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 uses of HDAC9 and its inhibitors

technical field

The invention belongs to the field of biotechnology, and in particular relates to the new application of HDAC9 and its inhibitor.

Background technique

At present, there is still a lot of research space for CaM acetylation regulators and non-histone deacetylation substrates of HDAC9. CaM acetylation regulating preparations also include medical and non-medical purposes. Medicinal preparations are mainly used to treat CaM-related diseases, while non-medical preparations can be used as in vitro regulating preparations, such as biological agents. Although some existing studies have provided some new products that can promote acetylation or deacetylation of CaM, they are still lacking and need to be improved.

CaM acetylation disorders may also cause memory impairment. Memory impairment is clinically manifested as memory impairment, aphasia, apraxia, agnosia, impairment of spatial ability, impairment of abstract thinking, personality and behavior changes, etc. It has a variety of different symptoms, including memory impairment diseases including spatial memory impairment, new objects Disorders of recognition and impairment of scene memory.

Memory impairment may lead to Alzheimer's disease, and Alzheimer's disease is also the cause of memory impairment. At present, it is particularly important to study the etiology of memory impairment and Alzheimer's disease. The memory function of patients is impaired, and the performance of the elderly is the most prominent. For example, memory loss, depression, or emotional disorders are common to most patients. Therefore, symptoms depend on the conditions of patients in different age groups.

Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs in old age or presenile age and is characterized by progressive cognitive and behavioral impairments. In the past few decades, most AD clinical trials have failed, and the development of new AD drugs has been slow. Currently, there are only a handful of drugs for the treatment of AD memory impairment. Memantine hydrochloride is a non-competitive NMDA receptor antagonist, which can appropriately improve AD cognitive impairment; cholinesterase inhibitors (donepezil, rivastigmine, galantamine, huperzine A) can appropriately improve AD cognitive impairment ; Aducanumab (aducanumab) is used to treat AD by clearing Aβ deposits; the first domestic AD drug, mannoturner (GV-971), treats AD by improving intestinal flora. At present, the specific pathogenesis of AD memory impairment is complex and diverse, and there is a lack of effective treatment methods in clinical practice. AD drugs currently on the market have shortcomings such as poor curative effect and large side effects. Aducanumab (aducanumab) is very expensive, has a low penetration rate, and has side effects of cerebral edema. Manna Turner GV-971 has little effect, and improves intestinal flora to treat AD. The mechanism is unknown.

Research on memory impairment is still lacking, and some treatments need to be perfected. At the same time, the in vitro acetylation reagents of CaM also need to be further studied and perfected.

Contents of the invention

In order to solve the above problems, the present invention adopts the following technical solutions:

Use of the sirtuin HDAC9 as an inhibitory target in the treatment of memory impairment.

Application of the activity inhibitor of sirtuin HDAC9 in the preparation of memory impairment disease medicine.

In some embodiments, the memory impairment disease comprises spatial memory impairment, novel object recognition impairment, and scene memory impairment.

In some embodiments, the memory impairment disease comprises AD memory impairment.

In some ways, inhibitors of sirtuin HDAC9 activity act as

A. Promote CaM acetylation,

B. Promote the phosphorylation of CaMKIIα downstream of CaM,

C. Promote CaMKIIα downstream GluR1 phosphorylation.

In some aspects, the inhibitor of the activity of the sirtuin HDAC9 comprises TMP269.

Application of CaM acetylation level detection reagent or HDAC9 activity level detection reagent in the preparation of Alzheimer's disease detection products.

In some embodiments, the Alzheimer's disease is AD memory impairment.

Application of the HDAC9 activity inhibitor in preparing and treating diseases induced by deacetylation of CaM protein.

Application of HDAC9 in the preparation of in vitro CaM protein deacetylation preparations for non-diagnostic therapy.

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

The beneficial effects of the present invention are:

The present invention mainly aims at the pathological mechanism of memory impairment (especially AD memory impairment), effectively increases the acetylation of Calmodulin (CaM) and the phosphorylation level of CaMKIIα/GluR1 in AD brain neurons, enhances related learning and memory, and then relieves AD memory obstacle.

Description of drawings

Figure 1 shows the pathological mechanism of abnormal reduction of calmodulin acetylation involved in Alzheimer's disease;

Figure 2 is a graph showing the results of plasma CaM acetylation in APP/PS1 transgenic mice;

Figure 3 is a graph showing the results of CaM acetylation in the forebrain of AD mice;

Figure 4 is a graph showing the results of HDAC9 deacetylating CaM in HEK293 cells;

Figure 5 is a graph showing the results of direct deacetylation of CaM in vitro by HDAC9;

Figure 6 is a graph showing the results of HDAC9 inhibitors increasing CaM acetylation and CaMKIIα/GluR1 phosphorylation in AD mice;

Figure 7 is a graph showing the results of HDAC9 inhibitors improving hippocampus-dependent spatial memory and novel object recognition ability;

Figure 8 is a graph showing the results of HDAC9 inhibitors improving hippocampus-dependent scene memory ability.

detailed description

The present invention is described further below:

The first aspect of this part introduces :

The application of sirtuin HDAC9 as an inhibitory target in the treatment of memory impairment can relieve memory impairment by inhibiting the activity of HDAC9.

Application of the activity inhibitor of sirtuin HDAC9 in the preparation of memory impairment disease medicine.

In one of them, the memory impairment disease includes spatial memory impairment, novel object recognition impairment and scene memory impairment. Spatial memory impairment, new object recognition impairment and scene memory impairment are common memory impairments. Spatial memory impairment and scene memory impairment are manifested as Information about spatial orientation and scene environment is dysfunctional. Memory impairment may occur in dementia, traumatic brain injury, memory impairment caused by stress, and Korsakoff syndrome symptoms.

Wherein two, described memory disorder disease comprises AD memory disorder, and it is mainly the symptom caused by dementia.

Inhibitors of the expression of the acetylase HDAC9 act as

A. Promote CaM acetylation,

B. Promote the phosphorylation of CaMKIIα downstream of CaM,

C. Promote CaMKIIα downstream GluR1 phosphorylation.

Therefore, HDAC9 has a therapeutic effect on memory impairment caused by the abnormal decrease of CaM acetylation, the abnormal decrease of CaMKIIα phosphorylation downstream of CaM, and the abnormal decrease of CaMKIIα downstream GluR1 phosphorylation. Among them, promotion includes raising the level and promoting it to maintain the normal level.

In some aspects, the inhibitor of the activity of the sirtuin HDAC9 comprises TMP269.

The second aspect of this part introduces :

Application of CaM acetylation level detection reagent or HDAC9 expression level detection reagent in the preparation of Alzheimer's disease detection products. When the expression level of HDAC9 increases, the level of CaM acetylation decreases, and the two are negatively correlated, thus the detection of Alzheimer's disease can also be realized by detecting the expression level of HDAC9 or the level of CaM acetylation.

Wherein, the Alzheimer's disease is AD memory impairment. When the expression level of HDAC9 is too high, the acetylation level of CaM decreases, which is a symptom of memory impairment in AD.

The third aspect of this part introduces :

Application of HDAC9 in the preparation of in vitro CaM protein acetylation preparations for non-diagnostic therapy. Existing products basically need to realize the acetylation of CaM protein in vivo for regulation, but the present invention proposes that CaM can be deacetylated directly through HDAC9 in vitro.

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

Application of the HDAC9 activity inhibitor in the preparation and treatment of diseases induced by abnormal acetylation of CaM protein. Diseases induced by abnormal acetylation of CaM protein include existing confirmed related diseases, for details, please refer to the prior art information.

The fourth aspect of this part is further explained in combination with specific research projects :

Compared with wild-type WT mice, APP/PS1 mice, a genetic model of AD, had significantly lower plasma and brain CaM acetylation levels, and for the first time identified the deacetylase HDAC9 of CaM, and the inhibition of HDAC9 activity can rescue the decreased brain activity of AD mice. CaM acetylation and CaMKIIα/GluR1 phosphorylation and impaired learning and memory. Based on the experimental results of APP/PS1 transgenic AD mice, the abnormal reduction of calmodulin acetylation is involved in the pathological mechanism of Alzheimer's disease: the abnormal reduction of CaM acetylation in the brain of AD mice damages the CaMKIIα/GluR1 phosphorylation signal transduction pathway, leading to Impairment of learning and memory eventually leads to the pathological progression of Alzheimer's disease (see Figure 1).

1. Abnormally decreased CaM acetylation in plasma and brain of AD mice

Detect the CaM acetylation level in the plasma of 7-month-old WT and APP/PS1 transgenic mice (AD mouse genetic model), the method is as follows, the total CaM in plasma is enriched by immunoprecipitation with pan-acetylated antibody (Ac-K antibody) Acetylated protein, and then use CaM monoclonal antibody to detect the content of acetylated CaM (Ac-CaM) in the total acetylated protein.

The results showed that the plasma CaM acetylation levels were significantly lower in APP/PS1 mice compared with WT mice (A and B in Figure 2), and the decrease in plasma CaM acetylation had already occurred in 5-month-old APP/PS1 mice (Figure 2 C and D), at this time, the AD model mice did not show the memory impairment phenotype, and the plasma CaM acetylation level of the 3-month-old mice did not change significantly (E and F in Figure 2). These results suggest that the abnormal reduction of plasma CaM acetylation may be a potential plasma early diagnostic marker for Alzheimer's disease.

Figure 2: The acetylation of plasma CaM in APP/PS1 transgenic mice was significantly reduced. A, C, E, are the western blot bands of CaM acetylation levels in plasma of 7-month-old, 5-month-old, 3-month-old WT and APP/PS1 transgenic mice, respectively. B, D, and F are the quantitative analysis of Ac-CaM/CaM in A, C, and E, respectively, and the control group is taken as 1 for normalization. *, p<0.05; **, p<0.01; t-test; n=3. Data are expressed as mean ± standard error.

Detecting the CaM/CaMKIIα/GluR1 signal transduction activation level in the forebrain tissue of 7-month-old WT and APP/PS1 mice (A in Figure 3), it was found that compared with WT mice, the CaM acetyl The phosphorylation of CaMKIIα downstream of CaM and the phosphorylation of GluR1 downstream of CaMKIIα were significantly reduced (B in Figure 3), and the phosphorylation of GluR1 downstream of CaMKIIα was significantly reduced (C and D in Figure 3), while the total proteins of CaM, CaMKIIα, GluR1, PSD95 and CaM acetyltransferase SRC3 were relatively low. change (E, F, G, H and I in Fig. 3). These results suggest that the abnormal reduction of brain CaM acetylation promotes the pathological development of Alzheimer's disease by impairing the CaMKIIα/GluR1 signal transduction pathway.

Figure 3: Decreased CaM acetylation in the forebrain of AD mice. A, Western blot bands of forebrain histones in the control and experimental groups. B, C and D, Quantitative analysis of Ac-CaM/CaM, p-CaMKIIα/CaMKIIα, p-GluR1/GluR1, respectively. E, F and G, quantitative analysis of CaM, CaMKIIα, GluR1/α-tubulin, respectively. H and I, Quantitative analysis of PSD95 and SRC3/α-tubulin, respectively. Normalize to 1 as the control. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; t-test; n=4. Data are expressed as mean ± standard error.

2. Screening and identification of CaM sirtuin 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 Figure 4). The knockdown efficiency of HDAC shRNA was verified by Western blot (A, C-G in Fig. 4). HDAC6 shRNA significantly increased α-tubulin acetylation levels (A and H in Figure 4), which is consistent with previous reports, and served as another positive control. Both HDAC9 and HDAC6 shRNA increased CaM acetylation, and HDAC9 was the main deacetylase of CaM, while HDAC4, HDAC7, and HDAC8 shRNA did not significantly increase CaM acetylation (A and B in Figure 4). As a negative control, HDAC9 shRNA did not increase α-tubulin acetylation (A and H in Figure 4), indicating the specificity of HDAC9 deacetylation of CaM.

Figure 4: HDAC9 is the major sirtuin of CaM. A, HEK293 cell lysate under different treatments or shRNA transfection, respectively probed with corresponding antibodies. TSA, an HDAC inhibitor, was used as a positive control. HDAC6 shRNA increases α-tubulin acetylation is another positive control. B-H, are respectively Ac-CaM/CaM, HDAC4/α-tubulin, HDAC6/α-tubulin, HDAC7/α-tubulin, HDAC8/α-tubulin, HDAC9/α-tubulin and Ac-tubulin/α-tubulin summary graph. Data were normalized to 1 for the control group. **, p<0.01; ***p<0.001; one-way ANOVA; n=3. Data are expressed as mean ± standard error.

CaM has three lysine sites (K22, K95, and K116) that can undergo acetylation and are extremely conserved among species (A in Figure 5). In order to verify whether HDAC9 can directly deacetylate CaM in vitro, the site-directed acetylation recombinant CaM protein was purified using the genetic code extension method (B in Figure 5). This method uses an engineered pyrrolysylpyrrolysyl-tRNA synthetase specific for acetylated lysines and its cognate tRNAPry to incorporate Ac-K at designated codons to generate site-specific acetylated proteins. To determine which of the three lysine sites in CaM is acetylated, site-specific antibodies against CaM acetylation were raised at K22, K95, and K116. As shown in C in Figure 5, through the detection of pan-acetylated antibodies and site-specific antibodies, Ac-3K-CaM, Ac-3K-CaM, Ac-K22, K95 and K116-CaM acetylated at three sites were successfully synthesized. Next, express and purify site-specific acetylated His-Ac-3K-CaM and His-Ac-K22/K95/K116-CaM together with Flag-tagged HDAC9 purified from HEK293 cells by Flag magnetic beads After incubation, the results showed that HDAC9 could directly deacetylate the three acetylation sites of K22, K95 and K116 of CaM in vitro. This is the first time that the deacetylase HDAC9 of CaM has been discovered and identified. At the same time, CaM is also the first non-histone deacetylation substrate of HDAC9 discovered so far, and it also makes up for the non-histone deacetylation substrate of HDAC9. Research blank.

Figure 5: HDAC9 directly deacetylates CaM in vitro. A, top, shows the position of acetyllysine in mouse CaM, EF, EF hands, numbers represent amino acids. Bottom, amino acid sequence alignment of CaM from different species. The red K indicates a lysine that can undergo acetylation. B, top, schematic diagram of the pCDF construction vector for expressing His-tagged acetylated CaM recombinant protein in E. coli. Bottom, Schematic of the genetic code expansion method for the synthesis of site-specific acetylated recombinant CaM proteins. C, Western blot bands of acetylated CaM proteins with His tags (His-Ac-3K-CaM and His-Ac-K22/K95/K116-CaM) expressed in E. coli, HDAC9 can be directly deacetylated in vitro There are three acetylation sites at K22, K95 and K116 of CaM.

3. Mechanism of Inhibiting HDAC9 in the Treatment of AD Memory Impairment

The decreased CaM acetylation in Alzheimer's disease model mice was caused by the increased expression of its sirtuin HDAC9. HDAC9 was abnormally increased and decreased in the brains of 5-7-month-old and 12-month-old APP/PS1 mice, respectively (A and B in Figure 6). HDAC9 inhibitor TMP269 was injected into the lateral ventricle of APP/PS1 transgenic AD mice, as shown in Figure 6 C-D, the Morris water maze (Morris water maze, MWM) learning and training can enhance the level of CaM acetylation in the forebrain tissue of AD mice, Compared with the Vehicle group, the intracerebroventricular injection of HDAC9 inhibitor (HDAC9 inhibitor, HDAC9 i) can further enhance the CaM acetylation level in the forebrain tissue of mice, and the results show that HDAC9 can deacetylate endosomes in the forebrain of AD mice CaM. Previous studies have shown that CaM acetylation can enhance the activation of CaMKIIα. The phosphorylation of CaMKIIα was detected using an antibody specific for the phosphorylation of the Thr286 site of CaMKIIα. The phosphorylation is an indicator of CaMKIIα activation. As shown in C and E in Figure 6, the phosphorylation of CaMKIIα increased Consistent with enhanced CaM acetylation. CaMKIIα can phosphorylate AMPA receptor subunit GluR1 Ser831 site. Consistent with the increased phosphorylation of CaMKIIα, p-GluR1 Ser831 was also further increased during MWM learning training in AD mice in HDAC9 i group (C and F in Figure 6). These results suggest that inhibition of HDAC9 can improve the CaM acetylation and CaMKIIα/GluR1 phosphorylation signaling cascades involved in water maze learning stimulation activation in AD mice.

Figure 6: HDAC9 inhibitors increase CaM acetylation and CaMKIIα/GluR1 phosphorylation in AD mice. A, Representative blots, dynamically expressed HDAC9 protein levels in the forebrain of APP/PS1 mice. B, Statistical diagram of HDAC9/α-tubulin. C, Water maze MWM training can increase the levels of Ac-CaM, p-CaMKIIα, and p-GluR1 in the forebrain of APP/PS1 transgenic AD mice, while intracerebroventricular injection of HDAC9 inhibitor (HDAC9 i) compared with Vehicle group, HDAC9 i can further increase Enhance the levels of Ac-CaM, p-CaMKIIα and p-GluR1 in mouse forebrain. D-F, the quantitative analysis of Ac-CaM/CaM, p-CaMKIIα/CaMKIIα, p-GluR1/GluR1, respectively. Data were normalized to 1 for the control. **, p<0.01; ***, p<0.001; one-way ANOVA; n=4. Data are expressed as mean ± standard error.

4. The effect of inhibiting HDAC9 in the treatment of AD memory impairment

HDAC9 inhibitor can improve the hippocampus-dependent learning and memory ability of APP/PS1 transgenic AD mice. The hippocampus-dependent spatial memory was studied using the Morris water maze (MWM) behavioral paradigm, which is the gold standard for detecting memory impairment in animal models of AD. During MWM training, compared with the Vehicle group, the HDAC9 i group had a stronger learning curve in the HDAC9 i group (A in Figure 7). During the platform test, compared with the Vehicle group, the AD mice in the HDAC9 i group spent more time in the target quadrant and the number of platform shuttles increased (B-D in Figure 7), and the swimming speeds of the AD mice in the Vehicle and HDAC9 i groups were similar (Figure 7 E). These results suggest that HDAC9 inhibitors can improve hippocampus-dependent spatial learning and memory in AD mice. Next, it was studied whether HDAC9 inhibitors could improve the ability of new object recognition (NOR) in AD mice. New object recognition is a hippocampus-dependent short-term memory behavioral paradigm, and it is a gold indicator for detecting memory impairment in AD animal models. Compared with AD mice in Vehicle group, the discrimination index of AD mice in HDAC9 i group was significantly enhanced, and the total exploration time of AD mice in Vehicle and HDAC9 i groups around new and familiar objects was similar (Figure 7F-G). These results suggest that HDAC9 inhibitors can improve hippocampus-dependent short-term memory in AD mice. The above results indicate that inhibition of HDAC9 can improve the hippocampus-dependent learning and memory ability of AD mice.

Figure 7: HDAC9 inhibitors improve hippocampus-dependent learning and memory. A, During MWM training, the learning curve of AD mice in the HDAC9 i group was enhanced in the intracerebroventricular injection HDAC9 inhibitor group (HDAC9 i) compared with the Vehicle group. *Genotype F(1,170)=8.842, p=0.0041, two-way ANOVA, n=15. Data are expressed as mean ± standard error. B, Swimming trajectories of AD mice in the Vehicle group and HDAC9i group during the platform test. C, Compared with AD mice in Vehicle group, AD mice in HDAC9 i group spent more time staying in the target quadrant. *, p=0.0216, two-way ANOVA, n=15. Data are expressed as mean ± standard error. D, Compared with the AD mice in the Vehicle group, the number of times the AD mice in the HDAC9 i group crossed the platform increased during the platform test. E, The swimming speeds of AD mice in Vehicle group and HDAC9i group were similar. F, Head heat maps around novel (N) and familiar (F) objects of AD mice in the Vehicle group and HDAC9i group. G, Similar total exploration time with N and F between AD mice in Vehicle group and HDAC9i group. H, Recognition index statistics show that AD mice in HDAC9i group have impaired recognition of novel objects. ns, not significant; *, p<0.05; ***, p<0.001; t-test; n=15. Data are expressed as mean ± standard error.

Finally, it was investigated whether HDAC9 inhibition could rescue the memory deficit in APP/PS1 mice in contextual fear memory, which is a hippocampus-dependent contextual memory behavior paradigm and a gold indicator for detecting memory impairment in AD animal models. The mice were subjected to scene-conditioned fear memory training (CS, conditioned stimulus, using a box with a specific background; US, unconditioned stimulus, using a 0.5mA plantar electric shock), and one day later, when the mice returned to the electric shock box, the freezing time was tested (fear memory) (A in Figure 8). 7-month-old WT and APP/PS1 mice treated with intracerebroventricular injection of vehicle or HDAC9 inhibitor TMP269 received contextual fear training and testing flow chart (B in Figure 8). Freezing times during exploration and immediately after training were similar in WT or APP/PS1 mice (Fig. 8C). Whereas WT or APP/PS1 mice injected with TMP269 during the test one day later had increased freezing time (fear memory) (Fig. 8, C), suggesting that HDAC9 inhibition rescued episodic fear memory in APP/PS1 mice. On the other hand, HDAC9 inhibition enhanced contextual fear memory in WT mice (Fig. 8C). Taken together, these results suggest that HDAC9 inhibition ameliorates hippocampus-dependent memory deficits in APP/PS1 mice.

Figure 8: HDAC9 inhibition improves contextual fear memory in 7-month-old AD mice. A, Schematic diagram of situational fear conditioning test. CS, conditioned stimulus, using a specific background box; US, unconditioned stimulus, using 0.5mA plantar shock. B, Schematic representation of behavioral grouping and testing. C, Intracerebroventricular injection of HDAC9 inhibitors enhances or rescues freezing time (fear memory) in WT or APP/PS1 mice, respectively, during a paired test. **p<0.01, two-way ANOVA followed by Sidak’s multiple comparisons test, 12 mice per group, data expressed as mean ± standard error.

The pathological mechanism of HDAC9-mediated CaM deacetylation in Alzheimer's disease provides a new molecular target for the treatment of Alzheimer's disease. The invention mainly aims at the pathological mechanism of AD memory impairment, effectively increases the acetylation level of Calmodulin (CaM) and the phosphorylation level of CaMKIIα/GluR1 in AD brain neurons, enhances related learning and memory, and then relieves AD memory impairment.

It will be clear 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 present invention. They all fall within the protection scope of the present invention. The protection scheme of the present invention shall be based on the appended claims of the present invention.

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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