CN111214661A

CN111214661A - Application of compound for inhibiting Htr3a and intracellular signal pathway thereof in preparation of drugs for treating and/or preventing AD

Info

Publication number: CN111214661A
Application number: CN202010132159.4A
Authority: CN
Inventors: 袁琼兰
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2020-02-29
Filing date: 2020-02-29
Publication date: 2020-06-02
Anticipated expiration: 2040-02-29
Also published as: CN111214661B

Abstract

The invention relates to application of a compound for inhibiting Htr3a and a downstream intracellular signaling pathway thereof in preparing a medicament for treating and/or preventing Alzheimer disease. The invention discovers that the expression of Htr3a of AD animals and AD patients with Alzheimer's disease transgenosis is obviously increased for the first time, and the intracellular calcium (Ca) of the AD animals and AD patients is increased²⁺) in AD transgenic animal and mouse, the functions of Htr3a are inhibited, the generation of Abeta-amyloid is reduced remarkably, and the intracellular signal pathway Ca is reversed²⁺the invention first considers that Htr3a and the intracellular signal path thereof play an important role in the generation of β -amyloid plaques of AD, and the regulation of the pathway CaN prevent β -amyloid plaques from being generatedProgressive development of AD. Therefore, the medicine aiming at the pathway can prevent the progress of AD and slow down the development of AD, and has great research and application prospects in the aspect of AD treatment.

Description

Application of compound for inhibiting Htr3a and intracellular signal pathway thereof in preparation of drugs for treating and/or preventing AD

Technical Field

The invention relates to the technical field of research on medicaments for treating or preventing Alzheimer disease, in particular to application of a compound for inhibiting Htr3a and an intracellular signal pathway thereof in preparation of medicaments for treating and/or preventing AD.

Background

Alzheimer's disease is insidious and is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment. The 2015 world Alzheimer's report states that about 4680 million Alzheimer's patients are in the world, and 1 new patient is found in 3 seconds on average, and the 2050 world patients are expected to break through 1 hundred million 3150 people. At present, more than 800 million patients with Alzheimer disease are treated in China. The prevalence of alzheimer's disease is 5% in people over the age of 65 years; the prevalence increases to 25% over the age of 85 years, and up to 60% in older people over the age of 95 years. As the population ages, AD patients carry a tremendous mental and economic burden on the family and society.

at present, the clinical drugs used for Alzheimer disease mainly comprise acetylcholinesterase inhibitor and glutamate NMDA receptor inhibitor, so far, Alzheimer disease patients have no specific effective drug to prevent the development of the disease, therefore, the development of the medicine for AD pathological process, reducing the generation of amyloid A beta protein and nerve damage caused by neuroinflammation, delaying and preventing the development of the disease has great application prospect and social value.

the pathogenesis of AD is based on two hypotheses, namely amyloid plaques formed by aggregation of amyloid beta (also called senile plaques) and the beta amyloid waterfall hypothesis of neuroinflammation, glial cell activation, synaptic transmission dysfunction and synapse and neuron loss caused by the amyloid plaques, and the tau protein hypothesis that tau hyperphosphorylation causes neurofibrillary tangles, destroys normal functions of neurons and synapses and causes neuron death.

However, there is currently no good way to slow the progression of AD as to how to stop it.

Disclosure of Invention

The invention aims to provide application of a medicament for inhibiting Htr3a and a downstream intracellular signal pathway thereof in preparing a medicament for treating and/or preventing Alzheimer disease.

The invention can provide a new treatment target for the treatment of the Alzheimer disease and is beneficial to the development of new drugs for the Alzheimer disease.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a method for inhibiting Htr3a and a downstream intracellular signal path C thereofa²⁺-the use of a compound of CaN-NFAT for the preparation of a medicament for the treatment and/or prevention of Alzheimer's disease.

7 subtypes of 5-hydroxytryptamine receptors are known in the art, and in the present application, Htr3a is the 3 rd subtype alpha subunit of 5-hydroxytryptamine receptor, also known as 5-hydroxytryptamine receptor 3 alpha subunit, the structure of which is known.

Htr3 is an ionotropic channel receptor consisting of 3 α subunits and 2 β subunits, which, when activated, allows Na⁺And Ca²⁺in-flow, the alpha subunit is its functional unit.

Blockers of Htr3a are currently: tropisetron (tropisetron), ondansetron (ondansetron), and the like, and is used for preventing vomiting after radiotherapy and chemotherapy.

Further, the present invention provides a method for inhibiting Htr3a and its downstream intracellular signaling pathway Ca²⁺-the use of a compound of CaN-NFAT for the manufacture of a medicament for reducing a β -amyloid production.

Further, the present invention provides a method for inhibiting Htr3a and its downstream intracellular signaling pathway Ca²⁺-the use of a compound of CaN-NFAT for the manufacture of a medicament for decreasing calcineurin (CaN) activity.

Further, the present invention provides a method for inhibiting Htr3a and its downstream intracellular signaling pathway Ca²⁺-the use of a compound of the CaN-NFAT for the preparation of a medicament for reducing the activity of the transcription factor NFAT.

The invention also provides application of the compound for inhibiting calcineurin (CaN) in preparing a medicament for treating and/or preventing Alzheimer disease.

The invention also provides application of the compound for inhibiting the transcription factor NFAT in preparing a medicament for treating and/or preventing Alzheimer disease.

The invention also provides a medicament for treating and/or preventing Alzheimer disease, which takes a compound inhibiting Htr3a and a downstream intracellular signal pathway CaN-NFAT thereof, a compound inhibiting calcineurin or a compound inhibiting transcription factor NFAT as a medicinal component.

The invention also provides a method for developing a medicament for treating and/or preventing Alzheimer disease, and the method is used for designing and screening compounds aiming at Htr3a and key sites of downstream intracellular signaling pathways thereof and has the possibility of being used as a new medicament for treating and/or preventing Alzheimer disease.

furthermore, the drug for inhibiting the functions of Htr3a or the downstream intracellular signaling pathway thereof can reduce the generation of A β -amyloid protein and reverse the intracellular signaling pathway Ca²⁺-alteration of CaN-NFAT with the possibility of being a new drug for the treatment and/or prevention of Alzheimer's disease.

The invention also provides application of Htr3a and a downstream intracellular signal pathway key site, and application of Htr3a and the downstream intracellular signal pathway key site as a new drug for designing, screening and treating and/or preventing Alzheimer disease.

In the experiments we have accidentally found adult APP/PS1AD model (AD) mice whose Htr3a receptor expression was significantly increased compared to littermate Wild Type (WT) mice. The present inventors further observed mice of 1 to 12 months of age using the western blot method, and as a result, found that the expression level of Htr3a was significantly increased in AD mice of 1 to 12 months compared to WT mice of the same age, which suggests that Htr3a may play a role in the onset of AD.

in order to deeply investigate the problem, the inventor uses Htr3a shRNA-AAV virus to carry out intracerebral microinjection on silent Htr3a, and observes after the animal survives for one month, protein hybridization experiments show that in an Htr3a shRNA-AAV virus injection group, the expression of Htr3a protein of an AD mouse is obviously reduced compared with that of an AD mouse given with an empty virus, the Htr3a of the AD mouse is successfully silenced, immunofluorescence staining and thioflavin staining are used for observing amyloid plaques of A β, and the result shows that the Htr3a silent AD mouse is obviously reduced compared with that of the AD mouse given with the empty virus, and the research results firstly prove that Htr3a is highly expressed in an APP/PS1AD transgenic animal, the inhibition of the expression of Htr3a reduces the amyloid plaques of the A β, and suggest that the Htr3a positive intermediate can play an important role in the generation of the APP β amyloid.

To verify whether the same was observed in AD mice in AD patients, the inventors further observed changes in Htr3a expression in the cortex and hippocampus of AD patients. It is appreciated that in the frontal cortex and hippocampus of AD patients, a significant increase in the level of Htr3a expression was observed compared to elderly of the same age using the western blot procedure.

The present inventors further investigated the intracellular signal change caused by Htr3 a. Importantly, intracellular signaling calcineurin CaN and nuclear transcription factor NFAT of Htr3a receptor were found to be significantly elevated in both AD patients and APP/PS1AD mice compared to normal controls. In AD mice, the function of Htr3a is silenced, and the activity of calcineurin CaN and the activity of transcription factor NFAT CaN be reversed.

the inventor of the invention discovers for the first time that Htr3a is obviously increased in AD patients and AD mice, reduces the function of Htr3a and CaN reduce the generation of A β amyloid plaques, Htr3a has similar intracellular signal change, namely the change of a CaN-NFAT signal pathway in the AD patients and the AD mice, and the results indicate that the Htr3a which is obviously up-regulated and the intracellular signal thereof play an important role in the generation of the A β amyloid plaques of AD, and the research and development of drugs for regulating the pathway are likely to find a new drug for AD treatment.

according to A β research result of Htr3a, A β invention further researches key sites of downstream intracellular signal pathways of Htr3a and clarifies A β effect of A β key sites in A β pathogenesis of AD, and A β research shows that A β regulation of Htr3a and A β downstream intracellular signal pathways thereof can obviously reduce A β amyloid plaques of A beta of AD and slow down A β disease course of AD.

compared with the prior art, the invention discovers that the expression of Htr3a of Alzheimer's Disease (AD) transgenic animals and AD patients is remarkably increased for the first time in experiments, and key proteins of intracellular signals of the Alzheimer's Disease (AD) transgenic animals and AD patients are remarkably increased in calcineurin and transcription factor NFAT (NFAT). in AD transgenic animal mice, Htr3a shRNA-AAV virus brain stereotaxis injection is used for inhibiting the function of Htr3a, so that the generation of Abeta-amyloid is remarkably reduced, and the change of CaN and transcription factor NFAT of an intracellular signal pathway is reversed.

the invention considers that the Htr3a receptor and the intracellular signal pathway thereof play an important role in the generation of AD A beta-amyloid, and the regulation of the pathway can influence the process of AD and prevent the progressive development of AD.

Drawings

FIG. 1: the results of western blot analysis of Htr3a of AD mice of different ages in example 1;

figure 1 shows that Cortical (CTX) and Hippocampal (HIP) Htr3a receptors were significantly increased in 1, 3, 10, 12 month old APP/PS1AD mice compared to wild-type WT mice.

FIG. 2: the result of the immunofluorescence chemical staining experiment of AD mouse Htr3a in example 1;

FIG. 2 shows that APP/PS1AD mouse Htr3a receptor positive cells (indicated by arrows) are significantly increased over WT mice at 10 months of age (10 m); GC: dentate gyrus cell layer (GC).

FIG. 3: htr3a silenced AD mice in example 2 Htr3a western blot analysis;

the analysis in fig. 3 shows that the expression of Htr3a silenced AD mice (AD-Htr3a shRNA-AAV) Htr3a is significantly lower than that of AD group (AD-con-AVV), while Htr3a expression of AD empty virus group is significantly higher than that of WT group (WT-con-AAV) mice.

FIG. 4: the result of immunofluorescence chemical staining experiments for Htr3a of Htr3a silenced AD mice in example 2;

FIG. 4 shows that Htr3a silenced AD mice (AD-Htr3a shRNA-AAV) have a significant reduction in cortical Htr3a positive cells (indicated by the arrow) compared to the AD-group (AD-con-AAV); the number of Htr3 a-positive cells in the AD group (AD-con-AVV) was significantly higher than that in the WT (WT-con-AAV) group.

FIG. 5 immunofluorescence staining of A β amyloid protein (with 6E10 antibody) in Htr3 a-silenced AD mice of example 3;

FIG. 5 shows that immunofluorescence staining of Htr3a silenced AD mice (AD-Htr3a shRNA-AAV) significantly reduced hippocampal amyloid A β (6E10 antibody) compared to AD group (AD-con-AAV), GFP transfected cells.

FIG. 6 results of Western blot analysis of amyloid A β protein in AD mice silenced with Htr3a in example 3;

FIG. 6Western blot analysis shows that hippocampal amyloid A β is significantly reduced in Htr3 a-silenced AD mice (AD-Htr3a shRNA-AAV) compared to AD group (AD-con-AAV).

FIG. 7 immunofluorescence staining analysis shows a significant reduction in hippocampal amyloid A β compared to AD group (AD-con-AAV) in AD mice silenced with Htr3a (AD-Htr3a shRNA-AAV), while cortex (Ctx) is not different.

FIG. 8A β amyloid protein was observed by thioflavine S (thioflavine S) staining of the AD mouse with Htr3a silenced in example 3;

FIG. 8 shows that hippocampal (Hip) thioflavin S staining of Htr3 a-silenced AD mice (AD-Htr3a shRNA-AAV) shows a significant reduction in amyloid A β compared to AD group (AD-con-AAV).

FIG. 9: iba immunofluorescence staining of Htr3a silenced AD mice in example 4 showed results;

FIG. 9 shows a significant reduction in the number of hippocampal (Hip) Iba-positive microglia in Htr3 a-silenced AD mice (AD-Htr3a shRNA-AAV) compared to AD group (AD-con-AVV) by immunofluorescent staining; while the AD group (AD-con-AVV) showed a significant increase in the number of Iba-positive microglia as compared with the WT (WT-con-AAV) group.

FIG. 10Htr3a silenced AD mouse calcium indicator Fluo-4 AM shows hippocampal intracellular calcium (iCa) of Htr3a silenced AD mice (AD-Htr3ashRNA-AAV)²⁺) Significantly lower than that of AD group (AD-con-AVV) which has intracellular calcium (iCa)²⁺) Significantly higher than that of WT (WT-con-AAV).

FIG. 11: htr3a silenced AD mouse Fluo-4 AM and Htr3a double-staining to observe Htr3a positive intermediate energy neuron with obviously increased intracellular calcium;

FIG. 11Fluo-4 AM and Htr3a double staining observations show that Htr3a positive multipotent neurons with significant intracellular calcium elevation (Htr3 a/iCa)²⁺): htr3a positive mesogenic neurons with significantly increased hippocampal intracellular calcium (Htr3 a/iCa) in Htr3 a-silenced AD mice (AD-Htr3a shRNA-AAV) (Htr3 a/iCa)²⁺) The number of the peptides is significantly reduced compared with that of the AD group (AD-con-AVV) of Htr3a/iCa²⁺The amount was significantly higher than that of WT (WT-con-AAV) group.

FIG. 12 shows Western blot analysis of the expression changes of CaN and active CaN (. DELTA.CaN) in AD mice in example 5;

FIG. 12Western blot analysis shows that the hippocampal △ CaN content of AD mice is significantly increased compared to WT mice.

FIG. 13: htr3a silenced AD mice NFAT immunofluorescence staining;

FIG. 13: immunofluorescent staining of hippocampal NFAT of Htr3 a-silenced AD mice showed that Htr3 a-silenced AD mice (AD-Htr3a shRNA-AAV) had a significantly lower number of hippocampal NFAT immunopositives than AD group (AD-con-AVV), while AD group (AD-con-AVV) had a significantly higher number of NFAT immunopositives than WT-group (WT-con-AAV).

FIG. 14 histochemical staining of frontal A.beta.and phosphorylated tau protein in AD patients in example 6;

FIG. 14 histochemical staining of frontal A β and phosphorylated tau in AD patients shows that there are some β amyloid plaques and phosphorylated tau in the cortex of AD patients.

FIG. 15Western blot analysis of cortical A β amyloid protein of the AD patients in example 6;

FIG. 15Western blot analysis shows that cortical A β amyloid is significantly increased in AD patients compared to the normal group.

FIG. 16: in example 6, western blot method for expression of Htr3a protein in AD patients;

FIG. 16Western blot analysis of Cortex (CTX) and Hippocampus (HIP) of AD patients with significantly higher Htr3a expression than in the normal group (CN).

FIG. 17: in example 6, cells positive for Htr3a from AD patients were visualized by immunohistochemical staining;

FIG. 17 immunohistochemical staining showed that there were some Htr3a positive cells in the hippocampal region of the brain of AD patients, and no positive cells were observed in the normal group.

FIG. 18 is a graph showing the analysis of the contents of calcineurin CaN and active CaN (△ CaN) and nuclear transcription factor of T cell NFAT in AD patients using western blot in example 6;

FIG. 18Western blot analysis total CaN, active CaN (△ CaN) and NFAT of Cortex (CTX) of AD patients were significantly elevated compared to the normal group.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

The APP/PS1 transgenic AD mice (purchased from southern university model animal center) used in this experiment were widely used AD model mice.

First, Htr3a expression in cortex and hippocampus of 1-12 month APP/PS1 transgenic AD mice was found to be significantly increased compared to wild-type (WT) mice by western blot analysis (fig. 1); secondly, with immunofluorescence staining, analysis of the results showed that cortex of APP/PS1 transgenic AD mice increased significantly Htr3a positive cells at 10 months, whereas Htr3a positive cells were barely detectable in WT mice (fig. 2).

Therefore, the expression level of Htr3a of APP/PS1 transgenic AD mice at 1-12 months is obviously increased compared with that of age-matched wild type mice.

In this embodiment, the specific method for western blot analysis is as follows:

littermates of APP/PS1AD, WT (wild type) mice were housed in the same housing for 1 month, 3 months, 10 months and 12 months, respectively. At each time point, the mouse was anesthetized excessively, the hippocampus and cerebral cortex were removed by decapitation, proteins were extracted with RIPA lysate (P0013B, Beyotime), and the protein concentration was measured with BCA protein concentration measurement kit (P0010, Beyotime). After separating proteins by 12% SDS-PAGE electrophoresis according to a total amount of 10. mu.g of protein per well, the gel was transferred to a PVDF membrane. Membranes were cut according to protein molecular weight, and the cut membranes were rinsed with 1 × TBST for 5min on a slow shaker at room temperature, followed by blocking the membranes in 5% BSA for 1h at room temperature. After membrane blocking, the primary antibody was incubated for 16h at 4 ℃ with murine 5HT3AR antibody (1: 500) and mouse anti-GAPDH antibody (1: 1000; AG019, Beyotime). After the primary antibody incubation was completed, the cells were washed with 1 × TBST for 10min in a rapid shaker at room temperature for 3 times. The membrane was immersed in the corresponding horseradish peroxidase-labeled anti-mouse (1: 1000; A0181, Beyo-time) secondary antibody, incubated for 2h on a slow shaker at room temperature, then washed with 1 XTSST on a fast shaker at room temperature for 10min with shaking for 3 times. The results were visualized with ECL chemiluminescence kit (P0018A, Beyotime) and visualized using an ImageQuant LAS4000mini chemiluminescence imaging analyzer (28955813, GEHealthcare Life Sciences). The relative expression level of 5-HT3AR was calculated by ImageJ software analysis using GAPDH as an internal reference.

In this example, the specific method of immunofluorescence staining is as follows:

WT mice and APP/PS1AD mice each 3, for immunofluorescence staining. The mice were anesthetized with a suitable amount of 10% chloral hydrate, the chest was opened to expose the heart, the liver was perfused through the left ventricle with physiological saline until it became white, after pre-fixation by perfusion with Phosphate Buffer (PB) containing 4% paraformaldehyde, the whole brain was removed, and post-fixation was carried out in the same fixative at 4 ℃ for 24 h. After the postfixation was completed, the whole brain was allowed to settle in a PB solution containing 20% sucrose for 24h at 4 ℃, then transferred to a PB solution containing 30% sucrose, and allowed to stand for 24h at 4 ℃ to completely settle. The tissue was coronal sectioned using a cryomicrotome to a thickness of 10 μm. Brain pieces were selected, rinsed three times in PBS on a slow shaker at room temperature for 10min each, and subsequently blocked with 5% BSA for 2h at room temperature. After blocking, the brain slices were immersed in the Htr3a antibody (1: 100) and incubated at 4 ℃ for 18 h. Brain pieces were rinsed in PBS on a slow shaker three times at room temperature for 10min each time. The brain slides were immersed in Cy 3-labeled secondary antibodies (1: 500; Beyotime) and incubated at room temperature in the dark for 2h, then rinsed three times for 10min each on a slow shaker. Staining with DAPI (1: 300; Beyo-time) for 5min, rinsing three times at room temperature on a slow shaker, and mounting after 10min each time. The photographs were observed under a microscope and counted for each brain slice of 5-HT3AR positive cells using Im-ageJ software.

Example 2

Silencing Htr3a in APP/PS1AD mice, reducing Htr3a observed effects on amyloid plaques in APP/PS1AD mice.

AAV-htr3a shRNA-GFP virus was prepared, and AAV-EGFP empty virus was used as a control. 6 half-month old WT mice and APP/PS1AD mice, bilateral, injected intra-hippocampal. The experiment was divided into three groups: (1) WT mice were given the same volume of AAV-EGFP (CON-AAV); (2) APP/PS1AD mice were administered AAV-EGFP (CON-AAV, titer 2.19X 10⁹V.G/each side; (3) APP/PS1AD mice were administered AAV-Htr3a shRNA-GFP virus (titer: 2.19X 10)⁹V.G/each side). Animals are raised for 4 weeks after injection, overnarcotized, brain tissue is taken, western blot protein hybridization experiment and immunofluorescence staining are carried out,the western blot protein hybridization experiments and methods for immunofluorescence staining refer to example 1.

Protein hybridization experiments show that Htr3a shRNA-AAV virus injection group, AD mouse Htr3a protein expression is reduced compared with control group AD mice (figure 3). In addition, the number of Htr3a positive cells was observed by immunofluorescent staining of the brain tissue injected with the virus. Immunostaining statistics showed that the number of positive cells in the AD empty virus group Htr3a was significantly greater than that in the WT empty virus group, whereas the AD Htr3ashRNA group, Htr3a positive cells, was significantly lower than that in the AD empty virus group (fig. 4). These results demonstrate that Htr3a silencing in APP/PS1AD mice is successful, reducing the expression of Htr3a in AD mice.

Example 3

study on whether reduction of expression of Htr3a in APP/PS1AD mouse has influence on amyloid A beta

to investigate whether reducing the expression of APP/PS1AD mouse Htr3a had an effect on a β amyloid, in this example a β amyloid plaques were analyzed using immunofluorescence staining (fig. 5) and western blot (fig. 6) assays for a β amyloid and thioflavin staining of a β amyloid (fig. 7).

the results show that Htr3a of APP/PS1 AD-silenced mice, amyloid plaques of A beta are significantly reduced compared with AD mice (administered with empty virus).

Example 4

study on whether silencing Htr3a, reducing A β amyloid plaques, and thereby reducing microglial activation and neuroinflammation

the inventors further observed that silencing Htr3a reduced amyloid plaques of A β, thereby reducing microglial activation and neuroinflammation, and that the number of microglia positive for hippocampal Iba in AD mice in the Htr3a shRNA-AAV virus-injected group was significantly reduced compared to AD mice as shown by immunofluorescence staining analysis (FIG. 8), and that the neuroinflammatory factor interleukin 1 β (Interleukin 1 β, IL-1 β) was also significantly reduced compared to AD mice (FIG. 9).

the research results prove that Htr3a is highly expressed in APP/PS1AD mice for the first time, Htr3a shRNA is used for reducing the Htr3a expression of APP/PS1AD mice, inhibiting the function of Htr3a, reducing A beta amyloid plaques, and relieving microglial cell activation and neuroinflammatory reaction.

Example 5

Study of intracellular signaling pathway of Htr3a

The present inventors further investigated the intracellular signaling pathway of Htr3 a. Htr3a is an ion channel type receptor that allows passage of extracellular calcium ions. Therefore, high expression of Htr3a may result in an increase in intracellular calcium. While Calcineurin (CaN) is a downstream target molecule of intracellular calcium and T-nuclear transcription factor (NFAT) is a substrate for CaN the inventors further observed changes in intracellular calcium and Calcineurin (CaN) and NFAT in WT mice and APP/PS1AD mice using the western blot method.

First, calcium changes in hippocampal cells of WT mice and APP/PS1AD mice were observed by staining with calcium indicator (Fluo-4 AM) (FIG. 10). The signal intensity of intracellular calcium is analyzed, and the result shows that the signal intensity of the intracellular calcium of the AD-con empty virome is obviously higher than that of the WT-con empty virome; the intracellular calcium signal intensity of AD mice (AD-Htr3a shRNA-AAV) in the Htr3a shRNA-AAV virus injection group is obviously lower than that of AD-con empty virus group, and the experimental results show that the silencing of Htr3a can reduce the intracellular calcium of APP/PS1AD mice for the first time.

The experiment further uses Htr3a antibody immunostaining and Fluo-4 AM staining double markers, and the result shows that the signal of the calcium in Htr3a positive mesonervous cells of APP/PS1AD mice is obviously enhanced compared with WT mice; while the signals of intracellular calcium of intermediate energy neurons positive for Htr3a of AD mice (AD-Htr3a shRNA-AAV) silencing Htr3a were significantly lower than those of AD mice (AD-con-AAV) (FIG. 11).

finally, changes in calcineurin CaN and the downstream nuclear transcription factor NFAT were observed in 8-month-old WT and APP/PS1AD mice Western blot analysis showing that the hippocampal calcineurin CaN activity was significantly increased in APP/PS1AD mice, as indicated by a significant increase in △ CaN content (FIG. 12).

This example also shows changes in the transcription factor NFAT by immunofluorescence staining. The results show that the NFAT positive products of AD-con empty virus group are significantly increased compared with WT-con group; while the AD mice in the Htr3a shRNA-AAV virus-injected group (AD-Htr3a shRNA-AAV) had significantly lower NFAT positive products than the D-con empty virus group (fig. 13). These results show that silencing APP/PS1AD mouse Htr3a can reduce the expression of NFAT increased by AD mouse for the first time.

Example 6

To verify whether the same was observed in AD mice in AD patients, this example further observed changes in Htr3a expression in the cortex and hippocampus of AD patients.

The experiment is carried out in the research center of neurological research of Xiangya medical college of China-south university. Taking out the brain from the skull of a brain donation volunteer by using a Standardized Operation Protocol (SOP) established by the national institutes of health through a corpse donation plan of Xiangya medical college, dividing the brain into a left brain and a right brain along a sagittal suture, generally placing the left brain into 4 percent paraformaldehyde prepared by phosphate buffer solution for fixation, cutting the left brain into brain slices with the thickness of 1cm along a coronal plane after the fixation for 2 to 4 weeks, taking pictures for filing, and using the part of brain tissues fixed by formalin for the research of immunohistochemistry; the right brain is cut into brain slices with the thickness of 1cm from frontal lobe to occipital lobe along the coronal position, and the brain slices are packaged and marked by a simple plastic packaging bag, and then are frozen and stored in an ultralow temperature refrigerator at minus 80 ℃ for western blot protein analysis. Western blot and histochemical staining procedures were the same as above.

AD patients and normal old (W) cortex and hippocampus were first examined for amyloid a β and phosphorylated tau-protein (P-tau) by histochemical staining (fig. 14) and Western blot (fig. 15) analysis to confirm that they were indeed AD patients.

The above experimental results confirm that the present invention indeed obtains brain tissue samples of normal elderly and AD patients. Based on this, we observed protein expression of Htr3a in brain tissues of normal group and AD patients by western blot method, and found that cortex and hippocampus of AD patients had significantly increased expression of Htr3a (fig. 16). In addition, immunostaining of brain tissue, normal brain tissue (W) did not detect Htr3a positive cells; while the cortex and hippocampus of AD patients showed some Htr3a positive cells (fig. 17).

The invention further studies the intracellular signaling changes caused by Htr3a in AD patients. In frontal cortex and hippocampus of AD patients, western blot analysis was performed to find that calcineurin CaN and nuclear transcription factor NFAT of T cell were significantly elevated compared to normal group (fig. 18).

according to a series of research results, the Htr3a is found to be remarkably increased in AD patients and AD model mice for the first time, the function of Htr3a is inhibited, the generation of A β can be reduced, and the recovery of cognitive function is promoted.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. Inhibiting Htr3a and its downstream intracellular signal path Ca²⁺-the use of a compound of CaN-NFAT for the preparation of a medicament for the treatment and/or prevention of Alzheimer's disease.

2. The inhibitor of Htr3a and the downstream intracellular signaling pathway Ca of claim 1²⁺-the use of a compound of CaN-NFAT in the manufacture of a medicament for the treatment and/or prevention of alzheimer's disease, characterized by the use of a compound that inhibits Htr3a and its downstream intracellular signaling pathway CaN-NFAT in the manufacture of a medicament for reducing a β -amyloid production.

3. According to claim1 said inhibitor Htr3a and its downstream intracellular signaling pathway Ca²⁺The application of the compound of CaN-NFAT in the preparation of medicines for treating and/or preventing Alzheimer disease, and is characterized in that the compound inhibiting Htr3a and the downstream intracellular signal pathway CaN-NFAT is applied to the preparation of medicines for reducing the activity of calcineurin phosphatase.

4. The inhibitor of Htr3a and the downstream intracellular signaling pathway Ca of claim 1²⁺-the use of a compound of the CaN-NFAT for the preparation of a medicament for the treatment and/or prevention of Alzheimer's disease, characterized in that it inhibits Htr3a and its downstream intracellular signaling pathway Ca²⁺-the use of a compound of the CaN-NFAT for the preparation of a medicament for reducing the activity of the transcription factor NFAT.

5. Use of a compound that inhibits calcineurin in the manufacture of a medicament for the treatment and/or prevention of alzheimer's disease.

6. Application of a compound for inhibiting a transcription factor NFAT in preparing a medicament for treating and/or preventing Alzheimer disease.

7. A drug for treating and/or preventing Alzheimer's disease, characterized in that it comprises, as a pharmaceutically active ingredient, a compound that inhibits Htr3a and its downstream intracellular signaling pathway, CaN-NFAT, a compound that inhibits calcineurin, or a compound that inhibits the transcription factor NFAT.

8. A method for developing a drug for treating and/or preventing Alzheimer's disease, which is characterized in that a compound aiming at Htr3a and the key site of the downstream intracellular signaling pathway thereof is designed and screened, so that the drug has the possibility of being used as a new drug for treating and/or preventing Alzheimer's disease.

9. the method for drug development for the treatment and/or prevention of Alzheimer's disease according to claim 8, wherein the drug inhibiting the function of Htr3a or its downstream intracellular signaling pathway is capable of reducing the production of A β -amyloid, and inversely, the production of A β -amyloidCa of intracellular signaling pathways²⁺-alteration of CaN-NFAT with the possibility of being a new drug for the treatment and/or prevention of Alzheimer's disease.

The application of Htr3a and the key sites of the downstream intracellular signaling pathway, which is characterized in that the Htr3a and the key sites of the downstream intracellular signaling pathway are applied to designing, screening and treating and/or preventing new drugs for Alzheimer disease.