Compounds for clearing and / or dissolving senescent cells and / or inhibiting cell senescence for treating mental disorders

Abstract

The present invention relates to compounds that can remove and / or dissolve senescent cells and / or inhibit cell senescence for use in treating mental disorders. m / m The mutant zebrafish all had some degree of mental disorders, and all had severe DNA damage and cellular senescence. m / m The symptoms of mental disorders in mutant zebrafish were significantly improved, and PPP2R2C was given m / m Symptoms of mental disorders were also significantly improved in zebrafish infected with the mutant ABT263, which has the ability to clear senescent cells. These results strongly suggest that clearing and / or dissolving senescent cells and / or inhibiting cellular senescence are effective treatments for mental disorders mediated by accumulated DNA damage and increased neuronal senescence. Compounds that clear and / or dissolve senescent cells and / or inhibit cellular senescence can be used to develop drugs for the prevention and treatment of mental disorders.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to compounds that can clear and / or dissolve senescent cells and / or inhibit cell senescence for use in treating mental disorders. Background Art

[0002] Mental disorder is a syndrome characterized by disturbances in individual cognition, emotion, or volitional behavior. Based on 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 factors such as central nervous system diseases, metabolic disorders, alcohol and substance abuse, poisoning, brain damage, encephalitis, etc. Patients often show symptoms such as impaired consciousness, perceptual disorders, emotional disorders, memory disorders, personality and behavioral changes, such as delirium, amnesia syndrome and dementia. Functional mental disorders are caused by the long-term interaction of biological, psychological and social environmental factors, resulting in hallucinations, delusions, emotional disorders and other symptoms, such as anxiety disorders and schizophrenia.

[0003] Protein phosphatase 2A (PP2A) is a serine / threonine phosphatase with a wide range of substrates, most of which are involved in transcription factors and protein kinases. It participates in numerous cellular biological functions, playing important roles in energy metabolism, DNA damage and repair, protein translation, cell cycle regulation, and signal transduction. Previous studies have shown that PP2A has tumor suppressor effects and is involved in regulating glucose and lipid metabolism. Abnormal PP2A function can lead to tumorigenesis, diabetes, obesity, and other conditions. Coronary atherosclerosis is also associated with abnormal PP2A mutations. It also participates in secondary pathological processes such as retinopathy and cardiomyopathy.

[0004] 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 have the ability to eliminate senescent cells.

[0005] Currently, there are no studies on the use of compounds that clear and / or dissolve senescent cells and / or inhibit cell senescence to treat mental disorders mediated by accumulated DNA damage and increased neuronal senescence by clearing and / or dissolving senescent cells and / or inhibiting cell senescence. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a compound for removing senescent cells and / or dissolving senescent cells and / or inhibiting cell senescence for use in preventing and treating mental disorders.

[0007] In a first aspect, the present invention provides the use of a compound that eliminates senescent cells and / or dissolves senescent cells and / or inhibits cell senescence in the preparation of a drug for preventing or treating mental disorders.

[0008] As a preferred example, the mental disorder is a mental disorder caused by accumulation of DNA damage and / or increase in senescent neurons.

[0009] More preferably, the accumulated DNA damage and / or increased neural cell senescence is caused by normal aging, or by decreased PP2A activity and / or expression.

[0010] As another preferred example, the mental disorder is selected from cognitive disorder, affective disorder and / or behavioral disorder.

[0011] More preferably, the phenotype of the mental disorder is a weakened ability to respond to the outside world, a weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, decreased cognitive function, decreased learning ability or decreased memory ability.

[0012] As another preferred example, the compound for eliminating senescent cells and / or dissolving senescent cells and / or inhibiting cell senescence is a small molecule compound or a biomacromolecule.

[0013] As another preferred example, the compound that eliminates senescent cells and / or dissolves senescent cells and / or inhibits cell senescence is selected from ABT263, a compound that causes a decrease in PP2A activity and / or expression, a compound that causes a decrease in p53 activity and / or expression, and a compound that causes a decrease in p21 activity and / or expression.

[0014] In a second aspect, the present invention provides a method for preventing and treating a mental disorder in a subject in need thereof, comprising the step of administering to the subject a compound that eliminates senescent cells and / or dissolves senescent cells and / or inhibits cell senescence.

[0015] As a preferred example, the mental disorder is a mental disorder mediated by accumulation of DNA damage and / or increased neuronal senescence.

[0016] More preferably, the accumulated DNA damage and / or increased neural cell senescence is caused by normal aging, or by decreased PP2A activity and / or expression.

[0017] As another preferred example, the mental disorder is selected from cognitive disorder, affective disorder and / or behavioral disorder.

[0018] More preferably, the phenotype of the mental disorder is a weakened ability to respond to the outside world, a weakened ability to explore new things, irritability, anxiety, depression, decreased social skills, decreased cognitive function, decreased learning ability or decreased memory ability.

[0019] As another preferred example, the compound for eliminating senescent cells and / or dissolving senescent cells and / or inhibiting cell senescence is a small molecule compound or a biomacromolecule.

[0020] As another preferred example, the compound that eliminates senescent cells and / or dissolves senescent cells and / or inhibits cell senescence is selected from ABT263, a compound that causes a decrease in PP2A activity and / or expression, a compound that causes a decrease in p53 activity and / or expression, and a compound that causes a decrease in p21 activity and / or expression.

[0021] Herein, "mental disorder" is a general term for varying degrees of impairment in mental activities, including cognition, emotion, behavior, and volition. It is known in the art that "cognition" encompasses three processes: sensation and perception, memory and attention, and thought processes. "Sensation and perception" refers to the use of the eyes, ears, nose, tongue, and skin to understand the world and understand the surrounding environment. This process is the brain's perception of the existence of objective external objects, that is, the reflection of external objects in the brain. "Memory" in "memory and attention" refers to the retention of previously perceived objects in the brain, and "attention" refers to the direction of a person's mental activity toward a specific object. "Thought processes" refer to the process by which the brain analyzes, synthesizes, and judges the information reflected by sensory input, drawing conclusions based on past experience. "Emotion" refers to a person's attitude and outward expression toward any object. "Behavior and volition" refers to the psychological process by which a person takes action to achieve a specific goal. Under normal circumstances, the three aspects of mental activity—cognition, emotion, and behavior and volition—are coordinated and synchronized, and are consistent with the external environment. When there is a mental disorder, it manifests as an incoordination between "cognition", "emotion" and "behavior and will", or the inconsistency between "cognition", "emotion" or "behavior and will" and the external environment.

[0022] Here, the phrase "mental disorders caused by the accumulation of DNA damage and / or increased neuronal senescence due to normal aging" refers to mental symptoms that affect healthy aging. The WHO believes that aging, at a biological level, is the gradual accumulation of various molecular and cellular damage, leading to a gradual decrease in physiological reserves and a general decline in various abilities, resulting in an increased risk of many diseases. However, these changes are neither linear nor uniform across the population; they are intertwined with age. For example, while some individuals over 70 may enjoy good health and a good mental state, the vast majority experience progressive frailty and require various supportive measures to meet their basic needs. The WHO defines mental state changes (or disorders) as including: changes in coping ability; behavioral changes; memory loss; aggression; depression, anxiety, and apathy; sleep difficulties; delusions and hallucinations; repetitive behaviors; wandering and getting lost; and changes in judgment. In the elderly, the presence of one or more of these symptoms indicates a decline in mental or psychological well-being. However, these mental state changes (or disorders) are distinct from Alzheimer's disease (AD), which is caused by β-amyloid (Aβ) deposition and tau tangles.

[0023] Herein, the "PP2A agonist" refers to a compound that directly or indirectly acts on PP2A and activates it, thereby producing a physiological response.

[0024] In this article, MPH is known as methylphenidate. DT-061 is a known PP2A agonist, also abbreviated as SMAP. FTY720 is a known PP2A agonist, also known as fingolimod.

[0025] The advantages of the present invention are:

[0026] The present invention found that old zebrafish and mice and PPP2R2C m / m The mutant zebrafish all had some degree of mental disorders and severe DNA damage and cellular senescence. Using MPH or other PP2A agonists could reverse the aging of zebrafish and mice and PPP2R2C. m / m The mutant zebrafish showed reduced symptoms of mental disorders and eliminated DNA damage and senescent neurons. m / m PPP2R2C mutant zebrafish with knockout of the aging-related gene p21 m / m Significant improvement in the symptoms of mental disorders in mutant zebrafish was observed, and administration of PPP2R2C m / mIn zebrafish, the ABT263 mutant, which has the ability to eliminate senescent cells, also showed significant improvement in symptoms of mental disorders. These results clearly demonstrate that eliminating and / or dissolving senescent cells and / or inhibiting cellular senescence are effective approaches for treating mental disorders, and compounds that eliminate and / or dissolve senescent cells and / or inhibit cellular senescence can be used to prepare drugs for the prevention and treatment of mental disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram showing the mutation of the zebrafish ppp2r2c gene using the CRISPR-Cas9 system. Yellow and red boxes indicate the target exon and domain, respectively. The red dashed line indicates the 4-nucleotide deletion in ppp2r2c. The black triangles indicate the locations of the PCR primers used to amplify the ppp2r2c mRNA.

[0028] Figure 2 : Primers ppp2r2c-p1, ppp2r2c-p2 and ppp2r2c-p3 (including ( Figure 1 ), RT-qPCR analysis of the ppp2r2c mutation region shown in ), RT-qPCR analysis of other regulatory subunit genes of PP2A (ppp2r2a, ppp2r2b, ppp2r2d), and RT-qPCR analysis of ppp2r5c in the brains of adult wild-type and homozygous ppp2r2c mutant fish indicated that the mRNA expression level of ppp2r2c in this model was specifically decreased, and the model was successfully constructed (n = 3 independent biological samples; unpaired two-sided t-test).

[0029] Figure 3 : Experimental design. m / m Fish (6 months old) were behaviorally tested with or without MPH treatment for 3 days.

[0030] Figure 4 Experimental protocol for the light stimulation test and parameters for recording and analysis during the light stimulation test. a) Experimental protocol for the light stimulation test; b) Representative images of adult fish locomotor activity during a 30-second light exposure; c) Quantification of burst duration during a 30-second light exposure (n = 9 per group).

[0031] Figure 5 : a WT and ppp2r2c with or without MPH treatment for 3 days during a 5-min test m / m Representative movement trajectories (grey lines) in the mirror attack test (6 months old). b Quantification of the number of mirror attacks within a 5-min interval (WT-vehicle, WT-MPH and ppp2r2c m / m -vehicle, n=9; ppp2r2c m / m-MPH, n=8).

[0032] Figure 6 a: WT and ppp2r2c cells treated with or without MPH for 3 days during a 30-min test in the open field test. m / m Representative movement trajectory of a 6-month-old fish (grey line), where the square represents the central area. b is the cumulative time the adult fish stayed 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.

[0033] Figure 7 : Photostimulation, mirror attack, and open field tests with different psychotropic drugs.

[0034] Figure 8 :For adults ppp2r2c m / m Zebrafish brains were subjected to a 3-day PP2A phosphatase assay (n=3 independent biological samples, each containing two brains).

[0035] Figure 9 : a is WT and ppp2r2c treated with or without DT-061, FTY720 and MPH+DT-061 m / m Representative graph of locomotor activity during a 30-second light period (6 months old). b Quantification of the duration of bursts during a 30-second light period (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).

[0036] Figure 10 : a is the 5-min test period, with or without DT-061, FTY720 and MPH+DT-061 treatment for 3 days, for WT and ppp2r2c m / m Representative movement trajectory (grey line) of the mirror attack test (6 months old). b is the quantification of the number of mirror attacks within a 5-min interval (WT-vehicle, n = 9; WT-DT-061, ppp2r2c m / m -vehicle and ppp2r2cm / 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).

[0037] Figure 11 : a is the open field test for 30 minutes. During the test, WT and ppp2r2c cells were treated with or without DT-061, FTY720 and MPH+DT-061 for 3 days. m / m Representative movement trajectory of 6-month-old fish (grey line). The box shows the central area. b is the cumulative time that adult fish stayed 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 -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).

[0038] Figure 12 Immunofluorescence analysis of WT and ppp2r2c cells in cells treated with or without DT-061, FTY720, and MPH+DT-061 for 3 days m / mNeuN (green) and γH2AX (red) in OT of 6-month-old rats (scale bar, 5 μm). Quantification of the percentage of γH2AX positive cells in neurons (NeuN+) and non-neurons (NeuN) (positive value represents the number of nuclei with at least 5 γH2AX foci in the nucleus / total number of nuclei, n=6 / group; * indicates statistical difference in NeuN+ group, # indicates statistical difference in NeuN- group).

[0039] Figure 13 :WT and ppp2r2c treated or not with DT-061, FTY720 and MPH+DT-061 for 3 days m / m Representative images of immunofluorescence co-staining of SA-β-gal and NeuN (green) in OT (6-month-old) (scale bar, 15 μm). Arrows point to SA-β-gal+ neurons. Determination of the percentage of neuronal SA-β-gal-positive neurons (NeuN+) and non-neurons (NeuN) (n=6 / group; * indicates statistical difference in NeuN+ group, # indicates statistical difference in NeuN- group). Data are mean ± SD, *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01, two-way ANOVA.

[0040] Figure 14 : Experimental design for behavioral testing of 6-month-old (adult) and 22-month-old (senile) fish, treated with or without MPH or DT-061 for 3 days.

[0041] Figure 15 Recording and analysis parameters during the light stimulation test. a: Representative images of locomotor activity during the 30-second light-on period. b: Quantification of burst duration during the 30-second light-on period (6 m, n = 8; 22 m, n = 15; 22 m MPH, n = 11; 22 m-DT-061, n = 9; one-way ANOVA).

[0042] Figure 16 a: Representative movement trajectories (gray lines) of adult fish during the 5-min test. b: Quantification of the number of mirror attacks within a 5-min interval (6m, n = 14; 22m, n = 15; 22m-DT-061, n = 12; one-way ANOVA).

[0043] Figure 17 a: Representative movement trajectory (gray line) during a 30-min open-field test. The box indicates the central zone. b: Cumulative time spent in the central zone by adult fish (3 m, n = 18, 22 m, n = 11; 22 m MPH, n = 10; 22 m-DT-061, n = 9; one-way ANOVA).

[0044] Figure 18: a is a representative confocal image of Neun (green) and γH2AX (red) co-staining in the brains of 22-month-old fish treated with or without MPH or DT-061 for 3 days (scale bar, 5 μm). b is a 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 two-sided t-test).

[0045] Figure 19 : a Representative images 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 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).

[0046] Figure 20 : PP2A phosphatase assay in aged fish treated with MPH or DT-061 (n=3 independent biological samples, two brains per group; one-way ANOVA). Data are expressed as mean ± SD, *P < 0.05, **P < 0.01.

[0047] Figure 21 : Design of behavioral experiments on 3-month-old and 14-month-old mice after MPH treatment.

[0048] Figure 22 : Number of transitions between the light and dark chambers in the light-dark transition test (n=5, 3m; n=8, 14m; n=8, 14m-MPH; one-way ANOVA).

[0049] Figure 23 : The longest latency in the Morris water maze test.

[0050] Figure 24 : The frequency of crossing the platform in the Morris water maze test in mice.

[0051] Figure 25 : Time spent in the correct quadrant in the Morris water maze test (3 m, n=4; 14 m, n=4; 14 m-MPH, n=5; one-way ANOVA).

[0052] Figure 26: a Representative confocal images of NeuN (green) and γH2AX (red) co-staining in the frontal and temporal lobes of 14-month-old mice treated with MPH or vehicle compared with 3-month-old mice (scale bar, 25 μm). b Quantification of γH2AX-positive nuclei in the frontal or temporal lobes of 3m, 14m, and 14m-MPH (3m, n=6; 14m, n=3; 14m-MPH, n=3; unpaired two-sided t-test).

[0053] Figure 27 a: Representative images of SA-β-gal staining in the 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: Quantification of the percentage of SA-β-gal-positive cells (n=3 / group; unpaired two-sided t-test).

[0054] Figure 28 : PP2A phosphatase assay in the frontal and temporal lobes of mouse brains (3m, n=5; 14m, n=8; 14m-MPH, n=8; unpaired two-sided t-test). Data are expressed as mean ± standard deviation, *P<0.05, **P<0.01.

[0055] Figure 29 : a is a representative result of locomotor activity during the 30-second light period of the light stimulation test. b is a quantification of the burst movement state during the 30-second light period (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).

[0056] Figure 30 :a is WT and ppp2r2c in the mirror attack experiment m / m Treatment with or without ABT263 for 3 days, and PPP2R2C m / m p53 - / - and ppp2r2c m / m p21 - / - Representative movement trajectory (grey line). b is the quantification of the number of mirror attacks within a 5-minute interval (ppp2r2c m / m p53 - / - , n = 11; WT, ppp2r2c m / m and ppp2r2c m / m p21 - / -Group, n = 10; WT-ABT263 and ppp2r2c m / m -ABT263, n = 9; one-way ANOVA).

[0057] Figure 31 : a is the 30-min open field test between WT and ppp2r2c m / m Treatment with or without ABT263 for 3 days, PPP2R2C m / m p53 - / - and ppp2r2c m / m p21 - / - Representative movement trajectories (gray lines). The box shows the central region. b is the quantification of the cumulative time spent in the central region (WT, ppp2r2c m / m and ppp2r2c m / m p21 - / - Group, n = 10; WT-ABT263, ppp2r2c m / m -ABT263 and ppp2r2c m / m p53 - / - , n = 9; one-way ANOVA). DETAILED DESCRIPTION

[0058] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0059] Example 1

[0060] 1. Experimental Methods

[0061] 1. All drug solutions were freshly prepared using PBS (MPH 1.08 mg / kg, sodium valproate 300 mg / kg, aripiprazole 0.6 mg / kg, ziprasidone 1.6 mg / kg, DT061 and FTY720 5 mg / kg). Zebrafish were given food soaked in drug solution for 3 days, and behavioral analysis was performed on the 4th day. 14- and 3-month-old C57B6 / J mice were produced and raised in the animal facility of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. This protocol was approved by the Animal Experiment Ethics Committee. 14- and 3-month-old C57B6 / J mice were gavage-administered MPH (12.3 mg / kg / d) for 14 days.

[0062] 2. Generation of CRISPR-Cas9 mutant zebrafish. ppp2r2c mutant zebrafish were generated using the CRISPR-Cas9 system. Guide RNAs targeting ppp2r2c exon 9 (sgRNA: 5′-GGGCAGAGATACC-3′ (SEQ ID NO: 1)) and cdkn1a / p21 exon 2 (sgRNA: 5′-GGTAATGGGCCGACTAGG-3′ (SEQ ID NO: 2)) were designed using ZiFiT Targeter software (http: / / zifit.partners.org / ZiFiT). F0 offspring were raised to 3 months of age and outcrossed with wild-type zebrafish to generate potential F1 indel mutations. Genomic DNA isolated from F1 zebrafish tails was PCR amplified and sequenced 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 used for genotyping: fwd 5′-TCTGTGATTGTGTG-3′ (SEQ ID NO: 5); rev 5′-GAGTGCACATCGTTC-3′ (SEQ ID NO: 6)).

[0063] 3. Immunofluorescence imaging of brain sections. Zebrafish tissues were isolated at 4°C and fixed with 4% PFA overnight, then dehydrated with 30% sucrose overnight. The tissues were embedded in OCT and sectioned at 5 minutes. Frozen sections 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 the primary antibody was performed overnight at 4°C, followed by three washes with 0.1% Tween-20 in PBS. Hybridization with the corresponding secondary antibody (Invitrogen) was performed at 37°C for at least 2 hours. Finally, the sections were incubated with 1× DAPI for 5 minutes at room temperature. Photos were taken using a confocal laser scanning microscope (SP8; Leica).

[0064] 4. PP2A phosphatase assay. Homogenates prepared from zebrafish brain, mouse frontal lobe, and temporal lobe were placed in 20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0, 10 μg / mL each of aprotinin, leupeptin, and pepstatin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride, and dissolved by adding 1% NP-40. The assay was then performed using a PP2A immunoprecipitation phosphatase assay kit (Millipore) according to the manufacturer's instructions. The absorbance at a wavelength of 650 nm (A650) was measured in a microtiter plate reader (BioTAK).

[0065] 5. Zebrafish behavior analysis.

[0066] An adult fish was placed in a standard mating tank (21 × 10 × 7.5 cm) containing system water at a depth of 6 cm and allowed to acclimate for 15 min before being transferred to an automated observation and video tracking system (ZebraLab; Viewpoint Life Sciences).

[0067] Light stimulation test: An adult fish was placed in darkness and allowed to adapt for 15 minutes. The lights were then turned on for 30 seconds, and activity was recorded using the ZebraLab quantification software module. The quantitative test focuses on the zebrafish's activity, which represents the total amount of movement the zebrafish made in the tank and its frequency. The software can automatically record the zebrafish's position and compare the previous position with the new position. Changed pixels are considered activity. We used wild-type fish to obtain two thresholds (a burst threshold of 100 and a freeze value of 20). The burst threshold indicates that 95% of the pixels moving changed below this value. The freeze value indicates that when the fish stopped moving, the pixel change was below this value. If the value of the movement surface was above the burst threshold, the activity was recorded as burst activity, and the software automatically recorded the duration of the activity.

[0068] Mirror aggression 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.

[0069] Open field test: Zebrafish activity was monitored continuously for 30 min, and the time spent in the central area (30% of the total area) was recorded and analyzed using video tracking software.

[0070] 6. SA-β-gal staining was performed using the Senescence β-galactosidase Staining Kit (Beyotime). Analysis was performed according to the manufacturer's instructions. Images were captured using a Zeiss A2 microscope.

[0071] 7. Mouse behavior measurement.

[0072] Morris water maze experiment: The Morris water maze experiment was conducted in a blue circular pool and platform with diameters of 120 cm and 10 cm respectively. The circular area is divided into 4 quadrants and a platform area. 4 pieces of paper of different shapes are hung around the pool. The experiment took a total of 6 days. During the first 5 days, the platform was fixed in a fixed position, and then the mice were placed in the pool with their heads facing the wall from each quadrant. The latency and distance of the mice entering the platform were recorded using the software of Shanghai Xinruan Information Technology Co., Ltd. as a judgment of the mice's learning ability. On the 6th day, the procedure was repeated after the platform was removed. The frequency with which the mice crossed the platform area, the time in the correct quadrant, and the average distance from the platform location were recorded as a judgment of their memory.

[0073] Light / dark transition test: We used an apparatus consisting of a lighted, open-top, opaque Plexiglas box (25 × 25 × 30 cm) connected to a black, closed-top, opaque Plexiglas box (20 × 20 × 30 cm). Animals were placed in the lighted box and allowed to move freely from the light to the dark compartment through a connecting door (12 × 5 cm) for 5 minutes. The light box was illuminated by a desk lamp. If all four paws were in the light box, the mouse was considered to be in the light box. The number of transitions between the light and dark compartments was recorded.

[0074] 2. Experimental Results

[0075] 1. Construction of a zebrafish model of mental and behavioral disorders ( Figure 1 、 Figure 2 ).

[0076] Figure 1 Schematic diagram showing the mutation of the zebrafish ppp2r2c gene using the CRISPR-Cas9 system. Yellow and red boxes indicate the target exon and domain, respectively. The red dashed line indicates the 4-nucleotide deletion in ppp2r2c. The black triangles indicate the locations of the PCR primers used to amplify the ppp2r2c mRNA.

[0077] Figure 2 : Primers ppp2r2c-p1, ppp2r2c-p2 and ppp2r2c-p3 (including ( Figure 1 ), RT-qPCR analysis of the ppp2r2c mutation region shown in ), RT-qPCR analysis of other regulatory subunit genes of PP2A (ppp2r2a, ppp2r2b, ppp2r2d), and RT-qPCR analysis of ppp2r5c in the brains of adult wild-type and homozygous ppp2r2c mutant fish indicated that the mRNA expression level of ppp2r2c in this model was specifically decreased, and the model was successfully constructed (n = 3 independent biological samples; unpaired two-sided t-test).

[0078] 2. MPH can reverse ppp2r2c m / m The model exhibits abnormal mental behavior ( Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 ).

[0079] Figure 3 : Experimental design. m / m Fish (6 months old) were behaviorally tested with or without MPH treatment for 3 days.

[0080] Figure 4 Experimental protocol for the light stimulation test and parameters for recording and analysis during the light stimulation test. a) Experimental protocol for the light stimulation test; b) Representative images of adult fish locomotor activity during a 30-second light exposure; c) Quantification of burst duration during a 30-second light exposure (n = 9 per group).

[0081] Figure 5 : a WT and ppp2r2c with or without MPH treatment for 3 days during a 5-min test m / m Representative movement trajectories (grey lines) in the mirror attack test (6 months old). b Quantification of the number of mirror attacks within a 5-min interval (WT-vehicle, WT-MPH and ppp2r2c m / m -vehicle, n=9; ppp2r2c m / m -MPH, n=8).

[0082] Figure 6 a: WT and ppp2r2c cells treated with or without MPH for 3 days during a 30-min test in the open field test. m / m Representative movement trajectory of a 6-month-old fish (grey line), where the square represents the central area. b is the cumulative time the adult fish stayed 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.

[0083] Figure 7 : Photostimulation, mirror attack, and open field tests with different psychotropic drugs.

[0084] 3. PP2A agonists improve ppp2r2c m / m behavioral disorders, restoring PP2A activity, eliminating DNA damage (DDR) and senescent neurons ( Figure 8 、 Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 ).

[0085] Figure 8 :For adults ppp2r2c m / m Zebrafish brains were subjected to a 3-day PP2A phosphatase assay (n=3 independent biological samples, each containing two brains).

[0086] Figure 9 : a is WT and ppp2r2c treated with or without DT-061, FTY720 and MPH+DT-061 m / m Representative graph of locomotor activity during a 30-second light period (6 months old). b Quantification of the duration of bursts during a 30-second light period (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).

[0087] Figure 10 : a is the 5-min test period, with or without DT-061, FTY720 and MPH+DT-061 treatment for 3 days, for WT and ppp2r2c m / m Representative movement trajectory (grey line) of the mirror attack test (6 months old). b is the quantification of the number of mirror attacks within 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).

[0088] Figure 11 : a is the open field test for 30 minutes. During the test, WT and ppp2r2c cells were treated with or without DT-061, FTY720 and MPH+DT-061 for 3 days. m / mRepresentative movement trajectory of 6-month-old fish (grey line). The box shows the central area. b is the cumulative time that adult fish stayed 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 -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).

[0089] Figure 12 Immunofluorescence analysis of WT and ppp2r2c cells in cells treated with or without DT-061, FTY720, and MPH+DT-061 for 3 days m / m NeuN (green) and γH2AX (red) in OT of 6-month-old rats (scale bar, 5 μm). Quantification of the percentage of γH2AX positive cells in neurons (NeuN+) and non-neurons (NeuN) (positive value represents the number of nuclei with at least 5 γH2AX foci in the nucleus / total number of nuclei, n=6 / group; * indicates statistical difference in NeuN+ group, # indicates statistical difference in NeuN- group).

[0090] Figure 13 :WT and ppp2r2c treated or not with DT-061, FTY720 and MPH+DT-061 for 3 days m / mRepresentative images of immunofluorescence co-staining of SA-β-gal and NeuN (green) in OT (6-month-old) (scale bar, 15 μm). Arrows point to SA-β-gal+ neurons. Determination of the percentage of neuronal SA-β-gal-positive neurons (NeuN+) and non-neurons (NeuN) (n=6 / group; * indicates statistical difference in NeuN+ group, # indicates statistical difference in NeuN- group). Data are mean ± SD, *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01, two-way ANOVA.

[0091] 4. MPH improved behavioral disorders in aged fish, restored PP2A activity, and eliminated DDR and senescent neurons ( Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 、 Figure 20 ).

[0092] Figure 14 : Experimental design for behavioral testing of 6-month-old (adult) and 22-month-old (senile) fish, treated with or without MPH or DT-061 for 3 days.

[0093] Figure 15 Recording and analysis parameters during the light stimulation test. a: Representative images of locomotor activity during the 30-second light-on period. b: Quantification of burst duration during the 30-second light-on period (6 m, n = 8; 22 m, n = 15; 22 m MPH, n = 11; 22 m-DT-061, n = 9; one-way ANOVA).

[0094] Figure 16 a: Representative movement trajectories (gray lines) of adult fish during the 5-min test. b: Quantification of the number of mirror attacks within a 5-min interval (6m, n = 14; 22m, n = 15; 22m-DT-061, n = 12; one-way ANOVA).

[0095] Figure 17 a: Representative movement trajectory (gray line) during a 30-min open-field test. The box indicates the central zone. b: Cumulative time spent in the central zone by adult fish (3 m, n = 18, 22 m, n = 11; 22 m MPH, n = 10; 22 m-DT-061, n = 9; one-way ANOVA).

[0096] Figure 18: a is a representative confocal image of Neun (green) and γH2AX (red) co-staining in the brains of 22-month-old fish treated with or without MPH or DT-061 for 3 days (scale bar, 5 μm). b is a 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 two-sided t-test).

[0097] Figure 19 : a Representative images 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 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).

[0098] Figure 20 : PP2A phosphatase assay in aged fish treated with MPH or DT-061 (n=3 independent biological samples, two brains per group; one-way ANOVA). Data are expressed as mean ± SD, *P < 0.05, **P < 0.01.

[0099] 5. MPH can improve behavioral disorders in aged mice, restore PP2A activity, and eliminate DDR and senescent neurons ( Figure 21 、 Figure 22 、 Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 、 Figure 27 、 Figure 28 ).

[0100] Figure 21 : Design of behavioral experiments on 3-month-old and 14-month-old mice after MPH treatment.

[0101] Figure 22 : Number of transitions between the light and dark chambers in the light-dark transition test (n=5, 3m; n=8, 14m; n=8, 14m-MPH; one-way ANOVA).

[0102] Figure 23 : The longest latency in the Morris water maze test.

[0103] Figure 24 : The frequency of crossing the platform in the Morris water maze test in mice.

[0104] Figure 25: Time spent in the correct quadrant in the Morris water maze test (3 m, n=4; 14 m, n=4; 14 m-MPH, n=5; one-way ANOVA).

[0105] Figure 26 : a Representative confocal images of NeuN (green) and γH2AX (red) co-staining in the frontal and temporal lobes of 14-month-old mice treated with MPH or vehicle compared with 3-month-old mice (scale bar, 25 μm). b Quantification of γH2AX-positive nuclei in the frontal or temporal lobes of 3m, 14m, and 14m-MPH (3m, n=6; 14m, n=3; 14m-MPH, n=3; unpaired two-sided t-test).

[0106] Figure 27 a: Representative images of SA-β-gal staining in the 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: Quantification of the percentage of SA-β-gal-positive cells (n=3 / group; unpaired two-sided t-test).

[0107] Figure 28 : PP2A phosphatase assay in the frontal and temporal lobes of mouse brains (3m, n=5; 14m, n=8; 14m-MPH, n=8; unpaired two-sided t-test). Data are expressed as mean ± standard deviation, *P<0.05, **P<0.01.

[0108] 6. ABT263 and other pathways that inhibit cellular senescence can improve PPP2R2C m / m Mental behavioral disorders ( Figure 29 、 Figure 30 、 Figure 31 ).

[0109] Figure 29 、 Figure 30 and Figure 31 WT and ppp2r2c were shown m / m Treatment with or without ABT263 for 3 days, PPP2R2C m / m p53 - / - (ppp2r2c m / m and p53 - / - hybridization) and ppp2r2c m / m p21 - / - (ppp2r2c m / m and p21 - / - Behavioral test results of six-month-old mice (obtained by crossbreeding).

[0110] Figure 29: a is a representative result of locomotor activity during the 30-second light period of the light stimulation test. b is a quantification of the burst movement state during the 30-second light period (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).

[0111] Figure 30 :a is WT and ppp2r2c in the mirror attack experiment m / m Treatment with or without ABT263 for 3 days, and PPP2R2C m / m p53 - / - and ppp2r2c m / m p21 - / - Representative movement trajectory (grey line). b is the quantification of the number of mirror attacks within a 5-minute interval (ppp2r2c m / m p53 - / - , n = 11; WT, ppp2r2c m / m and ppp2r2c m / m p21 - / - Group, n = 10; WT-ABT263 and ppp2r2c m / m -ABT263, n = 9; one-way ANOVA).

[0112] Figure 31 : a is the 30-min open field test between WT and ppp2r2c m / m Treatment with or without ABT263 for 3 days, PPP2R2C m / m p53 - / - and ppp2r2c m / m p21 - / - Representative movement trajectories (gray lines). The box shows the central region. b is the quantification of the cumulative time spent in the central region (WT, ppp2r2c m / m and ppp2r2c m / m p21 - / - Group, n = 10; WT-ABT263, ppp2r2c m / m -ABT263 and ppp2r2c m / m p53 - / - , n = 9; one-way ANOVA).

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. SEQUENCE LISTING <110> Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine <120> Compounds for clearing and / or dissolving senescent cells and / or inhibiting cell senescence for treating mental disorders <130> / <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 13 <212> DNA <213> Artificial sequence <400> 1 gggcagagat acc 13 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 ggtaatgggc cgactagg 18 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <400> 3 caggcagtgt tgaagat 17 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <400> 4 gtgctgagag gccactaa 18 <210> 5 <211> 14 <212> DNA <213> Artificial sequence <400> 5 tctgtgattg tgtg 14 <210> 6 <211> 15 <212> DNA <213> Artificial sequence <400> 6 gagtgcacat cgttc 15

Claims

1. Use of a compound for eliminating senescent cells and / or dissolving senescent cells and / or inhibiting cell senescence in the preparation of a drug for preventing or treating mental disorders, characterized in that: The mental disorder is a neurodegenerative disease different from that caused by β-amyloid protein deposition and Tau tangles; the compound for clearing senescent cells and / or dissolving senescent cells and / or inhibiting cell senescence is selected from: ABT263, FTY-720, or a combination thereof; the phenotype of the mental disorder is: irritability, depression or anxiety.

2. The use according to claim 1, characterized in that The mental disorder is a mental disorder caused by ppp2r2c gene mutation.

3. The use according to claim 1, characterized in that The drug includes a compound that eliminates senescent cells and / or dissolves senescent cells and / or inhibits cell senescence, and the compound that eliminates senescent cells and / or dissolves senescent cells and / or inhibits cell senescence is ABT263.

4. The use according to claim 1, characterized in that The phenotype of the mental disorder is depression or anxiety.

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