CN115463215A - New use of HDAC9 and inhibitors thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, particularly relates to HDAC9 and a new application of an inhibitor thereof, and particularly discloses an application of deacetylase HDAC9 as an inhibition target in treating dysmnesia. Application of an activity inhibitor of deacetylase HDAC9 in preparing medicines for treating dysmnesia diseases. The application of a CaM acetylation level detection reagent or an HDAC9 expression level detection reagent in the preparation of Alzheimer disease detection products. The invention mainly aims at the pathological mechanism of dysmnesia (especially AD dysmnesia), effectively increases the acetylation level of AD cerebral neuron Calmodulin (Calmodulin, caM) and the phosphorylation level of CaMKII alpha/GluR 1, enhances the relevant learning and memory, and further relieves the AD dysmnesia.

Description

New use of HDAC9 and inhibitors thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to novel application of HDAC9 and an inhibitor thereof.

Background

There is currently a large search for regulatory formulations of CaM acetylation as well as non-histone deacetylation substrates of HDAC 9. The CaM acetylation regulation preparation also comprises medical application and non-medical application, the preparation for medical application is mainly used for treating CaM related diseases, and the preparation for non-medical application can be used as an in-vitro regulation preparation, such as biological agents and the like. Although some existing researches provide some new products capable of promoting acetylation or deacetylation of CaM, the researches are still in short and need to be perfected.

CaM acetylation disorder and the like may also cause memory disorders. Memory disorders are manifested clinically as memory disorders, loss of speech, misuse, loss of recognition, impairment of spatial abilities, impairment of abstract thinking, personality and behavioral changes, etc., which have a variety of different conditions, including memory disorder diseases including spatial memory disorders, new object recognition disorders and scenic memory disorders.

The memory disorder may cause alzheimer's disease, which is also a cause of the memory disorder. At present, the study on dysmnesia and Alzheimer disease causes is particularly important, and the memory function of patients is weakened, which is the most prominent in the elderly. Such as memory decline, depression or affective disorders, are common to most patients and therefore the condition is seen in patients of different ages.

Alzheimer's Disease (AD) is a degenerative disorder of the central nervous system that occurs in the elderly or pre-senile stages and is characterized by progressive cognitive and behavioral disorders. In the past decades, clinical trials for AD have largely failed, new drugs for AD have been developed slowly, and the current drugs for treating AD memory disorder have several fingers. Memantine hydrochloride is a non-competitive NMDA receptor antagonist, and can be used for properly improving AD cognitive impairment; cholinesterase inhibitors (donepezil, rivastigmine, galantamine, huperzine a) can properly improve AD cognitive impairment; aducazumab (aducanumab) is used to treat AD by clearing a β deposits; the first AD medicament in China, namely the Manttner (GV-971), treats AD by improving intestinal flora. At present, the specific pathogenesis of AD dysmnesia is complex and diverse, and an effective treatment means is lacking clinically. The existing AD medicines in the market have the defects of poor curative effect, large side effect and the like. Aducaumab is very expensive, has a low prevalence rate, and has the side effect of cerebral edema. The mannotus sodium GV-971 has little curative effect, improves intestinal flora and treats AD, and has unknown mechanism.

The research on the memory disorder is still deficient, and some treatment means are still to be perfected. Meanwhile, the in vitro acetylation reagent of CaM also needs to be further researched and perfected.

Disclosure of Invention

In order to solve the problems, the invention adopts the following technical scheme:

use of the deacetylase HDAC9 as an inhibitory target in the treatment of memory disorders.

Application of an activity inhibitor of deacetylase HDAC9 in preparing medicines for treating dysmnesia diseases.

In some approaches, the memory impairment disorders include spatial memory impairment, new object recognition impairment, and scene memory impairment.

In some forms, the memory impairment disorder comprises AD memory impairment.

In some ways, the effect of inhibitors of deacetylase HDAC9 activity is manifested by

A. The acetylation of the CaM is promoted,

B. promote the downstream CaMKII alpha phosphorylation of CaM,

C. promotes downstream GluR1 phosphorylation of CaMKII alpha.

In some forms, the inhibitor of deacetylase HDAC9 activity comprises TMP269.

The application of a CaM acetylation level detection reagent or an HDAC9 activity level detection reagent in the preparation of Alzheimer disease detection products.

In some forms, the alzheimer's disease is an AD memory disorder.

Use of an inhibitor of HDAC9 activity for the preparation of a medicament for the treatment of a condition induced by deacetylation of CaM protein.

Use of HDAC9 for the preparation of an in vitro CaM protein deacetylation preparation for non-diagnostic therapy.

In some forms, the CaM protein is His-Ac-3K-CaM.

The invention has the beneficial effects that:

the invention mainly aims at the pathological mechanism of dysmnesia (especially AD dysmnesia), effectively increases the acetylation level of AD cerebral neuron Calmodulin (Calmodulin, caM) and the phosphorylation level of CaMKII alpha/GluR 1, enhances the relevant learning and memory, and further relieves the AD dysmnesia.

Drawings

FIG. 1 is a pathological mechanism of abnormal calmodulin acetylation reducing involvement in Alzheimer's disease;

FIG. 2 is a graph showing the result of the acetylation degree of CaM in plasma of APP/PS1 transgenic mice;

FIG. 3 is a graph showing the result of the acetylation degree of CaM in the forebrain of AD mice;

FIG. 4 is a graph showing the results of deacetylation of CaM in HEK293 cells by HDAC 9;

FIG. 5 is a graph showing the results of in vitro direct deacetylation of CaM by HDAC 9;

FIG. 6 is a graph showing the results of increasing CaM acetylation and CaMKII α/GluR1 phosphorylation in AD mice by HDAC9 inhibitors;

FIG. 7 is a graph showing the results of HDAC9 inhibitors improving hippocampal-dependent spatial memory and recognition of novel objects;

fig. 8 is a graph of the results of HDAC9 inhibitors improving hippocampal-dependent scene memory.

Detailed Description

The invention is further illustrated below:

first aspect of this section：

The application of deacetylase HDAC9 as an inhibition target in treating memory disorder can realize the alleviation of memory disorder by inhibiting the activity of HDAC 9.

Application of an activity inhibitor of deacetylase HDAC9 in preparation of medicines for treating dysmnesia diseases.

One of the memory disorder diseases comprises spatial memory disorder, new object recognition disorder and scene memory disorder, wherein the spatial memory disorder, the new object recognition disorder and the scene memory disorder are common memory disorders, and the spatial memory disorder and the scene memory disorder are expressed as functional abnormalities of information of a spatial direction and a scene environment. The symptoms of dementia, brain trauma, stress-induced dysmnesia and Coxsacket syndrome can all present with dysmnesia.

In another aspect, the memory impairment condition comprises AD memory impairment, which is a symptom primarily caused by dementia.

The effect of the inhibitor of the expression of the acetylase HDAC9 is shown in

A. The acetylation of the CaM is promoted,

B. promote the downstream CaMKII alpha phosphorylation of CaM,

C. promoting phosphorylation of GluR1 at the downstream of CaMKII alpha.

Thus, HDAC9 has therapeutic effects on memory disorders due to abnormal reduction in CaM acetylation, abnormal reduction in CaM downstream CaMKII α phosphorylation, and abnormal reduction in CaM downstream GluR1 phosphorylation. Wherein promoting comprises raising the level, and maintaining the normal level.

In some embodiments, the inhibitor of deacetylase HDAC9 activity comprises TMP269.

Second aspect of this section：

The application of the CaM acetylation level detection reagent or the HDAC9 expression level detection reagent in the preparation of Alzheimer disease detection products. When the expression level of HDAC9 is increased, the acetylation level of CaM is reduced, and the two are in negative correlation, so that the detection of the Alzheimer disease can also be realized by detecting the expression level of HDAC9 or the acetylation level of CaM.

Wherein the Alzheimer's disease is AD memory impairment. When HDAC9 expression levels were too high, caM acetylation levels decreased, both characterizing AD memory impairment.

Introduction to the third aspect of this section：

Application of HDAC9 in preparation of in vitro CaM protein acetylation preparation in non-diagnostic treatment. The existing products basically need to realize the acetylation of CaM protein in vivo for regulation, but the invention provides that the deacetylation of CaM can be realized in vitro directly through HDAC 9.

In some forms, the CaM protein is any one of His-Ac-3K-CaM, his-Ac-K22/K95/K116-CaM.

Application of HDAC9 activity inhibitor in preparation of medicines for treating diseases induced by CaM protein acetylation abnormality. The CaM protein acetylation abnormality induced symptoms comprise existing proved related symptoms, and reference can be made to the prior technical data.

The fourth aspect of this section will be further described with reference to specific research projects：

The AD genetic model APP/PS1 mice had significantly reduced plasma and brain CaM acetylation levels relative to wild-type WT mice, and the first identification of CaM deacetylase HDAC9, and inhibition of HDAC9 activity could rescue the reduced brain CaM acetylation and CaMKII α/GluR1 phosphorylation and impaired learning memory in AD mice. Based on the experimental result of APP/PS1 transgenic AD mice, abnormal acetylation of calmodulin is reduced to participate in the pathological mechanism of Alzheimer disease: abnormal reduction of CaM acetylation in AD mice brain compromises CaMKII α/GluR1 phosphorylation signaling pathways, leading to brain learning and memory impairment, ultimately leading to pathological progression of alzheimer's disease (see figure 1).

1. Abnormal reduction of plasma and brain CaM acetylation in AD mice

The level of acetylation of CaM in the plasma of 7-month-old WT and APP/PS1 transgenic mice (AD mouse genetic model) was determined by enriching total acetylated proteins in the plasma by immunoprecipitation with a pan-acetylated antibody (Ac-K antibody) and detecting the amount of acetylated CaM (Ac-CaM) in the total acetylated proteins with CaM monoclonal antibody.

As a result, it was found that plasma CaM acetylation levels were significantly decreased in APP/PS1 mice relative to WT mice (A and B in FIG. 2), and that plasma CaM acetylation decreased already in APP/PS1 mice at 5 months of age (C and D in FIG. 2), at which time memory impairment phenotypes were not observed in AD model mice, while plasma CaM acetylation levels were not significantly changed in 3-month-old mice (E and F in FIG. 2). These results suggest that abnormal reduction in plasma CaM acetylation may be a potential early plasma diagnostic marker for alzheimer's disease.

FIG. 2 is a schematic diagram: caM acetylation of APP/PS1 transgenic mice plasma is obviously reduced. A. C, E, western blot bands of CaM acetylation levels in plasma of 7-month, 5-month, 3-month WT and APP/PS1 transgenic mice, respectively. B. D and F are quantitative analysis of Ac-CaM/CaM in graphs A, C and E, respectively, and normalization was performed with reference to control group 1. * P <0.05; * P <0.01; t-test; n =3. Data are presented as mean ± sem.

The level of activation of CaM/CaMKII alpha/GluR 1 signal transduction in forebrain tissue of 7-month-old WT and APP/PS1 mice was measured (A in FIG. 3), and as a result, it was found that in APP/PS1 mice, caM acetylation in forebrain tissue was significantly reduced (B in FIG. 3), caMKII alpha phosphorylation downstream of CaM and GluR1 phosphorylation downstream of CaMKII alpha were significantly reduced (C and D in FIG. 3), and total protein of CaM, caMKII alpha, gluR1, PSD95 and CaM acetyltransferase SRC3 was relatively unchanged (E, F, G, H and I in FIG. 3), compared to WT mice. These results suggest that abnormal reduction of brain CaM acetylation promotes the pathological progression of alzheimer's disease by compromising the CaMKII α/GluR1 signaling pathway.

FIG. 3: AD mice had reduced acetylation of the forebrain CaM. A, control and experimental groups of forebrain tissue protein western blot bands. B. C and D, quantitative analysis of Ac-CaM/CaM, p-CaMKII alpha/CaMKII alpha and p-GluR1/GluR1, respectively. E. F and G, quantitative analysis of CaM, caMKII alpha, gluR 1/alpha-tubulin, respectively. H and I, quantitative analysis of PSD95 and SRC3/α -tubulin, respectively. Normalization was performed with control 1. ns, not significant; * P <0.05; * P <0.01; * P <0.001; t-test; n =4. Data are expressed as mean ± sem.

2. Screening and identification of CaM deacetylase HDAC9

shRNA interference was performed on several cytoplasmic HDACs in HEK293 cells. As a positive control, treatment with the HDAC inhibitor TSA significantly increased Ac-CaM (a and B in fig. 4). The knockdown efficiency of HDAC shRNAs was verified by Western blotting (A, C-G in FIG. 4). HDAC6 shRNA significantly increased the level of α -tubulin acetylation (A and H in FIG. 4), which is consistent with the previous report, as another positive control. Both HDAC9 and HDAC6 shRNA increased CaM acetylation, with HDAC9 being the most predominant deacetylase of CaM, while HDAC4, HDAC7, HDAC8 shRNA did not significantly exhibit increased CaM acetylation (a and B in fig. 4). As a negative control, HDAC9 shRNA did not increase α -tubulin acetylation (a and H in fig. 4), indicating the specificity of HDAC9 deacetylation CaM.

FIG. 4: HDAC9 is the CaM major deacetylase. A, HEK293 cell lysates were probed with the corresponding antibodies, respectively, under different treatments or shRNA transfection. TSA, HDAC inhibitor, used as positive control. Increasing α -tubulin acetylation by HDAC6 shRNA is another positive control. B-H, are statistical plots for each of the groups Ac-CaM/CaM, HDAC4/α -tubulin, HDAC6/α -tubulin, HDAC7/α -tubulin, HDAC8/α -tubulin, HDAC9/α -tubulin, and Ac-tubulin/α -tubulin, respectively. Data were normalized to control 1. * P <0.01; * P <0.001; one-way ANOVA; n =3. Data are presented as mean ± sem.

CaM has three lysine sites (K22, K95 and K116) that can be acetylated and is extremely conserved across species (a in fig. 5). To verify whether HDAC9 could directly deacetylate CaM in vitro, site-directed acetylation of recombinant CaM proteins was purified using a genetic codon expansion method (B in fig. 5). The method uses an engineered pyrrolysinyl pyrrolosyl-tRNA synthetase specific for acetylated lysine and its cognate tRNAPry to incorporate Ac-K into designated codons to produce a site-specific acetylated protein. To determine which of the three lysine sites in CaM was acetylated, site-specific antibodies were prepared against CaM acetylation at the K22, K95 and K116 sites. As shown in C in figure 5, three sites of acetylated Ac-3K-CaM and Ac-K22, K95 and K116-CaM were successfully synthesized by pan-acetylated antibody and site-specific antibody detection. Next, the expression of purified site-specific acetylated His-Ac-3K-CaM and His-Ac-K22/K95/K116-CaM was incubated with Flag-tagged HDAC9 purified from HEK293 cells by Flag magnetic beads, indicating that HDAC9 could directly deacetylate three acetylated sites at positions K22, K95 and K116 of CaM in vitro. This is the first discovery and identification of CaM deacetylase HDAC9, and CaM is also the first non-histone deacetylation substrate of HDAC9 found so far, and also fills the blank of research on non-histone deacetylation substrate of HDAC 9.

FIG. 5 is a schematic view of: HDAC9 deacetylates CaM directly in vitro. A, top, shows the position of the acetyl lysine in mouse CaM, EF hands, numbers represent amino acids. Bottom, alignment of amino acid sequences of CaM from different species. The red K represents lysine that can undergo acetylation. B, top, pCDF construction vector schematic for expression of His-tagged acetylated CaM recombinant protein in e. And at the bottom, synthesizing a schematic diagram of a genetic codon expansion method of the site-specific acetylated recombinant CaM protein. And C, expressing a western blot band of acetylated CaM proteins (His-Ac-3K-CaM and His-Ac-K22/K95/K116-CaM) with His tags in Escherichia coli, wherein HDAC9 can directly deacetylate three acetylated sites of K22, K95 and K116 of the CaM in vitro.

3. Mechanism of action of inhibiting HDAC9 in the treatment of AD memory disorders

The reduction in CaM acetylation in mice, a model of Alzheimer's disease, is caused by an increase in the expression of its deacetylase HDAC 9.HDAC9 was abnormally elevated and lowered in brains of APP/PS1 mice at 5-7 and 12 months of age, respectively (A and B in FIG. 6). The experiment shows that by injecting HDAC9 inhibitor TMP269 into lateral ventricle of APP/PS1 transgenic AD mice, as shown in C-D in FIG. 6, morris Water Maze (MWM) learning training can enhance the acetylation level of CaM in forebrain tissue of AD mice, while by injecting HDAC9 inhibitor group (HDAC 9 inhibitor, HDAC9 i) into lateral ventricle compared with Vehicle group, HDAC9 i can further enhance the acetylation level of CaM in forebrain tissue of AD mice, and the result shows that HDAC9 can deacetylate CaM in vivo in forebrain of AD mice. Previous studies showed that CaM acetylation can enhance CaMKII α activation, and CaMKII α phosphorylation, which is an indicator of CaMKII α activation, was detected using an antibody specific for CaMKII α Thr286 site phosphorylation, as shown by C and E in fig. 6, with elevated CaMKII α phosphorylation consistent with increased CaM acetylation. CaMKII alpha can phosphorylate the GluR1 Ser831 site of AMPA receptor subunit. Consistent with increased CaMKII α phosphorylation, p-GluR1 Ser831 was also further increased during MWM learning training in HDAC9 i group AD mice (C and F in fig. 6). These results indicate that inhibition of HDAC9 can improve both the CaM acetylation and CaMKII α/GluR1 phosphorylation signaling cascades involved in water maze learning stimulus activation in AD mice.

FIG. 6: HDAC9 inhibitors increased CaM acetylation and CaMKII α/GluR1 phosphorylation in AD mice. A, representative blot band, dynamically expressed HDAC9 protein level in the forebrain of APP/PS1 mice. B, statistical map of HDAC9/α -tubulin. C, the water maze MWM training can increase the levels of the APP/PS1 transgenic AD mice forebrain Ac-CaM, p-CaMKII alpha and p-GluR1, and compared with the Vehicle group through lateral ventricle injection HDAC9 inhibitor (HDAC 9 i), the HDAC9 i can further enhance the levels of the mice forebrain Ac-CaM, p-CaMKII alpha and p-GluR 1. D-F is the quantitative analysis of Ac-CaM/CaM, p-CaMKII alpha/CaMKII alpha and p-GluR1/GluR1 respectively. Data were normalized to control 1. * P <0.01; * P <0.001; one-way ANOVA; n =4. Data are presented as mean ± sem.

4. Effect of inhibiting HDAC9 on treating AD memory disorder

HDAC9 inhibitors can improve learning and memory ability of APP/PS1 transgenic AD mice depending on hippocampus. Spatial memory of hippocampal dependence was studied using the Morris Water Maze (MWM) behavioral paradigm, which is the golden index for the detection of dysmnesia in AD animal models. During MWM training, the learning curve of HDAC9 i group AD mice was enhanced compared to the Vehicle group by lateral ventricle injection of HDAC9 inhibitor group (HDAC 9 i) (a in fig. 7). During the plateau test, HDAC9 i group AD mice had increased residence time in the target quadrant and plateau shuttle times compared to the Vehicle group (B-D in fig. 7), which was similar to the swim speed of the Vehicle and HDAC9 i group AD mice (E in fig. 7). These results indicate that HDAC9 inhibitors can improve spatial learning and memory ability dependent on hippocampus of AD mice. Next, it was investigated whether HDAC9 inhibitors could improve New Object Recognition (NOR) ability of AD mice, which is a hippocampal-dependent short-term memory behavior paradigm that is a gold index for detecting dysmnesia in AD animal models. The discrimination index was significantly enhanced in HDAC9 i group AD mice compared to Vehicle group AD mice, which had similar total search times around new and familiar objects (F-G in fig. 7). These results indicate that HDAC9 inhibitors can improve hippocampal-dependent short-term memory in AD mice. The results show that inhibiting HDAC9 can improve learning and memory ability of AD mice depending on hippocampus.

FIG. 7: HDAC9 inhibitors improve hippocampal-dependent learning and memory. A, during MWM training, the learning curve of HDAC9 i group AD mice was enhanced compared to the Vehicle group by lateral ventricle injection HDAC9 inhibitor group (HDAC 9 i). * Genotype F (1,170) =8.842, p =0.0041, two-way ANOVA, n =15. Data are presented as mean ± sem. Swimming trajectories during platform testing of AD mice in the Vehicle and HDAC9 i groups. C, increased time spent in target quadrant in HDAC9 i group AD mice compared to Vehicle group AD mice. * P =0.0216, two-way ANOVA, n =15. Data are presented as mean ± sem. D, increased number of platform crossings during the HDAC9 i group AD mouse platform test compared to the Vehicle group AD mice. E, swimming speed of AD mice in the Vehicle group and the HDAC9 i group is similar. F, lateral ventricle injection Vehicle and HDAC9 i group AD mice heat maps of the head around new (N) and familiar (F) objects. Similar total search times with N and F between AD mice in the Vehicle group and HDAC9 i group. H, recognition index statistics indicate impaired recognition of new objects by HDAC9 i group AD mice. ns, not significant; * P <0.05; * P <0.001; t-test; n =15. Data are expressed as mean ± sem.

Finally, whether the memory defect of the APP/PS1 mouse in scene fear memory can be saved by HDAC9 inhibition is researched, and the scene fear memory is a scene memory behavior paradigm of hippocampal dependence and is a golden index for detecting AD animal model memory disorder. The mice were subjected to scene conditioned fear memory training (CS, conditioned stimulus, using a background-specific box; US, unconditioned stimulus, using a 0.5mA plantar shock), and after one day, the freezing time (fear memory) was tested when the mice returned to the shock box (a in fig. 8). 7-month-old WT and APP/PS1 mice treated with intraventricular injection of solvent or HDAC9 inhibitor TMP269 were subjected to a scene fear training and testing protocol (B in fig. 8). In WT or APP/PS1 mice, the freezing times during exploration and immediately after training were similar (C in fig. 8). Whereas WT or APP/PS1 mice injected with TMP269 during the one day post test increased freezing time (fear memory) (C in fig. 8), indicating that HDAC9 inhibition rescued the scene fear memory of APP/PS1 mice. On the other hand, HDAC9 inhibition enhanced the scene fear memory of WT mice (C in fig. 8). Taken together, these results indicate that HDAC9 inhibition ameliorates hippocampal-dependent memory deficits in APP/PS1 mice.

FIG. 8: HDAC9 inhibition improves contextual fear memory in 7-month-old AD mice. A, a scene fear condition reflection test schematic diagram. CS, conditioned stimulus, boxes with a specific background; US, unconventionalized stimulus, using a 0.5mA foot shock. B, schematic representation of behavioral grouping and testing. C, lateral ventricle injection of HDAC9 inhibitors enhanced or rescued the freezing time (fear memory) of WT or APP/PS1 mice, respectively, during the pairing test. * P <0.01, two-way ANOVA closed by Sidak's multiple complexes test, 12 mice per group, data are expressed as mean. + -. Standard error.

The pathological mechanism of CaM deacetylation mediated by HDAC9 in Alzheimer disease provides a new molecular target for treating Alzheimer disease. The invention mainly aims at the pathological mechanism of AD dysmnesia, effectively increases the acetylation level of AD cerebral neuron Calmodulin (CaM) and the phosphorylation level of CaMKII alpha/GluR 1, enhances the relevant learning and memory, and further relieves the AD dysmnesia.

It will be apparent to those skilled in the art that various modifications to the above embodiments can be made without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims (10)

1. Use of the deacetylase HDAC9 as an inhibitory target in the treatment of memory disorders.

2. Application of an activity inhibitor of deacetylase HDAC9 in preparation of medicines for treating dysmnesia diseases.

3. The use of claim 2, wherein the memory impairment diseases comprise spatial memory impairment, neo-object recognition impairment and scene memory impairment.

4. The use of claim 2, wherein the memory impairment disorder comprises AD memory impairment.

5. The use according to any of claims 2 to 4, wherein the inhibitor of deacetylase HDAC9 activity is indicated by the action

A. The acetylation of the CaM is promoted,

B. promote the downstream CaMKII alpha phosphorylation of CaM,

C. promotes downstream GluR1 phosphorylation of CaMKII alpha.

6. The use according to any one of claims 2 to 4, wherein the inhibitor of deacetylase HDAC9 activity comprises TMP269.

And 7, application of the CaM acetylation level detection reagent or the HDAC9 expression level detection reagent in preparation of Alzheimer disease detection products.

8. The use according to claim 7, wherein the Alzheimer's disease is AD memory impairment.

The application of HDAC9 in preparing in-vitro CaM protein acetylation preparations for non-diagnosis and treatment, or the application of HDAC9 activity inhibitors in preparing medicines for treating diseases induced by abnormal CaM protein acetylation.

10. The use of claim 9, wherein the CaM protein is any one of His-Ac-3K-CaM, his-Ac-K22/K95/K116-CaM.

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