CN113209301B

CN113209301B - Application of gamma Camk II expression promoter or stabilizer in preparation of anti-aging and anti-neurodegenerative drugs

Info

Publication number: CN113209301B
Application number: CN202110546149.XA
Authority: CN
Inventors: 马欢; 杨静; 瞿靖
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-03-22
Anticipated expiration: 2041-05-19
Also published as: CN113209301A

Abstract

The invention discloses an application of a gamma Camk II expression promoter or stabilizer in preparing a medicine for slowing down aging and/or resisting neurodegeneration. For those skilled in the art, the role of γ CaMKII in body aging is the first discovery by the inventors of the present application, and the present invention has significant meaning for developing anti-aging drugs.

Description

Application of gamma Camk II expression promoter or stabilizer in preparation of anti-aging and anti-neurodegenerative drugs

Technical Field

The invention relates to the field of molecular biology, in particular to application of a gamma Camk II expression promoter or stabilizer in preparation of a medicine for slowing down aging and resisting neurodegeneration.

Background

Aging and age-related diseases in humans are becoming the biggest challenges facing the world and placing a huge financial burden on each country. With the development of medical technology and changes in lifestyle, the average life expectancy of people has improved greatly over the past several decades, but unfortunately, the life expectancy of the health of people has not improved accordingly. With the increase of the life span of people, the incidence of age-related diseases such as cardiovascular diseases, metabolic disorders, cancers, neurodegenerative diseases, etc. is on an increasing trend. The most immediate challenge facing current geriatric medicine is therefore to identify a pharmacological strategy that promotes healthy aging (Healthspan) with the ideal goal of delaying the onset of the senile syndrome, not necessarily prolonging life itself.

The existing anti-aging strategies mainly have the following aspects: 1. pharmacological inhibition of the GH/IGF-1 axis 2. protein restricted and intermittent fasting diet 3. pharmacological inhibition of the TOR-S6K pathway 4. pharmacological modulation and application of spermidine and other epigenetic modulators to specific sirtuin proteins 5. pharmacological inhibition of inflammation 6. chronic use of metformin. These strategies either intervene primarily in the prolongation of life or only in the molecular pathways of specific systems of the body (such as the metabolic system or the immune system) and are not very effective in delaying the complications associated with aging, for example rapamycins can reduce the occurrence of tumors and cardiovascular diseases but can trigger immunosuppression, cataract formation, insulin resistance, etc.

Gamma CaMKII is a calcium/calmodulin dependent protein kinase, encoded by Camk2G gene, and its protein sequence is: SEQ ID NO. 1. The protein structure consists of an N-terminal kinase area, a Ca2+/CaM combined self-inhibition regulation area, a variable connection area and a C-terminal central area, has the molecular weight of about 60KD, mainly exists in a 12-polymer form in cells, and exists in different subunits in different organs.

The gamma CaMKII molecules are widely distributed in various organs of mice and human bodies, wherein the expression level in the brain is highest, and the gamma CaMKII molecules are distributed in heart, liver, muscle smooth muscle and immune system. The role of gamma CaMKII in neurons, and early researches suggest that the gamma CaMKII can be used as a CaM nuclear transport protein, the CaM is transported into cell nuclei to be released after a type I calcium ion channel is activated, and the free CaM can activate the CaMKIV and CREB in the nuclei, so that the gene transcription is regulated. The Camk2G gene was identified as a susceptibility gene to intellectual disability and its mutant forms were also identified in schizophrenic and epileptic patients. In other systems, studies have shown that γ CaMKII-associated pathway abnormalities are closely related to the development of obesity. And the abnormal activation of gamma CaMKII can accelerate the acute change of chronic myelocytic leukemia. However, the role of gamma CaMKII in body aging has never been reported.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a new application of gamma CaMKII. In order to achieve the purpose, the technical scheme of the invention is as follows:

one aspect of the invention relates to the use of a gamma Camk ii expression promoter or stabilizer in the preparation of a medicament for slowing aging and/or resisting neurodegeneration.

In a preferred embodiment of the invention, the gamma Camk ii expression promoter or stabilizer comprises an expressible vector with Camk2G gene and/or a small molecule drug targeting gamma Camk ii protein expression and stabilization.

In another aspect, the present invention relates to a method for preparing an animal model of aging, which is characterized by knocking out a gene expressing γ CaMKII.

In a preferred embodiment of the invention, the model animal is a mouse or a primate.

For those skilled in the art, the role of γ CaMKII in body aging is the first discovery by the inventors of the present application, and the present invention has significant meaning for developing anti-aging drugs.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1: gamma CaMKII antibody specificity detection

(A) Wild type mice (WT) and gamma CaMKII whole body knock-out (global gamma CaMKII KO) mice hippocampal brain slices gamma CaMKII (ab01 and ab02) (green) were stained with a representative image with NeuN (red) immunofluorescence double-labeled. (B) Statistical plot of gamma CaMKII immunofluorescence intensity in hippocampal neurons.

FIG. 2: variation of the amount of Gamma CaMKII protein in different organs of mice of different age groups

(A)2 months (2m) and 8 months (8m) mice hippocampal brain region gamma CaMKII (green) and PV (red) immunofluorescence staining representative and statistical plots. (B) Western Blot assay of spleen tissue gamma CaMKII protein levels in mice at 2 months (2m) and 6 months (6m) representative and statistical profiles of protein levels at different ages. (C) Western Blot assay results representative and statistical plots of gamma CaMKII protein levels in ovarian tissues of mice at 2 months (2m) and 12 months (12 m).

FIG. 3: debilitating phenotype associated with animal aging

Statistical scoring plots of eye secretions, cataracts, gait stability, hunchback, tumors, hair color, hair loss, dermatitis indices of Wild Type (WT), gamma CaMKII half-Hit (HET) and full-hit (KO) female and male rats at different age groups.

FIG. 4: typical map of debilitating phenotype associated with animal aging

Typical profile of the aging-associated phenotype (cataract, ocular secretions, dermatitis, tumors) in γ CaMKII full knock-out (KO) mice.

FIG. 5: reactome enrichment assay

(A) Gamma CaMKII half knock-out (HET) mice and wild type mice (WT) differential gene Reactome enrichment analysis result graphs; (B) gamma CaMKII full knock-out (KO) mouse and wild type mouse (WT) differential gene Reactome enrichment analysis result chart

Detailed Description

The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1:

(1) to determine the relationship between the key molecule in this pathway, gamma Camk II, and senescence, specific antibodies (ab01 and ab02) were prepared against it, where the ab01 antibody was directed against the gamma Camk II protein amino acid sequence 441-458: RTGNGSLVPEGRSPRDRTAP, immunizing rabbit with the peptide fragment as antigen, collecting serum, and purifying; the ab02 antibody was directed against the gamma Camk II protein amino acid sequence 369-386: RTGNGSLVPEGRSPRDRTAP (two antibodies were prepared by the company ThermoFisher Scientific using conventional methods), which are currently proprietary to the laboratory and not commercially available. After obtaining the antibody, in order to detect the specificity of the antibody, respectively perfusing and fixing a wild type mouse (WT) and a gamma Camk II whole body knockout mouse (KO), taking a brain, dehydrating the brain by 30% sucrose, slicing to obtain brain slices, taking the brain slices containing a hippocampal brain region, respectively co-staining the brain slices with an ab01 or ab02 antibody and a NeuN (neuron marker) antibody, shooting by using a confocal microscope to obtain an immunofluorescence picture, and analyzing and counting the expression condition of the gamma Camk II in the hippocampal brain region WT and the KO animal cerebral neurons. FIG. 1 shows the specificity of the antibody. The results show that significant protein signals can be detected in WT animals with ab01 or ab02, and in γ Camk ii knock-out animals these signals are clearly absent, indicating that the antibodies we produced are specific.

(2) Detecting the change of gamma Camk II protein quantity of different organs of the mouse along with the increase of age. The wild mouse is used as an observation object, different tissues of a young mouse, a middle aged mouse and an aged mouse (3 animals in each group) are respectively taken, and the amount of gamma Camk II protein is detected. Wherein, after the cerebral tissue is respectively perfused and fixed for 2 months and 8 months, the cerebral tissue is taken out and fixed, and then dehydrated, and the coronal section is 30 μm, and the results of the co-staining of gamma Camk II and PV by an immunofluorescence double-label method show that the amount of the gamma Camk II in the PV neurons of the cerebral cortex of the middle-aged mice is obviously reduced. The spleen tissues are anesthetized, the spleens of mice of different age groups (2 months, 4 months, 5 months, 6 months, 8 months and 17 months) are quickly taken out, RIPA lysate is added to the spleens, the tissues are cracked through ultrasonic disruption, the protein concentration is measured by a BCA protein quantitative method, then loading buffer is added to prepare protein loading solution, and the expression level of gamma Camk II in the spleens of the mice of different age groups is detected by a Western Blot method. The results showed a significant decrease in spleen tissue starting from 6 months from the mice. For female mouse ovarian tissues, 2-month and 12-month mouse ovarian tissues are respectively taken and lysed by RIPA lysate, the expression level of gamma Camk II in the female mouse is detected by a Western Blot method, and the result shows that the amount of gamma Camk II in the ovary of the female mouse in 12 months is obviously reduced compared with that of the female mouse in the young month. It can be seen that the amount of gamma Camk ii protein decreased in many organs (brain, spleen, ovary) of the body with age in the naturally aging mice. FIG. 2 shows the changes in gamma Camk II protein levels in different organs.

(3) To determine the relationship of γ CaMKII to body aging, γ CaMKII KO mice, which are systemic knock-out animals of γ CaMKII, were introduced, which were constructed in Eric n.olson laboratory in 2019 using the cre-loxp system (see Backs, Stein et al.2010). The observation animals were divided into 6 groups: male normal C57 mice (WT 24), male gamma CaMKII half-knock mice (HET 34), male gamma CaMKII full-knock mice (KO 18), female normal C57 mice (WT 16), female gamma CaMKII half-knock mice (HET 23), female gamma CaMKII full-knock mice (KO 15). Mice of different age groups (including female and male) were then observed for-3-4 months (3-4m),4-5 months (4-5m),5-6 months (5-6m), 8-9 months (8-9m),10-11 months (10-11m),11-12 months (11-12m) and age-related extrinsic debilitating phenotypes, and eight indices of eye secretions, cataracts, gait stability, kyphosis, tumors, hair color, hair loss, dermatitis, at different age groups per mouse were scored from 0 to 1, 0 for complete absence, 1 for most severe, 0.2 for a score scale, and if animal death indices were all 1.

The experimental results show that the extrinsic debilitating phenotype of γ CaMKII HET and KO mice, whether female or male, was scored higher than normal at each observation time point, indicating that it exhibited features of premature aging (see figure 3), with significant aging complications (e.g., large net, skin infection, cognitive impairment, obesity, hemiplegia, etc.), see figure 4.

(4) To further observe the molecular mechanism of the effect of gamma CaMKII knockout on the aging phenotype of animals, whole genome expression profiles (RNAseq) were analyzed by means of big data analysis. Normal C57 mice (WT), gamma CaMKII half-knock-out mice (HET) and gamma CaMKII full-knock-out mice (KO) hippocampal brain mRNA were extracted, respectively, and then RNAseq sequencing analysis was performed. Differential gene Reactome enrichment analysis is carried out on the HET group, the WT group, the KO group and the WT group, and the results show that the differential genes are enriched on a cell senescence pathway caused by DNA damage and a pathway for responding to external stimulation (figure 5), which indicates that the deletion of gamma CaMKII protein can cause the change of the cell internal pathway to cause cell senescence and further cause the premature senility of organisms.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, and these embodiments are within the scope of the invention.

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Claims

1. The application of a gamma Camk II expression promoter in preparing a medicine for slowing down aging is disclosed, wherein the gamma Camk II expression promoter comprises an expressible vector with a Camk2G gene.

2. A method for establishing an aging animal model is characterized in that a gene for expressing gamma CaMKII is knocked out.

3. The method of claim 2, wherein the animal is a mouse or primate.