Application of pharmaceutical preparations containing BAY 73-6691 or its derivatives in the preparation of drugs for treating brain injury diseases

Abstract

The application belongs to the field of biological medicine, and relates to application of a pharmaceutical preparation containing BAY 73-6691 or a derivative thereof in preparation of a medicine for treating brain injury diseases. A structural formula of the BAY 73-6691 is shown in the following formula. The pharmaceutical preparation comprises BAY 73-6691 and / or a pharmaceutically acceptable salt of BAY 73-6691. It is found in the application that BAY 73-6691 can improve ARCA brain injury through a ubiquitin phosphorylation double pathway, so that BAY 73-6691 can become a candidate drug for treating ARCA; a new drug mechanism and an action target of BAY 73-6691 are further explained; and the application can provide a new supplement for clinical application of new use of old drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the use of a pharmaceutical preparation containing BAY 73-6691 or a derivative thereof in the preparation of a drug for treating brain injury diseases. Background Art

[0002] Autosomal recessive cerebellar ataxia (ARCA) is a group of clinically and genetically heterogeneous central nervous system disorders characterized by progressive adult-onset ataxia. These disorders primarily cause cerebellar degeneration and atrophy, leading to impaired balance, coordination, and gait, or may be accompanied by other clinical symptoms such as cognitive impairment and dementia. The current prevalence is approximately 4 per 100,000 individuals. Most patients have a family history, while a minority present sporadically. The characteristic pathological changes are the degeneration and death of cerebellar Purkinje cells. Gordon Holmes syndrome (GHS [OMIM 212840]), a subtype of ARCA, presents primarily with ataxia that develops during adolescence and is associated with gonadal dysfunction. Earlier, we reported for the first time in humans that mutations in the CHIP protein-encoding gene, STUB1, can cause GHS, proving that STUB1 is the third pathogenic gene for GHS. Subsequent molecular genetics and animal model studies suggest that biallelic CHIP mutations leading to CHIP protein dysfunction are an important cause of ARCA. Similar to the human phenotype, CHIP mutation models also exhibit progressive clinical manifestations, primarily characterized by cerebellar degeneration and atrophy, leading to ataxia symptoms such as balance, coordination, and gait impairment, accompanied by other cognitive impairments and dementia phenotypes. Currently, clinical treatment options for ARCA are extremely limited, and its mortality and disability rates are extremely high. Supportive and symptomatic treatment is the primary treatment of choice. Therefore, further exploration of the pathogenic mechanisms of this disease and the identification of new therapeutic and neuroprotective targets are urgently needed.

[0003] STIP1 homology and U-box containing protein 1 (STUB1), also known as Carboxyterminus of Hsp70-interacting protein (CHIP), is encoded by the CHIP / STUB1 gene and located on chromosome 16 of the human genome. The functional domains of the CHIP protein mainly include a tetratricopeptide repeat (TPR) domain at the amino terminus that mediates molecular chaperone function, a coiled coil (CG) domain that promotes protein dimer formation, and a U-box domain at the carboxyl terminus that has E3 ubiquitin ligase activity. The TPR domain at the N terminus of the CHIP protein exerts a molecular chaperone function, mainly binding to heat shock proteins HSP70 or HSP90. HSP-CHIP can connect to substrate proteins, complete targeted modification of substrate proteins, inhibit chaperone protein folding activity, and promote physiological protein degradation and recycling. The U-box domain confers CHIP ubiquitin ligase activity, responsible for recruiting the ubiquitin-conjugating enzyme E2. Through a cascade reaction involving E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and CHIP, ubiquitin bound to E2 is transferred to a chaperone bound by the TPR domain and its substrate protein, or directly to a CHIP-bound substrate protein, thereby forming polyubiquitin chains on the substrate. Due to its dual properties as both a molecular chaperone and an E3 ligase, CHIP plays a crucial role in the protein quality control (PQC) system. CHIP maintains normal cellular function by promoting molecular chaperone-mediated degradation of abnormal proteins through the proteasome and autophagy-lysosome-dependent pathways. Animal models using genetic loss-of-function CHIP have reported impaired cardiac responses to hemodynamic or ischemic stress, accelerated aging, and neurodegeneration.

[0004] Mutations in the CHIP protein have been shown to be the cause of ARCA, making it a type of CHIP-related ataxia. Proteomic studies of the brain of animal models of CHIP-related hereditary ataxia have revealed a significant increase in PDE9A expression in brain tissue, with this expression progressively increasing with disease progression, suggesting that PDE9A may be involved in the pathogenesis of CHIP-related ataxia. Phosphodiesterases (PDEs) are a superfamily of enzymes responsible for hydrolyzing the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Based on their structural and functional properties, PDEs can be divided into 11 subfamilies (PDE1-PDE11). Of all PDE subfamilies, phosphodiesterase 9A (PDE9A) has the highest affinity for cGMP and is highly expressed in the cortex, hippocampus, basal ganglia, and cerebellum. Furthermore, PDE9A is the most highly expressed PDE isoform in brain tissue and is widely involved in cell signaling pathways through cGMP. cGMP signaling is important for many brain functions, such as synaptic plasticity, phototransduction, learning and memory, and stem cell differentiation. Therefore, upregulated PDE9A expression may inhibit cGMP-regulated cell signaling pathways. Inhibition of PDE9A activity has been reported in various diseases, including AD. Inhibition of PDE9A not only mitigates oxidative damage to hippocampal neurons caused by Aβ deposition but also has potential therapeutic effects in animal models of various diseases, including myocardial damage caused by heart failure and DMD.

[0005] PDE9A inhibitors, as an emerging class of drugs, are undergoing active research and development. While no drugs are currently approved for clinical use, these compounds are currently being explored in preclinical and clinical trials for the treatment of various diseases, particularly neurodegenerative disorders. Among them, PF-04447943 has entered Phase II clinical trials for Alzheimer's disease; BI 409306 has entered Phase II clinical trials for Alzheimer's disease and schizophrenia; while the earliest discovered compound, BAY 73-6691, is still in the early stages of preclinical development. BAY 73-6691, a widely used PDE9A inhibitor, inhibits PDE9A, preventing the breakdown of cGMP and thereby increasing cGMP levels. Designed to be highly selective for PDE9A, BAY 73-6691 has minimal inhibitory effect on other PDE family members. This high selectivity helps minimize nonspecific drug effects and side effects. Previously, in animal models, BAY 73-6691 was reported to have potential effects on cognitive function and therefore may have an ameliorative effect on Alzheimer's disease.

[0006] However, the drug development process for BAY 73-6691 is complex, and its translation into clinical applications faces significant challenges. Currently, detailed data on its pharmacokinetics, bioavailability, and in vivo stability are very limited, and its pharmacological effects remain relatively unknown. Therefore, it is unclear what diseases BAY 73-6691 might be effective for as a drug. Summary of the Invention

[0007] In order to further study the therapeutic effect of BAY 73-6691 and the correlation between BAY 73-6691 and ARCA brain injury, the present invention proposes the use of a pharmaceutical preparation containing BAY 73-6691 or its derivatives in the preparation of a drug for treating brain injury diseases.

[0008] The technical solution of the present invention is achieved as follows:

[0009] Use of a pharmaceutical preparation containing BAY 73-6691 or its derivatives in the preparation of a drug for treating brain injury diseases, wherein the structural formula of BAY 73-6691 is:

[0010] The above-mentioned pharmaceutical preparations include BAY 73-6691 and / or a pharmaceutically acceptable salt of BAY 73-6691.

[0011] The pharmaceutically acceptable salt of BAY 73-6691 is any one of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, propionate, hexanoate, heptanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate, 3-phenylpropionate, pivalate, tert-butylacetate, dodecyl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate and stearate.

[0012] The above-mentioned brain injury diseases are secondary brain injuries caused by ARCA.

[0013] The above-mentioned brain damage disease is autosomal recessive spinocerebellar ataxia.

[0014] The above-mentioned CHIP inactivation mutations will lead to increased PDE9A expression in ARCA brain tissue, and the PDE9A content will progressively increase as the disease progresses.

[0015] The above-mentioned autosomal recessive spinocerebellar ataxia is caused by impaired mitochondrial autophagy in Purkinje cells.

[0016] The present invention has the following beneficial effects:

[0017] 1. This study found that BAY 73-6691 can improve ARCA brain damage through a dual ubiquitin phosphorylation pathway. Therefore, BAY 73-6691 may become a candidate drug for the treatment of ARCA; it is also expected to provide new supplements for the clinical application of old drugs in new applications.

[0018] 2. BAY 73-6691 regulates the cGMP / cAMP pathway in the central nervous system and participates in the regulation of the CHIP-related protein quality control system, explaining the new pharmacological mechanism and target of BAY 73-6691.

[0019] 3. BAY 73-6691 inhibits PDE9A activity, maintaining the balance of CHIP-mediated cGMP / cAMP signaling interactions and ameliorating the pathological changes in ARCA-induced brain damage caused by impaired mitochondrial autophagy in Purkinje cells. Improving the imbalance in the intracellular cGMP / cAMP pathway offers potential therapeutic and brain-protective effects for ARCA. It also enhances CHIP's ability to clear damaged and misfolded proteins and inhibit proteotoxicity, potentially improving immunity, aging, metabolic stress, and numerous human diseases, including cardiac and neurological disorders.

[0020] 4. BAY 73-6691 inhibits the increased PDE9A aggregation and increases CHIP protein expression to improve mitochondrial damage and maintain the balance of mitochondrial autophagy; by inhibiting the toxic damage mediated by PDE9A protein aggregation, it treats the clinical phenotype of ARCA. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1Bidirectional degradation regulation of PDE9A and CHIP proteins. a: 4 μg of Flag-CHIP plasmid was co-transfected with 0, 4, 8, or 16 μg of V5-His-PDE9A into 293T cells, and western blot analysis was performed to analyze CHIP-regulated PDE9A expression. b: 4 μg of V5-His-PDE9A plasmid was co-transfected with 0, 4, 8, or 16 μg of Flag-CHIP into 293T cells, and western blot analysis was performed to analyze PDE9A-mediated CHIP changes. c: Changes in PDE9A mRNA expression after cells were transfected with different CHIP mutant plasmids; T246M: homozygous mutation at p.T246M; K30A: homozygous mutation at p.K30A; CHIP+Bay: cells overexpressing CHIP and then adding 200ug / ml BAY 73-6691 (PDE9A inhibitor); sh-CHIP-1, sh-CHIP-2, and sh-CHIP-3: cells interfering with CHIP expression; d: Changes in PDE9A expression after cells were transfected with CHIP mutant plasmids; e: Changes in CHIP mRNA expression after cells were transfected with PDE9A mutant plasmids; PDE9A+Bay cells overexpressing PDE9A and then adding BAY 73-6691; sh-CHIP: cells interfering with CHIP expression; f: Changes in PDE9A expression after changes in PDE9A protein expression; g: cells were transfected with HA-CHIP and Flag-PDE9A plasmids, 50nM Bafilomycin A1 (autophagy inhibitor) and 100 nM Bozteomib (protease inhibitor, PS-341) were used to determine the half-life and degradation pathways of CHIP and PDE9A proteins. h: Bidirectional degradation of CHIP and PDE9A following co-transfection of HA-CHIP and Flag-PDE9A into cells. i: Degradation of PDE9A and CHIP by PDE9A, CHIP, and BAY 73-6691. j: Schematic diagram of the PDE9A and CHIP protein structures. ΔTPR: TRP domain knockout; ΔU-box: U-box domain knockout. k: Detection of the interaction domains between PDE9A and CHIP proteins by co-immunoprecipitation. Antibodies: V5-rabbit, Flag-mouse. l: PDE9A and CHIP plasmids were co-transfected into 293T cells, and immunofluorescence was used to observe the cytoplasmic colocalization of PDE9A and CHIP. Data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0023] Figure 2Predicted docking model for PDE9A and HSP70; a: HSP70 β-SBD domain bound to PDE9A; NBD: N-terminal nucleotide binding domain; β-SBD: β-sandwich substrate binding domain; α-SBD: C-terminal α-helical lid. b: Top1 predicted model for hydrogen bonding between the β-SBD and PDE9A. PDE9A CYS338 and HSP70 TYR183 are linked by hydrogen bonds. Probable docking models were predicted using gram-x software (http: / / vakser.bioinformatics.ku.edu / resources / gramm / grammx). Prediction results are visualized using PDBePISA and Dockeasy (www.dockeasy.cn).

[0024] Figure 3 CHIP directly mediates K63- and K27-linked polyubiquitination of PDE9A. a: Schematic representation of the structures of ubiquitin mutants. b: Schematic representation of the structures of homozygous CHIP p.T246M and p.K30A mutations. c: In vitro ubiquitination assay, E1 and E2: UbcH5, ATP: 1X ATP buffer. d: CHIP-mediated ubiquitination of PDE9A by different E2 enzymes. Ube2V-Ubc1, UbcH5D and UbcH5a can all synthesize ubiquitinated chains in vitro; e: CHIP and PDE9A in vivo ubiquitination experiment, Antibody: V5-rabbit, HA-mouse; f and g: In vivo ubiquitination experiments verify that different ubiquitin chain mutants participate in CHIP ubiquitination modification of PDE9A; K4, K6, K1, K2, K29 chains all produce polyubiquitin chains, while K63R and K27R ubiquitin chain synthesis is impaired; f: In vivo ubiquitination experiment detects the structure of CHIP ubiquitinated PDE9A, CHIP domain deletion ubiquitination chain synthesis is impaired; g: In vivo ubiquitination experiments show that CHIP Both p.T246M and p.K30A site mutations prevent the ubiquitination of PDE9A; hi: The cAMP activator Forskolin (50uM) was added to the K63- and K27-linked CHIP-mediated ubiquitination of PDE9A in vivo, and the in vitro ubiquitination assay showed that the ubiquitination reaction of CHIP to PDE9A changed after cAMP activation.

[0025] Figure 4Inhibiting PDE9A activity and increasing exogenous CHIP protein ameliorates the clinical phenotype of ARCA. (a) Behavioral assessment timeline for BAY 73-6691-treated and AAV-CHIP-injected groups. The BAY 73-6691-treated group included 4-month-old male BAY 73-6691 rats, wild-type littermates, homozygous Hom mutant rats, and homozygous Hom mutant rats. The AAV-CHIP-injected group included AAV-CHIP rats, wild-type littermates, Hom mutant rats, and AAV-NC rats. (b) and (j) Maximum wheel speed tolerated by rats in a running wheel test. (c) and (k) Time required for rats to complete a complete crossover in a balance beam test. (d) and (l) Gait analysis showing rat footprints, with forelimbs in red and hindlimbs in blue. LFSL: left front step length, RFSL: right front step length, LRSL: left rear step length, RRSL: right rear step length, LFRO: left front-to-back overlap, RFRO: right front-to-back overlap, FBW: front-base width, RBW: rear-base width, e and m: quantitative analysis of rat gait; f and n: water maze experiment trajectory diagram, after the hidden platform was hidden, the rats explored the platform trajectory after entering the water from the third quadrant; g and o: after the hidden platform was hidden, the latency of the rats to explore the platform after entering the water from the third quadrant was monitored for 5 consecutive days; h and p: on the sixth day of the water maze, after the platform was removed, the proportion of time the rats stayed in the quadrant after entering the water from the third quadrant; i: after the platform was removed, the number of times the rats crossed the platform within 60 s after entering the water from the third quadrant; q: after the platform was removed, the number of times the rats crossed the platform within 5 min after entering the water from the third quadrant; data are expressed as mean ± SD, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, n s P>0.05.

[0026] Figure 5 PDE9A inhibits CHIP Serine 20 phosphorylation; a: Phospho S20 antibody was used to detect CHIP phosphorylation in the cerebellum and hippocampus of male rats in the Bay group and the wild-type and Hom groups; b: CHIP phosphorylation levels in the cerebellum and hippocampus of male rats in the AAV-CHIP, AAV-NC, wild-type and Hom groups; c: CHIP expression levels were altered in 293T cells transfected with CHIP mutant plasmids and different PDE9A expression plasmids, and CHIP phosphorylation levels were detected by western blot. Data are expressed as mean ± SD, and were analyzed by ANOVA (Tukey's multiple comparisons). ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0027] Figure 6To reduce the toxic damage of PDE9A aggregation and CHIP to cells; a: AnnexinV / PI flow cytometry apoptosis assay was used to detect the apoptosis of 293T cells when the expression of PDE9A and CHIP proteins changed. AnnexinV was used to detect early cell apoptosis, and the nucleic acid dye PI was used to detect late apoptosis because it penetrates the cell membrane. Q1 was mechanical necrotic cells, Q2 was late apoptotic cells, Q3 was normal cells, and Q4 was early apoptotic cells. The sum of Q2+Q4 was selected for quantitative statistics of apoptosis; b: CCK8 assay was used to detect the cell proliferation activity when the expression of PDE9A and CHIP proteins changed. After adding CCK8 reagent to each group, the cells were incubated at 37℃ for 2h and the absorbance was measured. The detection time gradient was selected as 24h, 48h, 72h, 96h, and 120h.

[0028] Figure 7 Figure 7: BAY 73-6691 and exogenous CHIP improve ARCA brain histopathology. (a) and (b) Tissue immunofluorescence quantification of the Purkinje cell marker protein calbindin-D28K in cerebellum tissue to determine changes in cerebellar Purkinje cell number. (c) and (d) Automated capillary electrophoresis detection of autophagy proteins LC3B and P62 expression in the cerebellum and hippocampus of mice in each group. (e) Transmission electron microscopy observation of mitochondrial changes in rat cerebellum. In the Hom group, mitochondria (M, enclosed in red dashed circles) were fewer in number, with locally blurred and damaged membranes, extensive cristae fragmentation and dissolution, and extensive matrix dissolution or vacuolation. Autolysosomes (ASS, red arrows) were also observed under the microscope. (N: nucleus, Go: Golgi apparatus, Lip: lipofuscin, RER: rough endoplasmic reticulum, AP: autophagosome). Data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0029] Figure 8 To investigate the effects of PDE9A aggregation and CHIP depletion on hippocampal neuronal structure, researchers studied rats homozygous for the STUB1 p.T246M mutation (Hom, 6.25 months of age), rats exogenously administered CHIP via adeno-associated virus (AAV-CHIP, 7 months of age), rats in which PDE9A activity was inhibited by BAY 73-6691 (Bay, 6.25 months of age), and male littermate rats of the same age (Wt, 6.25 months of age). Hippocampal neurons in each group were observed using Nissl staining. In the Wt, Bay, and AAV-CHIP groups, numerous Nissl bodies were observed, with well-defined centrally located nuclei. In contrast, the Hom group showed a decrease in the number of neurons in the CA1 region of the hippocampus, disorganized neuron structure, a reduced number of Nissl bodies, and absent or displaced nuclei.

[0030] Figure 9 Figure 2: Single-cell sequencing cell clustering; a: Schematic diagram of single-cell transcriptome analysis. There were 4 model groups, with three rats in each group: STUB1 p.T246M homozygous mutant rats (Hom, 6.25 months old), exogenously injected AAV-CHIP rats (AAV-CHIP, 7 months old), PDE9A inhibitor BAY 73-6691 treated rats (Bay, 6.25 months old), and wild-type male littermates (Wt, 6.25 months old); b and c: Cell clustering diagrams. Cells were divided into 12 groups based on marker genes of rat hippocampal and cerebellar cells: granule cells (Reln, Rbfox3, Neurod1), oligodendrocytes (Mpb), glutamatergic neurons (Aldoc), GABAergic neurons (Gad2, Pnoc, Tcf7l2, Lhx9), GABA interergic neurons (Pax2, Lbx1), pyramidal cells (N ell1, Npy2r, Slc4a8, B3gat2), oligodendrocyte precursor cells (Pdgfra, Olig1, Olig2), endothelial cells (C1qa, C1qb, C1qc), Bergmann glia (Gdf10, S100b), glial cells (Tmem119), Purkinje cells (Gad1, Car8, Calb1, Pcp2, Necab2, Pcp4), astrocytes (Clu, Apoe); d: Volcano plot showing the expression of specific marker genes in 12 cell groups.

[0031] Figure 10 PDE9A participates in CHIP-mediated cGMP / cAMP signaling interactions; ac: KEGG enrichment analysis of differentially expressed genes in total cells between Wt and Home, Bay and Home, and AAV-CHIP and Home. The most relevant ARCA signaling pathway is highlighted in red. d: Aucell pathway activity analysis shows cGMP-PKG pathway expression in total cells from the four rat groups. eg: GSEA pathway activity analysis shows changes in the cGMP-PKG pathway in total cells between Wt and Home, Bay and Home, and AAV-CHIP and Home; h: Aucell pathway activity analysis shows cAMP signaling pathway expression in the four rat groups. ik: GSEA analysis of changes in the cAMP signaling pathway in total cells between Wt and Home, Bay and Home, and AAV-CHIP and Home.

[0032] Figure 11Figure 2: Bidirectional post-translational modifications of PDE9A and CHIP. Figure 2: KEGG enrichment analysis of differentially expressed genes in Purkinje cells compared to Wt vs. Hom, Bay vs. Hom, and AAV-CHIP vs. Hom cells. The most relevant signaling pathways are highlighted in red. Figure 3: Aucell pathway activity analysis revealed alterations in the cGMP-PKG signaling pathway, ubiquitin-mediated proteolysis, mitochondrial autophagy, cAMP signaling pathway, oxidative phosphorylation, and autophagy signaling pathways in Purkinje cells from the four rat groups.

[0033] Figure 12 The expression of pathway signaling proteins; a and b: Western blot detection of cAMP-PKA signaling protein expression in the cerebellum and hippocampus of mice in each group; d: ELISA detection of cGMP content in the cerebellum and hippocampus of male rats injected intraperitoneally with BAY 73-6691 and in the wild-type and Hom groups of the same littermates; c: The cAMP activator Forskolin (50uM) was added to the K63- and K27-linked CHIP-mediated PDE9A ubiquitination in vivo, and the in vitro ubiquitination assay showed that after cAMP activation, the ubiquitination reaction of CHIP to PDE9A changed. e: ELISA detection of cGMP content in the cerebellum and hippocampus of male rats injected with AAV-CHIP into the tail vein, wild-type littermates, Hom group of littermates, and homozygous mutant mice injected with empty AAV (AAV-NC) into the tail vein; f and g: Automated capillary electrophoresis detection of PKG signaling protein expression in the cerebellum and hippocampus of mice in each group; Data are expressed as mean ± SD, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0034] Figure 13 Schematic diagram of the main results of this study. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Experimental Materials:

[0037] Plasmids and antibodies

[0038] All plasmids in this experiment were purchased from Shanghai Genetech Co., Ltd.:

[0039] pCMV-PDE9A-3xflag; pCMV-PDE9A-eGFP; pCMV-PDE9A-V5-6his; pCMV-PDE9A(del236-557aa)-V5-6his; pCMV-STUB1-3xflag; pCMV-STUB1-HA; pCMV-STUB1(del226-300aa)-3xflag; pCMV-STUB1(del26-aa127)-3xflag; pCMV-STUB1(T246M)-3xflag; pCMV-STUB1(K30A)-3xflag; pCMV-HA-ubiquitin-WT; pCMV-HA-ubiquitin-K48; pCMV-HA-ubiquitin-K48R;

[0040] pCMV-HA-ubiquitin-K63;

[0041] pCMV-HA-ubiquitin-K63R;

[0042] pCMV-HA-ubiquitin-K11;

[0043] pCMV-HA-ubiquitin-K11R; pCMV-HA-ubiquitin-K27; pCMV-HA-ubiquitin-K27R; pCMV-HA-ubiquitin-K29; pCMV-HA-ubiquitin-K29R; pCMV-HA-ubiquitin-K0; shRNA-PDE9A; shRNA-STUB1-1; shRNA-STUB1-2; shRNA-STUB1-3.

[0044] List of antibodies:

[0045] Anti-PDE9A-rabbit (Proteintech, China); Anti-PDE9A-mouse (Santacruz, USA); anti-STUB1-rabbit (Abcam, USA); anti-STUB1-mouse (Santacruz, USA); anti-HA-tag-mouse (Abcam, USA); anti-DYKDDDDK-mouse (Proteintech, China); anti-V5-rabbit (Proteintech, China); anti-GFP-tag-rabbit (Abcam, USA); anti-ubiquitin-mouse (Abcam, USA) USA); anti-PRKG1-rabbit (Proteintech, USA); anti-PRKG2-rabbit (Proteintech, China); anti-CREB1-mouse (Proteintech, China); anti-cAMP-Rabbit (Proteintech, China); anti-PRKACA-Rabbit (Proteintech, China); anti-Calbindin-rabbit (Abcam, USA); anti-GAPDH-mouse (Proteintech, China); anti-pho-CHIP (Abmart, China); HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, China); HRP-conjugated Affinipure Goat Anti-mouse IgG (H+L) (Proteintech, China); Goat Anti-Rabbit IgG H&L (abcam, USA); Goat Anti-mouse IgG H&L (abcam, USA).

[0046] Cell culture and plasmid transfection

[0047] HEK293T cells (cell bank, Chinese Academy of Sciences) were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (Hyclone, China). All plasmids were transfected into 293T cells using the lp3000 transfection kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Medium was changed 6 hours after transfection. Medium changes included the addition of 20 μM bafilomycin A1, a protease inhibitor called bozteomib, and 200 μg / μl of the PDE9A inhibitor BAY 73-6691. Medium was removed at designated times for further analysis.

[0048] Experimental animal husbandry and treatment

[0049] All animal experimental operations and treatments in this study were approved by the Ethics Committee of the Animal Experiment Center of Zhengzhou University. Specific pathogen-free (SPF) breeding and feeding were carried out in the Animal Center of Zhengzhou University. The animals used in this experiment were the offspring of CHIP p.T246M mutant rats from previous experiments. Rat tail DNA was extracted using a kit.

[0050] CHIP positive chain: 5'-CTCATGGGCAGGCTCTGGTATGG-3',

[0051] PCR reaction was performed with the primer 5'-GAGCAGTTCAGAACCCATCATCAGG-3' of the antisense strand of CHIP, and the product was sequenced by Sanger sequencing at Shangya Company for genotyping. +15 Month-old male mutant rats were intraperitoneally injected with 200 μg / kg of protease inhibitor BAY 73-6691 daily for 15 consecutive days, and subsequent experiments were performed after 7 days. 5-month-old male homozygous mutant rats were selected for exogenous overexpression of CHIP in brain tissue. The rats were anesthetized with 1.8% isoflurane and injected with 1.2*10 12 vg / ml HBAAV2 / BBB-CHIP virus (Hanbi Biotechnology, China) was added and the rats were maintained in the same environment. Male homozygous mutant rats from the same litter were injected with the same dose of HBAAV2 / BBB-flag as a control. Subsequent experiments were performed when the rats were 7 months old. The control groups included male normal wild rats and homozygous mutant rats from the same litter.

[0052] Monocyte RNA sequencing and biological information analysis

[0053] Rats in the PDE9A inhibitor treatment group (6 +7 months old), male normal wild rats of the same litter (6 +7 months old), homozygous mutant rats (6 +7Three rats were included in each of the HBAAV2 / BBB-CHIP group (7 months old) and the intravenously injected HBAAV2 / BBB-CHIP group (3 rats). After 20 minutes of inhalation anesthesia with 1.8% isoflurane, fresh rat hippocampus and cerebellum tissues were quickly extracted. After single-cell sorting at Nanjing Xingeyuan Company, three samples in each group were used to capture mRNA from hippocampal neurons and cerebellar neurons in a 1:1 ratio to construct single-cell libraries. The libraries were sequenced using the MGISEQ-2000 platform PE150 sequencing strategy by Wuhan BGI Genomics Co., Ltd.; the sequencing results were compared with , and the original Fastq format sequencing files were processed using the CeleScope standardized process to obtain a 10X format single-cell gene expression matrix. Further processing and analysis of the scRNA-seq datasets were performed in R software. Data quality control was first performed, filtering cells with more than 2500 or less than 500 unique genes expressed, mitochondrial counts >15%, and gene coverage <3 cells, i.e., cells with empty droplets and low-quality mitochondrial contamination were removed. Data were then normalized using the SCTransform function and harmony (v.0.1.0) software to remove batch effects between samples. Seurat was used for dimensionality reduction, clustering, and grouping, and marker gene visualization was used to annotate cell subpopulations. Finally, FindMarkers was used to calculate differentially expressed genes, with a threshold of log2FC > 0.25 and p < 0.05. Two-sample T-tests were performed. KEGG enrichment analysis of differentially expressed genes was performed using the KEGG Pathway and Module data of rattus norvegicus (rat) from ClusterProfiler software. GSEA analysis was used to identify expression trends of reference gene sets between groups. AUCell was used for single-cell pathway activity analysis.

[0054] Real-time fluorescent quantitative polymerase chain reaction

[0055] RNA was extracted using a RNA extraction kit and Prime Script TM RNA was reverse-transcribed into cDNA using the RT reagent kit with gDNA Eraser (Takara, Japan) according to the manufacturer's instructions. The cDNA was diluted 20-fold and then probed for CHIP and PDE9A mRNA expression in different cell groups using primers for CHIP and PDE9A. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed using a RT-qPCR kit according to the manufacturer's instructions, with triplicate wells per group. Primers included:

[0056] CHIP sense strand: 5′-ATCCCCGACTACCTGTGTGGC-3′ and antisense strand: 5′-TGCTCCTCGATGTCCTTGCG-3′;

[0057] PDE9A sense strand: 5′-TTGACTCCTCGACGCGATGTTC-3′ and antisense strand: 5′-CTGAGGGTGACAGGGTTGATGC-3′;

[0058] GAPDH sense strand: 5′-GGCGCTGAGTACGTCGTGGAGT-3′ and antisense strand: 5′-GGGCAGAGATGATGACCCTTTTG-3′.

[0059] Western blotting

[0060] A certain amount of rat brain tissue, hippocampal tissue and collected cells were taken and RIPA lysis buffer, PMSF, protease inhibitors, and phosphatase inhibitors were added at a ratio of 1000:10:10:10. The cells were then fully lysed in an ultrasonic lysator to prepare protein samples. The protein concentration of each group was unified by BCA protein quantification. For western blotting, the protein samples were diluted with 5XSDS protein loading buffer and then heated at 100°C for 5 minutes. BeyoGel was used. TM Protein electrophoresis and membrane transfer were performed using Plus4-20% PrecastPAGE Gel for Tris-Gly System with 15-well (Beyotime, China). Primary antibodies were selected as protein markers and incubated overnight at 4°C (Flag-mouse 1:5000, PDE9A-mouse 1:5000, CHIP-rabbit 1:10000, v51:5000, HA 1:20000, ubiquitin 1:1000, cAMP 1:2000, PRCAKA 1:1000, GAPDH 1:5000, β-tubulin 1:5000). The next day, the membranes were washed and incubated with secondary antibodies (HRP-conjugated Affinipure Goat Anti-Rabbit 1:5000, HRP-conjugated Affinipure Goat Anti- Anti-mouse 1: 5000), add ECL chemiluminescent A+b mixed solution and develop under an exposure instrument;

[0061] The final concentration of protein samples used in fully automated capillary electrophoresis experiments was 2 μg / μl per well. Standard procedures were performed according to the instructions of the Chemiluminescence training Kit (ProteinSimple, USA) on a SimpleWestern Jess (ProteinSimple). Protein cartridge 25 and size range 12–230 kDa were selected, and primary antibodies were diluted to the specified concentrations (PDE9A-rabbit 1:50, CHIP-rabbit 1:100, PRKG1-rabbit 1:30, PRKG2-rabbit 1:25, P62-rabbit 1:25, LC3B-rabbit 1:40, β-tubulin-rabbit 1:100).

[0062] Enzyme-linked immunosorbent assay

[0063] The same concentration of mouse and hippocampal tissue protein lysate in each group was used to quantitatively detect the cGMP content in the tissue using Cyclic GMP Complete ELISA Kit (abcam, USA).

[0064] Protein half-life determination

[0065] 293T cells transfected with CHIP-HA alone, PDE9A-flag alone, or co-transfected with CHIP and PDE9A plasmids were replaced with culture medium containing different reagents for 24 hours, and 40uM cycloheximide was added. Cells were collected at different time points of 0h, 2h, 4h, 6h, 8h, and 10h for western blotting to detect the expression of PDE9A and CHIP proteins.

[0066] Co-immunoprecipitation

[0067] 293T cells transfected with plasmids were collected and lysed with NP-40 lysis buffer (Beyotime, China), PMSF, protease inhibitors and phosphatase inhibitor mixture in a ratio of 1000:10:10:10 to obtain 5 μg protein samples; protein was pre-adsorbed with BeyoMag TMProtein A+G beads (Beyotime, China) were added for 1 h, and the magnetic beads were discarded. The tag protein antibodies (V5-rabbit 10 μg, GFP-rabbit 10 μg) were added and incubated at 4°C on a side-by-side shaker for 12 h. Then, new 100 μl of magnetic bead mixture was added and incubated at 4°C overnight. After 12 h, the magnetic beads were washed 4 times with NP-40 lysis mixture for 5 min, and then 20 μl of 5XSDS protein loading buffer and 30 μl of NP-40 lysis mixture were added. After heating at 100°C for 5 min, the supernatant was collected and the target protein was detected by western blotting.

[0068] Immunostaining

[0069] Cell immunofluorescence: cells were plated in 24-well plates, fixed with 4% paraformaldehyde (Beyotime, China) for 15 min, permeabilized with 0.3% TritonX-100 (PBS) (Beyotime, China) for 20 min, blocked with 5% fetal bovine serum albumin (PBS) (Beyotime, China) for 1.5 h, incubated with primary antibodies at 4°C overnight (CHIP-mouse 1:500, PDE9A-rabbit 1:500), and incubated with fluorescent secondary antibodies at room temperature for 1 h ( 488 1:500, 594 1:500), and DAPI (Beyotime, China) was added dropwise and the slides were mounted.

[0070] Tissue immunofluorescence: rats were anesthetized with 1.8% isoflurane for 20 minutes. The brain was removed by cardiac perfusion with 4% paraformaldehyde, embedded in paraffin and sliced ​​with a thickness of 4um. First, dewaxed with environmentally friendly dewaxing solution for 30 minutes, dehydrated with gradient alcohol (100% I, 100% II, 95%, 85%, 75% for 5 minutes each), and then antigen repaired with 50X antigen repair solution. The cells were permeabilized with 0.3% TritonX-100 (PBS) at 37°C for 20 minutes, blocked with 5% BSA (PBS) for 1.5 hours, and incubated with primary antibodies (Calbindin-rabbit 1:300, CHIP-mouse 1:500, PDE9A-rabbit 1:500) at 4°C. After 12 hours, the cells were incubated with fluorescent secondary antibodies at room temperature for 1 hour ( 488 1:500, 594 1:500), DAPI was added to seal the slides, and fluorescence images were taken under an automatic fluorescence microscope (Leica, Germany).

[0071] In vivo and in vitro ubiquitination experiments

[0072] In vivo ubiquitination: CHIP-flag, PDE9A-v5-his, and HA-ubiquitin (the above human ubiquitination wild-type and mutant plasmids) plasmids were transfected into 293T cells. Co-interaction was performed using V5 antibody (v5-rabbit 10 μg) coupled to magnetic beads. Western blotting analysis was performed using the following primary antibodies: flag-mouse 1:5000, HA-mouse 1:2500, ubiquitin-mouse 1:1000.

[0073] In vitro ubiquitination: In vitro ubiquitination assays were performed using 0.3 μM E1, 3.4 μM E2, 250 μM Ubiquitin, and 10X ATP assay buffer (200 mM HEPES-KOH, 50 mM MgCl2, 20 mM ATP, 5 mM DTT) (RD, USA). A 10-μl system was prepared and incubated at 37°C for 1 hour. SDS was added, followed by boiling, and Western blotting was performed. E2 ligases used were Ube2V-Ubc13, UbcH5D, and UbcH5a (RD, USA).

[0074] Cell proliferation and cytotoxicity (CCK8 assay)

[0075] 1000 cells were seeded per well of a 96-well plate, and the cell proliferation activity was detected at 24 h, 48 h, 72 h, 96 h, and 120 h using Cell Counting Kit-8 (Dojindo, Japan) according to the instructions.

[0076] Flow cytometry

[0077] Get 1x10 per group 6 Filter 1x10 cells using a cell strainer 5 Single cells were stained using FITC Annexin V Poptosis Detection Kit I (BD, USA) and analyzed on a flow cytometer (Beckman, Germany).

[0078] electron microscope

[0079] Cerebellar tissues from 6.5-month-old PDE9A-treated rats, littermate CHIP p.T246M homozygous mutant rats, littermate wild-type male rats, and 7.3-month-old CHIP-overexpressing rats were collected and fixed in 1% osmium chloride (Ted Pella Inc, China) for 2 h in electron microscopy tissue fixative (Solebol, China). After washing, the tissues were dehydrated through a gradient of alcohol, infiltrated, embedded, and polymerized. Sections were then sectioned and stained with a copper grid in 2% uranyl acetate saturated alcohol solution for 8 min in the dark. The sections were then washed three times with 70% alcohol and ultrapure water, respectively, and stained with 2.6% lead citrate solution in the absence of carbon dioxide for 8 min. The sections were then washed three times with ultrapure water and observed under a transmission electron microscope.

[0080] Nissl stain

[0081] After dewaxing and antigen retrieval, tissue paraffin sections were stained using the Nissl Stain Kit (Cresyl Violet Method) (Solaibao, China).

[0082] Behavioral testing

[0083] Water maze experiment

[0084] The water maze hidden jump test was performed using SANS animal behavior analysis system software (Sansbio, China). A cylindrical barrel with a diameter of 150 cm and a height of 60 cm was selected and divided into the first quadrant (NE), the second quadrant (SE), the third quadrant (SW) and the fourth quadrant (NW). The liquid reached 40 cm and the temperature was maintained at 22°C. A small cylinder with a diameter of 12 cm was placed vertically at a position 30 degrees clockwise near the center circle of NW as the experimental platform. At the beginning of the experiment, the height of the experimental platform was adjusted to 1.5 cm above the liquid level. The exploration time of the water maze positioning and navigation experiment was set to 60 s, and the standing time on the platform was set to 5 s. Each rat entered the water maze from the NE quadrant to the barrel wall, and from the outer circle of the SE, SW and NW quadrants in turn. From the first day to the fifth day, the experimental platform was immersed 1.5 cm below the horizontal plane. The rest of the operations were the same as before and re-recorded. On day 6, the experimental platform was removed, and the exploration time was set to 60 seconds. Mice were placed in the water maze facing the wall, from the NE, SE, SW, and NW quadrants. The number of platform explorations and the ratio of exploration time in the NE quadrant to the total exploration time were recorded within 60 seconds to measure cognitive and memory abilities.

[0085] Wheel experiment

[0086] Motor coordination in rats was assessed using a rotarod (Softmaze). Rats were trained for four consecutive days, three times daily with 30-minute intervals between sessions, reaching a maximum speed of 40 rpm / min. Testing was performed on the fifth day and recorded. Rats were placed on the rotarod at an initial speed of 4 rpm / min. The speed was gradually increased to 40 rpm / min over 5 minutes. The speed of the rotarod corresponding to the second fall was recorded. If the rat grasped the rotarod, a cumulative two instances were considered falls.

[0087] Balance beam experiment

[0088] Referring to previous experimental reports, a wooden stick with a length of 150 cm and a diameter of 18 mm was made, and the test height was 40 cm. Rats were trained to walk from the starting point to the end point without stopping three times a day, with an interval of 2 minutes each time. The training lasted for 4 consecutive days, and the time taken for the rats to walk the entire balance beam was recorded on the fifth day.

[0089] Gait analysis

[0090] A rat runway with a length of 2m, a width of 20cm, and a height of 20cm on both sides was placed on a horizontal experimental table. Clean white paper was laid on the bottom of the runway. Blue non-toxic dye was applied to the rat's front paws and red dye was applied to the rat's hind paws. The rat was placed at the end of the runway. The gait trajectory of the rat from the end to the front was observed for two consecutive days. The gait trajectory was collected on the third day and calculated and analyzed.

[0091] in conclusion

[0092] 1. CHIP and PDE9A regulate each other's expression and interact with each other

[0093] PDE9A protein is composed of 593 amino acids, including a regulatory site at the amino terminal and a catalytic group at the carbon terminal. CHIP protein mainly plays a role in the protein quality control system through the TPR-Ubox dual domain. To explore the interaction between PDE9A protein and CHIP protein, we first exogenously increased the expression of PDE9A protein in cells. As the expression of PDE9A increased, the expression of CHIP protein gradually decreased ( Figure 1 a); On the contrary, as the expression of exogenous CHIP increased, the expression of PDE9A protein gradually decreased ( Figure 1 b); The results showed that there was a negative regulatory relationship between PDE9A and CHIP. There was no significant correlation change in PDE9A mRNA after CHIP mRNA expression changed ( Figure 1 c), and when the corresponding CHIP protein expression level changes, the PDE9A protein level immediately shows the opposite change ( Figure 1 d); Similarly, changes in PDE9A mRNA levels did not affect CHIP mRNA expression ( Figure 1 e), and when the corresponding PDE9A protein expression level changed, the CHIP protein expression also showed the opposite change ( Figure 1 f); Therefore, the mutual negative regulation between PDE9A and CHIP occurs post-transcriptionally. The half-life of CHIP protein is 6 hours. After the addition of the protease inhibitor Bortzeomib, its half-life is significantly increased, indicating that CHIP protein is hydrolyzed through the protease pathway; while the half-life of PDE9A protein is 4 hours. After the addition of the autophagy inhibitor BafilomycinA1, its protein degradation is significantly delayed, suggesting that PDE9A protein is degraded through the autophagy pathway ( Figure 1 g). CHIP protein can promote the autophagic degradation of PDE9A, shortening the half-life of PDE9A to 2h. At the same time, PDE9A can enhance the proteolysis of CHIP protein, thereby shortening the half-life of CHIP protein to 4h ( Figure 1 h, Figure 1 i), while the PDE9A inhibitor BAY 73-6691 prolongs the half-life of CHIP protein to 8h by promoting PDE9A degradation ( Figure 1 i). To further determine the binding domain of PDE9A protein and CHIP protein, a plasmid lacking the TPR domain and Ubox domain of CHIP protein was constructed ( Figure 1 j), after double transfection with PDE9A in 293T cells, immunoprecipitation was performed. The results showed that PDE9A could interact with the TPR domain of CHIP protein and bind to the U-box domain of CHIP protein, indicating that PDE9A protein may participate in the molecular chaperone activity of CHIP, and CHIP protein mainly interacts with PDE9A through the TPR structure ( Figure 1 k). In addition, immunofluorescence showed that CHIP protein and PDE9A protein were co-localized in the cytoplasm ( Figure 1 l).

[0094] The CHIP protein is primarily composed of three domains: TPR, GC, and Ubox. After removal of the TPR domain, neither the GC nor the Ubox domain interacted with PDE9A. However, after removal of the U-box domain, PDE9A significantly interacted with the TPR domain, indicating that PDE9A primarily binds to the TPR domain of the CHIP protein. TPR is a simple repeating sequence composed of 34 amino acids, typically forming a helix-turn-helix folding structure. Each repeat is linked by a short loop, forming an internal groove that serves as the primary structure for TPR-binding peptides. Based on the structural characteristics of TPR proteins, five main binding modes between TPR structures and interacting proteins mediate protein-protein interactions: short peptide binding to the TPR groove, long peptide binding to the TPR groove, and TPR hinge formation and binding of the folding domain to the TPR protein. According to the Protein Data Bank (PDB), CHIP proteins bind to the TPR groove via short peptides, and the peptides that form this binding mechanism are controlled by a specific sequence: the peptide is highly negatively charged (MEEVD) and must be located at the C-terminus. The most well-known TPR-binding peptide is the co-chaperone Hsp70-Hsp90 organizing protein (HOP). CHIP binds to HSP70 to mediate proteasomal and lysosomal degradation of substrate proteins; however, substrate proteins bound to CHIP-HSP90 are only degraded through the proteasome. Since CHIP mediates the autophagy-lysosomal degradation of PDE9A, PDE9A connects to the TPR domain of CHIP by binding to the chaperone HSP70. Hsp70 is one of the most common molecular chaperones that prevents aggregation, promotes the formation of native conformations, and dissolves and refolds aggregated proteins. Hsp70 consists of a 45 kDa N-terminal nucleic acid-binding domain (NBD) and a 25 kDa C-terminal substrate-binding domain (SBD). The SBD contains a β-sandwich domain (β-SBD) at the substrate binding site and a flexible α-helical lid domain (α-SBD) that regulates the affinity of misfolded proteins. This suggests that PDE9A may directly bind to the β-SBD domain of HSP70 (Table 1):

[0095] Table 1. Prediction results of the position and distance of hydrogen bonds between PDE9A and HSP70

[0096]

[0097]

[0098] We used GRAMM-X software to perform docking between PDE9A (3QI4) and HSP70 (4B9Q), predicted 10 possible models, and visualized the top 1 predicted model in PDBePISA. The results showed that hydrogen bonds H accounted for the largest proportion in interfacing residues, indicating that PDE9A may form an H-bond link with HSP70 in the β-SBD domain ( Figure 2 ).

[0099] 2. CHIP mediates K63- and K27-linked ubiquitination of PDE9A

[0100] The Ubox domain at the C-terminus of the CHIP protein gives CHIP, as an E3 ubiquitin ligase, important functions in substrate recognition, catalytic ubiquitin transfer, and determining the diversity and selectivity of ubiquitinated substrates. It can catalyze the connection of various substrate proteins with the seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) of the ubiquitin molecule, leading to the formation of different polymerized ubiquitin chains. After the translation of the substrate protein, it undergoes ubiquitination modification, thereby promoting ubiquitin-regulated substrate protein activity to regulate cell autophagy, mitochondrial function, lysosome biogenesis, and other corresponding biological processes. CHIP-mediated K48, K29, and K11 chain ubiquitination reactions activate protease degradation signals, K63 chain ubiquitination participates in the autophagic lysosomal degradation of proteins and the activity regulation of different signaling proteins, while K27 chain ubiquitination mediates changes in mitochondrial function ( Figure 3 a).

[0101] Ubiquitination experiments were conducted to explore how CHIP regulates the ubiquitination of PDE9A. In vitro ubiquitination experiments showed that PDE9A can be modified by CHIPE3 ligase as a ubiquitination substrate ( Figure 3 c), and E2 conjugating enzymes Ube2V-Ubc13, UbcH5D and UbcH5a can recognize PDE9A together with CHIP protein and ubiquitinate it ( Figure 3 d). UbcH5a and UbcH5D have little selectivity in catalyzing the ubiquitination of PDE9A, while Ube2V1 / Ube2V2 target ubiquitin K63 and catalyze the formation of specific K63-linked ubiquitin chains. Furthermore, in vivo ubiquitination experiments with PDE9A and CHIP proteins in 293T cells also confirmed that PDE9A can be ubiquitinated by CHIP in vivo ( Figure 3 e) Then, a series of Ub mutant plasmids were used to identify the ubiquitin lysine residues required for PDE9A-catalyzed Ub chain formation. In the Ub plasmids with single-site retention, K48A, K63, K11, K27, and K29 ​​chains were all generated, while K11 ubiquitin chains were synthesized less frequently ( Figure 3f, Figure 3 g), indicating that all of these sites can participate in CHIP-mediated PDE9A ubiquitination. However, in vivo ubiquitination reactions in Ub plasmids with single-site inactivation were significantly impaired compared to the plasmids with single-site activity retention, indicating that CHIP directly catalyzes K63- and K27-linked PDE9A ubiquitination. The PDE9A protein comprises 593 amino acids and contains 30 arginine residues. Using the targeted E3 ligase prediction software (GPS-Uber), CHIP was specifically selected as a potential E3 ubiquitin ligase for analysis of its potential ubiquitination sites on PDE9A. The results predict that CHIP catalyzes polyubiquitination at arginine 194 in PDE9A (Table 2).

[0102] Table 2. Prediction of CHIP ubiquitination sites on PDE9A

[0103]

[0104] Position: amino acid site, k: arginine; RING: RING-type E3 ligase

[0105] As an E3 ligase, CHIP has the ability to connect target proteins and specific E2s. Due to the presence of its specific TPR domain, its linkage with target proteins is diverse. Currently, there are three main ways for CHIP to link target proteins: molecular chaperone-mediated or misfolded protein degradation in cells: CHIP directly binds to substrate proteins to cause ubiquitin-proteasome system (UPS)-mediated substrate protein degradation; molecular chaperone HSP90-linked protease pathway and molecular chaperone HSP70-dependent lysosomal pathway; how CHIP transfers ubiquitin to PDE9A and catalyzes the degradation reaction of PDE9A. It has been previously confirmed that PDE9A interacts with the TPR domain, and CHIP promotes the degradation of PDE9A through the autophagy-lysosomal pathway, indicating that PDE9A protein is linked to CHIP by binding to HSP70. In addition, because both the CHIP TPR domain and the U-box domain interact with PDE9A, we need to further determine the specific details of CHIP ubiquitination modification of PDE9A. When we removed the CHIP protein TPR domain and U-box domain respectively, the CHIP protein ubiquitination reaction on PDE9A was affected. The CHIP TPR domain and U-box domain were changed, the molecular chaperone and E3 ligase functions were inactivated, and CHIP could not ubiquitinate PDE9A ( Figure 3h). Similarly, plasmids with CHIP protein T246M mutation and K30A mutation were transfected for in vivo ubiquitination. The results were completely consistent with those after TPR domain deletion and U-box domain deletion. The inactivating mutations of CHIP protein can disrupt the CHIP ubiquitination reaction of PDE9A ( Figure 3 b, Figure 3 i) These results suggest that CHIP recruits K63 and K27 chains to transfer to PDE9A protein bound to HSP70, promoting cascade ubiquitination of PDE9A and then completing degradation of PDE9A through autophagy-lysosome.

[0106] 3. Both the PDE9A inhibitor BAY 73-6691 and exogenous expression of CHIP protein can improve symptoms in CHIP mutation animal models

[0107] Previous work in this study has shown that CHIP mutation rat models, consistent with human CHIP mutation cases, exhibit progressive ataxia and cerebellar neuronal degeneration and death. Concomitantly, PDE9A protein expression in the brains of these animals is significantly increased, and this expression increases progressively with disease progression. This finding suggests that upregulation of PDE9A protein expression may be a key driver of the pathogenicity of CHIP mutations, but the mechanism remains unclear. Bay73-6691, a PDE9A inhibitor, has previously demonstrated significant improvements in memory impairment by increasing the retention of long-term potentiation (LTP), enhancing hippocampal synaptic plasticity, and mitigating damage to hippocampal neurons caused by toxic protein aggregation. Bay73-6691 has been primarily used in studies to improve symptoms of Alzheimer's disease, including memory loss and cognitive impairment.

[0108] Based on previous work, we speculated whether PDE9A inhibition might have therapeutic effects on CHIP mutant animal models. In further experiments, we selected 5.5-month-old T246M homozygous mutant male mice and injected them intraperitoneally with Bay73-6691 200ug / kg for 15 consecutive days. At 6 months of age, we compared them with untreated homozygous mutant male mice from the same litter, wild male mice from the same litter, and 4-month-old homozygous mutant male mice without clinical symptoms for behavioral testing ( Figure 4 a) The test results confirmed that after treatment with Bay73-6691, the balance coordination ability and gait of rats were alleviated, including the tolerance of rats in the wheel test ( Figure 4 b); The time required to cross the balance beam is shortened ( Figure 4 c); and the gait distance increases and the uniformity improves ( Figure 4 de), In addition, the rats' memory was significantly improved, and the time it took to find the platform in the water maze test was shortened ( Figure 4fg), the percentage and number of crossing platforms increased significantly ( Figure 4 These results suggest that Bay73-6691 may treat the clinical phenotype of autosomal recessive spinocerebellar ataxia by inhibiting PDE9A protein aggregation-mediated toxic damage.

[0109] The inactivating mutation at the p.T246M site of the STUB1 gene leads to the inactivation of the CHIP protein, which reduces the expression of CHIP protein in brain tissue. The reduction of CHIP protein can lead to the typical clinical manifestations of cerebellar ataxia caused by SCAR. Therefore, we also gave exogenous CHIP gene expression as a positive treatment control. In the experiment, we also injected 1.2*10 AAV virus carrying the CHIP gene, which can pass through the blood-brain barrier, into the caudal vein of 5.5-month-old rats. 11 vg / rat, after CHIP protein was completely overexpressed in the brain tissue of 7-month-old rats, behavioral observation showed that the balance coordination ability and gait of the CHIP overexpression group rats were alleviated compared with the homozygous mutant rats in the same litter ( Figure 4 a, Figure 4 jm), memory and cognitive abilities were significantly improved ( Figure 4 nq). This indicates that CHIP protein significantly improves ataxia gait and cognitive memory impairment. These results suggest that both BAY73-6691 treatment and exogenous CHIP gene expression therapy have therapeutic effects on CHIP mutation models.

[0110] 4. PDE9A inhibits PKG to directly phosphorylate CHIP. The PDE9A inhibitor BAY 73-6691 increases the phosphorylation level and expression of CHIP protein.

[0111] Recent studies have revealed that protein kinase G (PKG), a major effector of the second messenger cGMP, can directly activate the phosphorylation of CHIP-S19 (human, mouse S19) within the highly conserved residues of the TPR domain. This increases the binding affinity of CHIP to Hsp70 and reduces CHIP degradation by proteases after ubiquitination, thereby prolonging the half-life of CHIP. PKG then mediates CHIP-pS19 protein clearance of ubiquitin-insoluble proteins. We hypothesized that PDE9A reduces CHIP expression and shortens its half-life in vivo by modulating CHIP phosphorylation levels. Furthermore, we hypothesized that PDE9A inhibitors could inhibit PDE9A levels, thereby increasing CHIP phosphorylation and enhancing CHIP-HSP70 binding, further promoting PDE9A binding to CHIP-HSP70 and its ubiquitination and degradation.

[0112] The detection of CHIP-S20 site-specific phosphorylation antibody in the cerebellum and hippocampus of rats with CHIP p.T246M inactivation mutation showed a decrease in p-CHIP protein. The inhibition of PDE9A by Bay-736691 significantly increased the phosphorylation level of CHIP protein S20 ( Figure 5 a), while exogenously increasing CHIP protein levels, p-CHIP increased ( Figure 5 b) In vitro cell experiments also confirmed that p-CHIP expression decreased after CHIP Ubox inactivation mutation, and P-CHIP levels decreased significantly with increased PDE9A protein or decreased CHIP protein ( Figure 5 c) Because PDE9A lacks a cGMP binding domain, its catalytic activity is not enhanced by cGMP. Therefore, PDE9A inhibitors increase PKG levels by raising baseline cGMP concentrations. Consequently, PDE9A reduces PKG levels, thereby reducing phosphorylated CHIP protein, shortening its half-life and thereby increasing CHIP protein hydrolysis.

[0113] 5. PDE9A inhibitor BAY 73-6691 treatment improves CHIP mutation-induced mitochondrial autophagy and apoptosis

[0114] PDE9A regulates CHIP phosphorylation changes through cGMP-PKG signaling, thereby reducing the expression of CHIP protein in vivo. The reduction of CHIP protein inhibits the k63 chain and K27 chain tandem ubiquitination levels of PDE9A, inhibits the lysosomal degradation of PDE9A, and thus increases the aggregation of intracellular PDE9A. Previous results found that rats with the CHIP p.T246M site mutation mainly had pathological damage to cerebellar Purkinje cells, nuclear condensation, dendritic swelling, and a significant decrease in the number of Purkinje cells compared with normal wild-type rats; we inferred that increased aggregation of PDE9A and decreased CHIP protein can both lead to cell destruction and apoptosis. Therefore, the AnnexinV / PI flow apoptosis experiment was used to detect apoptosis under abnormal PDE9A and CHIP protein. The results showed that the CHIP p.T246M mutation, decreased CHIP protein, and increased PDE9A protein could all lead to varying degrees of apoptosis in 293T cells. After treatment with BAY 73-66791 or increasing CHIP protein expression, the level of cell apoptosis was significantly reduced ( Figure 6 a). Similarly, in CCK8 cell experiments, it was found that increased PDE9A and CHIPp.T246M mutation significantly reduced cell proliferation activity, while after BAY 73-66791 treatment or increased CHIP protein, the proliferation activity of 293T cells was significantly improved ( Figure 6b). Therefore, the increased aggregation of PDE9A and the reduction of CHIP protein caused by the CHIP p.T246M mutation can reduce cell viability and affect normal cell proliferation. On the other hand, tissue immunofluorescence observation showed that after BAY 73-66791 treatment, the Purkinje marker protein calbindin was significantly increased in the cerebellum of rats with the CHIP p.T246M mutation, and the number of Purkinje neurons increased ( Figure 7 a); After exogenous addition of CHIP protein, the number of Purkinje neurons increased significantly and the number of neuronal dendrites was significantly improved compared with the CHIP p.T246M mutant rats of the same littermate and age ( Figure 7 b); Nissl staining of the hippocampus of rats with CHIPp.T246M mutation showed that the number of neurons in mutant rats was reduced and their arrangement was disordered. After treatment with BAY 73-66791 and the addition of exogenous CHIP protein, the number and arrangement of neurons improved ( Figure 8 ).

[0115] CHIP plays a crucial role in mitochondria. Mitochondria lacking CHIP exhibit altered oxidative stress. When CHIP possesses intact TPR domains and E3 ubiquitin ligase activity, CHIP regulates autophagy in damaged mitochondria (mitophagy). Furthermore, a decrease in CHIP inactivation increases the number of abnormal CHIP-associated mitochondria by inhibiting LC3B-mediated autophagy. 30 CHIP-mediated K63-linked polyubiquitin chains often modify substrates for degradation in the autophagy-lysosomal pathway, playing a complementary protective role in maintaining protein homeostasis by directing abnormal proteins and organelles to autophagosomes. K27-linked ubiquitin chains, on the other hand, primarily mediate autophagy and are recognized by the autophagy receptor p62, leading to the formation of autophagosomes, ultimately initiating mitophagy and alleviating oxidative stress. The formation of tandem ubiquitin chains linked by K63 and K27 primarily promotes autophagy to selectively degrade aggregated proteins and nonfunctional mitochondria, initiating a signaling cascade that activates mitophagy. PDE9 is localized to mitochondria, and inhibition of its activity can activate mitochondrial oxidative respiration and reduce mitochondrial swelling. Therefore, we want to know whether the loss of CHIP E3 ligase activity mediates the formation of K63 and K27 chain ubiquitination of PDE9A, and whether the reduction and loss of CHIP function accompanied by increased PDE9A aggregation will lead to abnormal mitochondrial functional structure and disruption of mitochondrial autophagy balance. p62, also known as SQSTM1, can connect LC3 and ubiquitinated substrates, which are then incorporated into autophagosomes and degraded in autolysosomes. When autophagy is inhibited, autophagosomes accumulate, p62 levels increase, and LC3B decreases. Coincidentally, in rats with CHIP U-box inactivation mutations, P62 increased and LC3B decreased in the hippocampus and cerebellum tissues. However, after BAY 73-6691 treatment and increased CHIP protein in male rats of the same littermate, P62 decreased while LC3B expression increased ( Figure 7 c, Figure 7 d); Further transmission electron microscopy revealed that compared with rats with CHIP U-box inactivation mutation, the number of mitochondria in the cerebellum tissue was relatively small, the membrane was partially blurred and damaged, the cristae structure was largely broken and dissolved, the matrix was largely dissolved or vacuolated, and autophagosome structures were visible under the microscope; the number of mitochondria in the cerebellum tissue of rats after adding PDE9A inhibitors and increasing the CHIP protein content in brain tissue was relatively abundant, with uneven sizes and relatively intact membranes ( Figure 7 e). This suggests that CHIP reduction and functional loss accompanied by increased PDE9A aggregation disrupt mitochondrial structure and function, inducing mitophagy impairment. BAY 73-66791 inhibits PDE9A aggregation and increases CHIP protein expression, ameliorating mitochondrial damage and maintaining mitophagy balance.

[0116] 6. Single-cell sequencing results suggest that dysregulation of cGMP and cAMP signaling pathways is the main pathway change in CHIP mutations

[0117] Studies have shown that CHIP mutations (SCAR) primarily present with spinocerebellar ataxia and cognitive impairment, suggesting a close involvement of the cerebellum and the hippocampus, which is closely associated with cognition, in the disease. Single-cell RNA sequencing (scRNA) can reveal disease pathology at the single-cell level. Therefore, to clarify the regulatory relationship between PDE9A and CHIP at the single-cell level, this study performed single-cell RNA sequencing on hippocampal and cerebellar tissue from a rat disease model.

[0118] We obtained four groups of scRNA results, including CHIP mutant group (Hom), CHIP overexpression group (AAV-CHIP), PDE9A treatment group (Bay) and normal age-matched male rat group (Wt), capturing 19279, 25238, 23177 and 13473 single cells, respectively ( Figure 9 a). The expression of root marker genes divided 81,167 single cells into 12 cell subpopulations, including granule cells, oligodendrocytes, glutamatergic neurons, GABAergic neurons, GABAergic interneurons, pyramidal cells, oligodendrocyte precursor cells, endothelial cells, Bergmann glia, microglia, Purkinje cells, and astrocytes ( Figure 9 bc). Heat maps show that each cell subpopulation expresses specific genes.

[0119] KEGG enrichment analysis of differentially expressed genes in all cell subpopulations found that the differentially expressed genes in the Hom group compared with the Wt group were enriched in the cAMP signaling pathway, cGMP-PKG signaling pathway ( Figure 10 a). AUCell and GSEA analysis showed that compared with the Wt group, the Hom group showed significant inhibition of the cGMP-PKG signaling pathway and significant activation of the cAMP signaling pathway ( Figure 10 be).

[0120] Purkinje cells are the neuronal subpopulation mainly affected by CHIP mutations. We further explored the changes in Purkinje neurons using single-cell sequencing. Through cell ratio analysis, we found that the proportion of Purkinje cells in the Hom group was significantly lower than that in the Wt group, while the proportion of Purkinje cells increased after Bay and AAV-CHIP treatment, suggesting that CHIP mutations can lead to a decrease in the number of Purkinje cells, and Bay and AAV-CHIP treatment can restore the number of Purkinje cells and improve symptoms to a certain extent. Similar to the overall cell analysis, the KEGG enrichment analysis results of Purkinje cell differential genes showed that the differential genes in the Hom group compared with the WT group were also enriched in ubiquitin-mediated proteolysis, cAMP signaling pathway, spinocerebellar ataxia, cGMP-PKG signaling pathway ( Figure 11 a).

[0121] Previous studies have shown that CHIP regulates the cAMP-PKA pathway. The PKA holoenzyme is composed of two catalytic subunits (PKAc) and two regulatory subunits (PKAr). The PKA holoenzyme is inactive and only activates through the second messenger cAMP, which alters the conformation of the regulatory subunits to release the active subunit PKAc. 27 The catalytic subunit α of protein kinase A (PRKACA) is the predominant isoform of PKAc, expressed in most tissues and primarily used to visualize the activity of PKAc within tissues.

[0122] Previous studies have reported that CHIP can mediate the ubiquitination and degradation of PKAc. To verify the results of single-cell sequencing, we detected increased expression of PRKACA in the cerebellum and hippocampus of CHIP mutant rats ( Figure 12 ab), we further added Forskolin to the ubiquitination reaction of PDE9A and CHIP in vivo to increase cAMP levels by activating adenylate cyclase. The results showed that increasing cAMP concentration reduced CHIP-mediated K63-chain and K27-chain PDE9A ubiquitination, suggesting that cAMP-induced PKAc upregulation may compete with PDE9A for CHIP ubiquitination and inhibit PDE9A degradation ( Figure 12 c).

[0123] 7. AAV-CHIP and the PDE9A inhibitor BAY 73-6691 participate in the bidirectional regulation of cGMP and cAMP signaling and play a therapeutic role

[0124] To determine whether overexpression of CHIP could rescue the pathological changes caused by its mutation, we used mononuclear cell sequencing and found that overexpression of CHIP could eliminate the pathway abnormalities caused by CHIP mutation. That is, compared with the AAV-CHIP group, the activity of the cGMP-PKG signaling pathway in the Hom group was significantly downregulated, while the activity of the cAMP signaling pathway was significantly activated ( Figure 10 bc, Figure 10 fh). At the same time, Purkinje cell pathway activity analysis also supported the above results ( Figure 11 b, Figure 11 In summary, the results once again confirmed that CHIP can activate the cGMP / PKG signaling pathway and inhibit cAMP signaling, thereby maintaining the intracellular cGMP / cAMP homeostasis.

[0125] It is known that CHIP mutations can lead to abnormal formation of K63 and K27 polyubiquitin chains on PDE9A, thereby affecting the degradation of PDE9A in autophagic lysosomes. Moreover, PKG can mediate the reduction of p-CHIP and the reduction of CHIP half-life caused by the increase of PDE9A. In view of the above-mentioned role of PDE9A in CHIP mutations, and the fact that the BAY 73-6691 inhibitor can reduce PDE9A activity, we speculate that BAY 73-6691 may have an ARCA therapeutic effect after CHIP mutation. Therefore, we performed mononuclear cell sequencing on the Hom group treated with BAY 73-6691. The results were consistent with expectations. The Bay group showed a significant therapeutic effect on the Hom group, just like overexpressing CHIP ( Figure 10 c, Figure 10 ik). The results of Purkinje cell analysis showed that the differentially expressed genes in the Bay group compared with the Hom group were mainly enriched in oxidative phosphorylation, cAMP signaling pathway and protein hydrolysis reaction ( Figure 11 c), supporting the involvement of PDE9A in CHIP-mediated ubiquitination reactions, and CHIP may be involved in PDE9A-mediated phosphorylation modifications. It is worth noting that Aucell analysis results showed that the reduction of CHIP's E3 ubiquitin ligase activity led to a decrease in CHIP expression levels, which in turn affected the proteolytic reaction mediated by CHIP as an E3 ligase, while increasing intracellular cAMP activity. The activation of the ubiquitin-mediated proteolytic reaction mediated by CHIP was inhibited, and the corresponding PDE9A protein was inhibited. The decrease in PDE9A promoted autophagy and mitochondrial autophagy ( Figure 11 fh). These results support that CHIP promotes the degradation of PDE9A in autophagolysosomes through K63 and K27 chain ubiquitination, while cAMP competitively inhibits the ubiquitination of PDE9A. In addition, after BAY 73-6691 treatment, the cGMP-PKG signaling pathway and oxidative phosphorylation were significantly activated ( Figure 11 c, Figure 11 i) supports that PDE9A inhibits CHIP-S19 phosphorylation and reduces the levels of CHIP and p-CHIP in Purkinje cells.

[0126] To comprehensively assess the effects of CHIP mutation, CHIP overexpression, and BAY 73-6691 treatment on the animal model, we used heat maps to identify genes involved in the cAMP signaling pathway, the cGMP-PKG signaling pathway, oxidative phosphorylation, ubiquitin-mediated proteolysis, mitophagy, and autophagy pathways. Furthermore, compared to the Hom group, the wild-type, CHIP overexpression, and Bay 73-6691 treatment groups showed high consistency. These results are highly consistent with previous findings and further confirm that PDE9A and CHIP are potential therapeutic targets for ARCA.

[0127] It is known that cGMP, PKG1, and PKG2 are key molecules in the cGMP-PKG signaling pathway, while cAMP and PRKACA are key molecules in the cAMP signaling pathway. To verify the accuracy of the above conclusions, we further detected these key molecules in the cerebellum and hippocampus of each rat group using Western blotting. The results showed that CHIP, cGMP, PKG1, and PKG2 were decreased in the Hom group, while PDE9A was increased. After treatment with overexpression of CHIP and Bay, the levels of these proteins were reversed ( Figure 12 dg); In addition, cAMP and PRKACA increased significantly in the Hom group, but this increase was downregulated with the increase of CHIP protein and the inhibition of PDE9A ( Figure 12 ab), again confirming that CHIP ubiquitination reduces PKAc expression in CHIP-inactivated mutant animals, and PKAc competitively increases PDE9A protein. In summary, the above results effectively suggest that CHIP can mediate the interaction between cGMP and cAMP signals by reducing the affinity of PDE9A for hydrolyzing cGMP.

[0128] At the same time, we compared the pathogenic pathways of other cell subpopulations with those of Purkinje cells. The results showed that differentially expressed genes in five cell subpopulations—granule cells, Bergmann glia, glutamatergic neurons, oligodendrocyte precursor cells, and oligodendrocytes—were significantly enriched in the cAMP and cGMP-PKG signaling pathways, demonstrating pathway impairments similar to those in Purkinje cells. Although overexpression of Bay and CHIP abolished abnormal changes in some cell subpopulations, AUCell analysis only supported similar alterations in these pathways between granule cells and Purkinje cells, while these changes were not significant in other cell subpopulations, suggesting that different biological mechanisms may be involved.

[0129] In summary, the mononuclear cell sequencing results not only support the fact that CHIP mutation leads to abnormal formation of polyubiquitin chains at K63 and K27 on PDE9A and affects the degradation of PDE9A in autophagic lysosomes, but also that PKG mediates the increase of PDE9A, leading to a decrease in p-CHIP and a decrease in CHIP half-life. It also confirms that CHIP overexpression and BAY 73-6691 can improve the imbalance of the intracellular cGMP / cAMP pathway, thereby having potential therapeutic and brain protective effects on ARCA.

[0130] Implementation effect examples

[0131] The ability of CHIP to enhance the clearance of damaged / misfolded proteins and inhibit proteotoxicity is associated with immunity, aging, metabolic stress, and numerous human diseases, including cardiac and neurological disorders. Biallelic CHIP mutations are the direct cause of ARCA. Here, we reveal that upregulation of PDE9A protein expression is a key pathological mechanism underlying CHIP mutations. CHIP and PDE9A mutually regulate expression and interact, an interaction mediated by the CHIP TPR domain. The CHIP TPR domain likely binds to Hsp70 to link PDE9A, while the CHIP U-box domain recruits K63- and K27-linked ubiquitination modifications to PDE9A, mediating its autophagic and lysosomal degradation.

[0132] CHIP mutations prevent PDE9A from undergoing autophagic degradation and instead accumulate in the cell. Increased PDE9A protein hydrolyzes cGMP, inhibiting CHIP phosphorylation at the S20 site by PKGs and reducing CHIP protein half-life, leading to a decrease in CHIP protein. Simultaneously, CHIP ubiquitination by PKAc is reduced, which in turn enhances cAMP signaling. Upregulated cAMP further increases PKAc levels, competitively inhibiting PDE9A degradation through ubiquitination. Abnormal PDE9A aggregation and decreased CHIP protein disrupt the balance of mitochondrial autophagy, ultimately leading to cell death. Inhibition of PDE9A activity with the PDE9A inhibitor BAY 73-6691 can ameliorate these pathological outcomes. Given the important role of CHIP in cardiac, skeletal muscle, neurodevelopment, and tumorigenesis, our results have therapeutic implications and suggest that PDE9A inhibitors are a promising potential therapeutic approach for CHIP mutation-associated ataxias.

[0133] Our study also found that CHIP plays an important role in maintaining the balance of cAMP and cGMP. Using single-cell sequencing technology, we found that in the CHIP mutation model, the cGMP-PKG signaling interaction was in an inhibitory state, while the cAMP signaling pathway was in an activated state. Exogenous overexpression of CHIP or the application of PDE9A inhibitors can upregulate CHIP expression, increase cGMP, PKG1 and PKG2, and decrease PDE9A. With the increase of CHIP protein and the inhibition of PDE9A activity, cAMP and PKA were significantly downregulated. Our results suggest that the interaction between CHIP and PDE9A is a potential connecting molecule for the bidirectional regulation of cGMP and cAMP pathways ( Figure 13 ).

[0134] The signaling interactions between the intracellular second messengers cAMP and cGMP play a crucial role in cell signal transduction and maintaining normal biological function. cAMP can stimulate protein kinase A (PKA), opening calcium channels and initiating cAMP-activated enzyme exchange protein (Epac), activating downstream pathways. cGMP, as a second messenger, induces PKG, whose expression is limited to vascular tissue, lung, and brain tissue. In neurons, cAMP and cGMP can mediate antagonistic cell behaviors induced by in vitro factors, such as ion channel regulation, cell volume regulation, and axon guidance. Local cAMP and cGMP in the undifferentiated axons of neurons can promote and inhibit axon formation, respectively, and they also have opposing effects during dendrite formation. These two signaling pathways antagonize and restrict each other, jointly regulating the normal physiological function of cells. In cardiomyocytes, cAMP hydrolysis is regulated by cGMP. In PDE2, cGMP stimulates cAMP hydrolysis through allosteric stimulation; in PDE3, cGMP competitively inhibits cAMP hydrolysis. However, PDE9A is a specific and selective inhibitor of cGMP, selectively hydrolyzing only cGMP and inhibiting the cGMP / PKG pathway. Therefore, in neurons, CHIP is crucial for PDE9A's bidirectional regulation of cGMP and cAMP, mediating their interaction. Inhibiting PDE9A activity with BAY 73-6691 can maintain the balance of CHIP-mediated cGMP / cAMP signaling and improve the pathological changes in ARCA brain injury caused by impaired mitochondrial autophagy in Purkinje cells.

[0135] ARCA presents diverse pathological manifestations, the most common of which include neuronal atrophy and degeneration, demyelination, and Purkinje cell loss, coupled with glial proliferation, leading to widespread degeneration of the cerebellar hemispheres, vermis, and middle and inferior cerebellar peduncle. Therefore, in an animal model of ARCA, we first demonstrated that CHIP and PDE9A in Purkinje cells could be therapeutic targets for ARCA. To further investigate the role of CHIP in ARCA pathology, we used mononuclear cell subset analysis to investigate molecular changes in different cell types in the cerebellum and hippocampus. Although we found that granule cells share similar pathogenic mechanisms with Purkinje cells and can be ablated by BAY73-6691 and CHIP overexpression, granule cells are widely distributed in the hippocampus and cerebellum and play an important role in maintaining hippocampal cognitive function and cerebellar homeostasis, we propose that neuronal abnormalities, including granule and Purkinje cells, play a major role in ARCA caused by CHIP mutations, complementing previous studies. In addition, several glial cells, such as Bergmann glia, oligodendrocyte precursor cells, oligodendrocytes, and glutamatergic neurons, exhibited different pathway activity from that of Purkinje cells, suggesting that different pathological mechanisms may be involved. These findings may be related to the pathogenesis of other CHIP-mediated diseases previously reported.

[0136] In recent years, PDE9 inhibitors have been widely used in research related to neurodegenerative diseases. Among them, PF-04447943 has entered Phase II clinical trials for Alzheimer's disease; BI-409306 has entered Phase II clinical trials for Alzheimer's disease and schizophrenia. However, the earlier discovered PDE9A inhibitor Bay 736691 is still in the early stages of preclinical development, and it is unclear whether BAY 73-6691 is suitable for human trials or will remain a laboratory research tool. This study demonstrates that the PDE9A inhibitor BAY 73-6691 can ameliorate neurological damage in a CHIP mutation model through a dual ubiquitin phosphorylation pathway, potentially becoming a candidate drug for the treatment of CHIP-related diseases, providing a new approach for the treatment of related diseases, particularly CHIP mutation ataxia.

[0137] Therefore, it is obvious to those skilled in the art that pharmaceutically acceptable salts of BAY 73-6691 can also achieve the above-mentioned technical effects. The pharmaceutically acceptable salts of BAY 73-6691 are any one of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, propionate, hexanoate, heptanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate, 3-phenylpropionate, pivalate, tert-butylacetate, dodecyl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, and stearate.

[0138] The above acids are all common acid substances in the human body, so the salts containing these acids are easily absorbed by the human body and then exert their effects.

[0139] This study also suggests that the PDE9A inhibitor BAY 73-6691 regulates the cGMP / cAMP pathway in the central nervous system and participates in regulating the CHIP-related protein quality control system, explaining the possible new drug mechanism and target of the PDE9A inhibitor BAY 73-6691.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of BAY 73-6691 and / or a pharmaceutically acceptable salt of BAY 73-6691 in the preparation of a medicament for treating autosomal recessive spinocerebellar ataxia, wherein the structural formula of BAY 73-6691 is: 。 2. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt of BAY 73-6691 is any one of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, propionate, hexanoate, heptanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate, 3-phenylpropionate, pivalate, tert-butylacetate, dodecyl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate and stearate.

3. The use according to claim 2, characterized in that: The autosomal recessive spinocerebellar ataxia is caused by inactivating mutations in CHIP.

4. The use according to claim 3, characterized in that: CHIP inactivating mutations lead to increased PDE9A expression in ARCA brain tissue, and the PDE9A content increases progressively as the disease progresses.

5. The use according to claim 4, characterized in that: The autosomal recessive spinocerebellar ataxia is caused by a disorder of mitochondrial autophagy in Purkinje cells.

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