutic drugs.

3. PP2A agonist improves ppp2r2c ^m/m behavior disorder, restores PP2A activity, eliminates DNA damage (DDR) and aging nerve cells (Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13).

Figure 8: 3-day PP2A phosphatase assay in adult ppp2r2c ^m/m zebrafish brains (n = 3 independent biological samples, each containing two brains).

Figure 9: a is a representative graph of locomotor activity of WT and ppp2r2c ^m/m (6 months old) treated with or without DT-061, FTY720 and MPH+DT-061 during 30 seconds of light. b Quantification of burst state duration during 30 s illumination (WT, n = 9; WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n = 6; WT-FTY- 720 and ppp2r2c ^m/m -FTY-720, n=10; WT-MPH+DT-061 and ppp2r2c ^m/m- MPH+DT-061, n=11).

Figure 10: a is representative of the mirror challenge test of WT and ppp2r2c ^m/m (6 months old) with or without DT-061, FTY720 and MPH+DT-061 treatment for 3 days during the 5 min test period Sexual movement trajectory (grey line). b Quantification of the number of mirror attacks in 5 min intervals (WT-vehicle, n = 9; WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n = 6; WT-FTY- 720 and ppp2r2c ^m/m -FTY-720, n=10; ppp2r2c ^m/m- MPH+DT-061, n=11; WT-MPH+DT-061, n=12).

Figure 11: a is the representative movement of WT and ppp2r2c ^m/m (6 months old) with or without DT-061, FTY720 and MPH+DT-061 treatment for 3 days during the 30-minute open-field test trajectory (gray line). The box shows the central area. b is the cumulative time of adults staying in the central area (WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m- DT-061, n=9; WT-vehicle, n=10; WT-FTY -720 and ppp2r2c ^m/m -FTY-720, n=11, WT-MPH+DT-061 and ppp2r2c ^m/m- MPH+DT-061, n=6). c is the average speed of adult fish (WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n=9; WT-vehicle, n=10; WT-FTY-720 and ppp2r2c ^{m/m m} -FTY-720, n=11, WT-MPH+DT-061 and ppp2r2c ^m/m -MPH+DT-061, n=6). d is the total swimming distance of adult fish (WT-DT-061, ppp2r2c ^m/m -vehicle and ppp2r2c ^m/m -DT-061, n=9; WT-vehicle, n=10; WT-FTY-720 and ppp2r2c ^m/m -FTY-720, n=11, WT-MPH+DT-061 and ppp2r2c ^m/m -MPH+DT-061, n=6).

Figure 12: Immunofluorescence detection of NeuN (green) and γH2AX (red) in OT of WT and ppp2r2c ^m/m (6 months old) treated with or without DT-061, FTY720 and MPH+DT-061 for 3 days (Scale bar, 5 μm). Quantification of the percentage of γH2AX positivity in neurons (NeuN+) and non-neurons (NeuN) (positive values indicate the number of nuclei/total number of nuclei with at least 5 γH2AX foci in the nucleus, n=6/group; * indicates the statistical difference of the NeuN+ group , # indicates the statistical difference of the NeuN-group).

Figure 13: SA-β-gal and NeuN (green) immunofluorescence in OT of WT and ppp2r2c ^m/m (6 months old) treated with or without DT-061, FTY720 and MPH+DT-061 for 3 days Representative images of staining (scale bar, 15 μm). Arrows point to SA-β-gal+ neurons. Determination of neuronal SA-β-gal positive neuronal (NeuN+) and non-neuronal (NeuN) percentages (n=6/group; * indicates statistical difference of NeuN+ group, # indicates statistical difference of NeuN- group). Data are mean±SD, *P<0.05, **P<0.01, #P<0.05, ##P<0.01, two-way analysis of variance.

4. MPH improved the behavior disorder of aged fish, restored PP2A activity, and eliminated DDR and senescent nerve cells (Fig. 14, Fig. 15, Fig. 16, Fig. 17, Fig. 18, Fig. 19, Fig. 20).

Figure 14: Experimental design for behavioral testing of 6-month-old (adult) and 22-month-old fish (old fish), with or without MPH or DT-061 treatment for 3 days.

Figure 15: Recording and analysis parameters during photostimulation test. a is a representative graph of locomotor activity during the 30-s turn-on period. b Quantification of burst state duration during a 30-s light-on period (6m, n = 8; 22m, n = 15; 22m MPH, n = 11; 22m-DT-061, n = 9; single factor variance analysis).

Figure 16: a is a representative locomotion trajectory (gray line) of an adult fish during a mirror attack test during a 5-minute trial. b Quantification of the number of mirror attacks in 5-min intervals (6m, n = 14; 22m, n = 15; 22m-DT-061, n = 12; one-way ANOVA).

Figure 17: a is a representative locomotion trace (gray line) during a 30 min open field test. The box shows the central area. b is the cumulative residence time of adult fish in the central area (3m, n=18, 22m, n=11; 22m MPH, n=10; 22m-DT-061, n=9; one-way analysis of variance).

Figure 18: a Representative confocal images of Neun (green) and γH2AX (red) co-staining in brains of 22-month-old fish treated with or without MPH or DT-061 for 3 days (scale bar, 5 μm). b, quantification of γH2AX-positive nuclei (positive values indicate at least 5 γH2AX foci) (WT-vehicle, n=4; WT-MPH, n=7; WT-DT-061, n=3; unpaired bilateral t test).

Figure 19: a is a representative image of SA-β-gal staining of 22-month-old WT fish treated with or without MPH or DT-061 for 3 days (scale bar, 15 μm). b is the quantification of the percentage of SA-β-gal positive cells (WT-vehicle and WT-MPH, n=6; WT-DT-061, n=4; one-way ANOVA).

Figure 20: PP2A phosphatase assay after treatment of aged fish with MPH or DT-061 (n = 3 independent biological samples, each group containing two brains; one-way ANOVA). The data are expressed as mean ± standard deviation, *P<0.05, **P<0.01.

5. MPH can improve the behavior disorder of aged mice, restore PP2A activity, clear DDR and aging nerve cells (Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28).

Figure 21: Behavioral experimental design of 3-month-old and 14-month-old mice treated with MPH.

Figure 22: Number of transitions between light and dark chambers in light-dark switching experiments (n=5, 3m; n=8, 14m; n=8, 14m-MPH; one-way ANOVA).

Figure 23: Maximum latency in the Morris water maze test.

Figure 24: Frequency of platform crossing in the mouse Morris water maze experiment.

Figure 25: Time spent in the correct quadrant in the Morris water maze test (3m, n=4; 14m, n=4; 14m-MPH, n=5; one-way ANOVA).

Figure 26: a is the co-staining of Neun (green) and γH2AX (red) in the frontal and temporal lobes of 14-month-old mice compared with 3-month-old mice after treatment with MPH or vehicle Representative confocal images (scale bar, 25 μm). b Quantification of 3m, 14m and 14m-MPH frontal or temporal γH2AX-positive nuclei (3m, n = 6; 14m, n = 3; 14m-MPH, n = 3; unpaired two-sided t-test).

Figure 27: a is a representative image of SA-β-gal staining in frontal and temporal lobes of 14-month-old WT mice treated with or without MPH compared with 3-month-old mice (scale bar, 25 μm) . b is the quantification of the percentage of cells positive for SA-β-gal staining (n=3/group; unpaired two-sided t-test).

Figure 28: PP2A phosphatase assays in frontal and temporal lobes of mouse brain (3m, n=5; 14m, n=8; 14m-MPH, n=8; unpaired two-sided t-test). The data are expressed as mean ± standard deviation, *P<0.05, **P<0.01.

6. ABT263 and other pathways inhibiting cellular senescence can improve ppp2r2c ^m/m mental and behavioral disorders (Figure 29, Figure 30, Figure 31).

Figure 29, Figure 30 and Figure 31 show WT and ppp2r2c ^m/m treated with or without ABT263 for 3 days, ppp2r2c ^m/m p53 ^-/- (ppp2r2c ^m/m hybridized with p53 ^-/- ) and ppp2r2c ^m/ Behavioral test results of ^m p21 ^-/- (ppp2r2c ^m/m crossed with p21 ^-/- ) at six months of age.

Figure 29: a is a representative result of locomotor activity during the 30-second light period of the photostimulation test. b, quantification of burst movement states during 30 s of light exposure (ppp2r2c ^m/m p53 ^-/- , n = 11; WT, ppp2r2c ^m/m and ppp2r2c ^m/m p21 ^-/- , n = 10; WT- ABT263, n=9; ppp2r2c ^m/m -ABT263, n=8; one-way ANOVA).

Figure 30: a is WT and ppp2r2c ^m/m treated with or without ABT263 for 3 days in the mirror attack experiment, and the representative trajectory of ppp2r2c ^m/m p53 ^-/- and ppp2r2c ^m/m p21 ^-/- (grey Wire). b is the quantification of the number of mirror attack times in 5-min intervals (ppp2r2c ^m/m p53 ^-/- , n = 11; WT, ppp2r2c ^m/m and ppp2r2c ^m/m p21 ^-/- groups, n = 10; WT-ABT263 and ppp2r2c ^m/m -ABT263, n = 9; one-way ANOVA).

Figure 31: a is the representative trajectory of ppp2r2c ^{m/m p53 -/- and ppp2r2c m/m p21 -/- in} WT and ppp2r2c ^m/m treated with or without ABT263 for 3 days in a 30-minute open field test (gray line). The box shows the central area. b Quantification of cumulative time spent staying in the central region (WT, ppp2r2c ^m/m and ppp2r2c ^m/m p21 ^-/- groups, n = 10; WT-ABT263, ppp2r2c ^m/m -ABT263 and ppp2r2c ^m/m p53 ^-/- , n=9; one-way ANOVA).

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

Claims (20)

1. Application of PP2A agonist in preparing medicine for preventing and treating mental disorders caused by normal aging.
2. The application according to claim 1, wherein the mental disorder is selected from cognitive impairment, emotional disorder and/or behavioral disorder, and the phenotype of the mental disorder is that the ability to respond to the outside world becomes weak, and the ability to explore new things Weakness, irritability, anxiety, depression, decreased social skills, decreased cognitive function, decreased ability to learn, or decreased memory.
3. The use according to claim 1, characterized in that the PP2A agonist is MPH, DT-061 or FTY720.
4. Application of PP2A agonist in preparation of medicine for preventing and treating mental disorders caused by PP2A activity and/or expression reduction.
5. The application according to claim 4, characterized in that the mental disorder caused by the reduction of PP2A activity and/or expression is ADHD, bipolar disorder, schizophrenia or depression and anxiety.
6. The use according to claim 4, characterized in that the mental disorder is selected from cognitive disorders, affective disorders and/or behavioral disorders.
7. The application according to claim 6, wherein the phenotype of the mental disorder is weakened ability to respond to the outside world, weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, cognitive Decreased functioning, decreased ability to learn, or decreased memory.
8. The use according to claim 4, characterized in that the PP2A agonist is MPH, DT-061 or FTY720.
9. Application of PP2A agonist in preparation of experimental reagents for reducing DNA damage, removing senescent cells, dissolving senescent cells or inhibiting cell senescence.
10. The PP2A activity and/or expression amount is used as a biomarker to prepare a reagent or kit for diagnosing age-related mental disorders.
11. Application of compounds for removing senescent cells and/or dissolving senescent cells and/or inhibiting cell senescence in the preparation of drugs for preventing and treating mental disorders.
12. The application according to claim 11, wherein the mental disorder is a mental disorder mediated by accumulation of DNA damage and/or increased aging of nerve cells, and the accumulation of DNA damage and/or increased aging of nerve cells is caused by normal age Increased, or reduced PP2A activity and/or expression.
13. The use according to claim 11, characterized in that the mental disorder is selected from cognitive disorders, affective disorders and/or behavioral disorders.
14. The application according to claim 13, wherein the phenotype of the mental disorder is weakened ability to respond to the outside world, weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, cognitive Decreased functioning, decreased ability to learn, or decreased memory.
15. The application according to claim 11, characterized in that, the compound for removing senescent cells and/or dissolving senescent cells and/or inhibiting cell senescence is a small molecule compound or a biomacromolecule.
16. The application according to claim 15, characterized in that, the compound for removing senescent cells and/or lysing senescent cells and/or inhibiting cell senescence is selected from ABT263, a compound that causes PP2A activity and/or expression to increase, and causes p53 Compounds that decrease activity and/or expression, and compounds that cause a decrease in p21 activity and/or expression.
17. A method for preventing and treating mental disorders in a subject in need, characterized by comprising the step of administering to the subject a compound for eliminating senescent cells and/or dissolving senescent cells and/or inhibiting cell senescence.
18. The method according to claim 17, wherein the mental disorder is a mental disorder mediated by accumulation of DNA damage and/or increased aging of nerve cells, and the accumulation of DNA damage and/or increased aging of nerve cells are caused by normal age Increased, or reduced PP2A activity and/or expression.
19. The method according to claim 17, characterized in that the mental disorder is selected from cognitive disorders, affective disorders and/or behavioral disorders.
20. The method according to claim 17, characterized in that, the compound for removing senescent cells and/or lysing senescent cells and/or inhibiting cell senescence is selected from ABT263, compounds that cause PP2A activity and/or increased expression, and cause p53 Compounds that decrease activity and/or expression, and compounds that cause a decrease in p21 activity and/or expression.

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