Use of a DEK protein in the preparation of a medicament for activating dormant neural stem cells

Abstract

The application discloses application of DEK protein in preparation of a drug for activating dormant neural stem cells, and utilizes the DEK protein to activate the dormant neural stem cells, promotes neural regeneration and generates newborn neurons, realizes relieving the exhausted neural generation activity in the brain aging process, restarts neurogenesis, makes it equivalent to the neurogenesis level in the young brain, and in the brain stroke damage repair, the DEK protein can activate endogenous dormant neural stem cells, generates a large number of progeny neurons and astrocytes, accelerates the application of the damage repair process. The application relieves the application of the brain aging and the brain stroke damage repair. Overexpression of DEK in the adult dormant neural stem cells does not destroy the steady state of the neural stem cell library, and does not cause exhaustion of the neural stem cell library in a period of up to 8 months. The application has great application value in the clinic of various human neurodegenerative and injury diseases.

Description

(I) Technical Field

[0001] This invention relates to the application of DEK protein in the preparation of drugs that activate dormant neural stem cells, particularly the use of DEK protein to promote neurogenesis and generate new neurons in neurodegenerative and injury diseases, thereby achieving a cure for brain aging and stroke, and its clinical use in the treatment of various neurodegenerative and injury diseases. (II) Background Technology

[0002] Neurodegenerative (brain aging) and injury-related diseases, such as Alzheimer's disease, Parkinson's disease, stroke, and craniocerebral and spinal cord injuries, result in the death of functional neurons in different areas of the nervous system, leading to functional impairments in motor, language, and cognitive functions. Because apoptotic nerve cells cannot be replenished after neurodegenerative lesions, neurological diseases are among the leading causes of death and disability. Currently, there is no effective treatment for areas of complete cell death (such as the dark areas in ischemic stroke). Whether neurogenesis can occur in the adult brain is highly controversial. Although neural stem cells exist in the adult brain, they are in a dormant state for a long time, and their biological functions are not yet clear. How to activate dormant adult neural stem cells, promote neurogenesis to generate new neurons, and repair and replenish damaged or dead functional neurons has always been a major challenge in the effective treatment of neurodegenerative and injury-related diseases.

[0003] Neurogenesis in mammals primarily originates from neural stem cells in the subependymal region of the lateral ventricle and the subgranular layer of the dentate gyrus of the hippocampus. These stem cells proliferate and produce transitional neural progenitor cells and neuroblasts, migrating laterally to the olfactory bulb and radially along the granular cell layer, eventually differentiating into various neuronal subtypes. However, adult neural stem cells are largely dormant, and their biological functions under various neurodegenerative and injury conditions, as well as the regulatory mechanisms of their dormancy and activation, remain unclear. Once activated, dormant neural stem cells can generate activated neural stem cells with proliferative and differentiation capabilities, thus maintaining the stem cell pool. Through cell proliferation, migration, and differentiation, they can produce functional neurons and glial cells, thereby maintaining and reconstructing the function of the nervous system. Therefore, promoting neural regeneration by activating dormant neural stem cells holds great promise for the treatment of neurodegenerative diseases. Recent studies have found that various molecules regulating neural stem cell dormancy are closely related to the Notch signaling pathway. Among them, the HES family of Notch pathway effector molecules regulates the induction of neural stem cells into a dormant state by inhibiting Ascl1 gene expression and the degradation of the ASCL1 ubiquitination ligase HUWE1 (Ables, JL, Breunig, JJ, Eisch, AJ, and Rakic, P. (2011). Not(ch) just development: Notch signalling in the adult brain. Nat. Rev. Neurosci. 12, 269–283.). However, Notch knockout can activate dormant neural stem cells but quickly leads to the depletion of the stem cell pool, failing to achieve sustained neural regeneration.

[0004] To overcome the research bottleneck in the activation regulation of dormant neurons, we used Artemia salsa, an organism that lives in high-altitude salt lakes and can produce dormant embryos, and discovered that DEK protein can promote the transformation of heterochromatin to euchromatin structure by binding to chromatin, thereby epigenetically regulating the activation of dormant cells and causing Artemia salsa to break embryonic dormancy (Wen-HuanJia, An-Qi Li, Jing-Yi Feng, Yan-Fu Ding, Sen Ye, Jin-Shu Yang and Wei-JunYang (2019). DEK terminates diapause by activation of quiescent cells in the crustacean Artemia. Biochemical Journal 476 (12), 1753–1769.).

[0005] This invention constructs a mouse research model that activates dormant neural stem cells through conditional overexpression of DEK. It was found that conditional overexpression of DEK can activate dormant neural stem cells and exhibit a highly significant and durable neural regeneration capacity. Neural regeneration was observed in the brains of aged mice and stroke-affected mice, thereby alleviating brain aging in aged mice and showing a significant repair effect on stroke-affected mice. (III) Summary of the Invention

[0006] The purpose of this invention is to provide an application of DEK protein in the preparation of drugs that activate dormant neural stem cells, particularly by activating adult dormant neural stem cells through endogenous and exogenous DEK protein, promoting neural regeneration in the brains of adults, aged mice, and stroke-injured mice, and having a significant effect on slowing down brain aging in the elderly and repairing stroke damage.

[0007] The technical solution adopted in this invention is: In a first aspect, the present invention provides the application of DEK protein in the preparation of drugs that activate dormant neural stem cells.

[0008] Furthermore, the DEK protein has the conserved nucleic acid sequence shown in SEQ ID NO.1.

[0009] Furthermore, the DEK protein has more than 95% similarity to the amino acid sequence shown in SEQ ID NO.2.

[0010] Furthermore, the DEK protein corresponds to SEQ ID NO.1, has the conserved amino acid sequence (380 amino acids) shown in SEQ ID NO.2, and the encoding gene sequence is shown in SEQ ID NO.1 (the full-length DNA is 1143 bp).

[0011] The application of the DEK protein described in this invention in the preparation of drugs for activating dormant neural stem cells involves overexpressing endogenous DEK protein in dormant neural stem cells or delivering exogenous DEK protein into dormant neural stem cells to achieve the purpose of activating dormant neural stem cells.

[0012] Secondly, this invention provides an application of the DEK protein in the preparation of drugs for the prevention or treatment of brain nerve injuries, including stroke, Alzheimer's disease, Parkinson's disease, etc. The drug is prepared by expressing and purifying the DEK protein in vitro and then loading it onto exosomes, or by loading DEK onto a genetically engineered virus. The application involves using the DEK protein, expressed and purified in vitro and then loaded onto exosomes, as a formulation, or using a genetically engineered virus loaded with DEK as a means of gene therapy.

[0013] Thirdly, this invention provides an application of DEK protein in the preparation of a reagent for activating dormant neural stem cells, the reagent comprising endogenous and exogenous DEK proteins. The activation involves overexpressing endogenous DEK protein in dormant neural stem cells or delivering exogenous DEK protein into dormant neural stem cells to achieve the purpose of activating dormant neural stem cells. The reagent can be used for clinical treatment and basic scientific research of neurodegenerative and injury diseases.

[0014] The mechanism by which DEK protein activates dormant neural stem cells in this invention is as follows: Overexpression of DEK protein in mouse neural stem cells can activate dormant neural stem cells and promote neurogenesis. Specifically, DEK protein activates dormant neural stem cells by downregulating the expression of Notch, FOXO3, P53, and P21 genes and upregulating the expression of MYC and Wnt3A genes. This promotes the proliferation of these cells, generating a large number of neuroblasts. These neuroblasts then migrate laterally to the olfactory bulb and radially along the granule cell layer, eventually differentiating into different neuronal subtypes. These newly generated neurons further improve brain aging in aged mice and promote the repair of brain damage caused by stroke.

[0015] Compared with existing clinical treatments for brain aging and stroke, the beneficial effects of this invention are mainly reflected in the following: Neural stem cells in the adult brain are in a dormant state for a long time and cannot be activated to generate new neurons under aging and injury conditions. This is the main reason why neurodegenerative and injury-related diseases cannot be completely cured. Currently, clinical treatment for neurodegenerative diseases still focuses on slowing down the damage and apoptosis of neurons. For neuroinjury-related diseases, treatment focuses on protecting and restoring cells through eliminating inflammation, rebuilding blood supply and repairing the blood-brain barrier, and anti-oxidation. Currently, there is no effective treatment approach that generates new neurons. This invention utilizes DEK protein to activate dormant neural stem cells, promote neurogenesis, and generate new neurons. This alleviates the depleted neurogenesis activity during brain aging, restarts neurogenesis, and brings it back to a level comparable to that of a young brain. Furthermore, in stroke injury repair, DEK protein can activate endogenous dormant neural stem cells, generating a large number of progenitor neuronal blasts and astrocytes, accelerating the injury repair process. Applications include alleviating brain aging and repairing stroke injuries. Overexpression of DEK in adult dormant neural stem cells does not disrupt the homeostasis of the neural stem cell pool and does not cause the neural stem cell pool to fail for up to 8 months.

[0016] This invention enables the activation of dormant neural stem cells in mice through conditional overexpression of DEK protein, promoting neurogenesis in the brains of aged and stroke-injured mice, thereby alleviating brain aging and repairing nerve damage caused by stroke. Figure 1-3 In this experiment, significant new neurons were observed in the brains of aged mice and stroke-affected mice.

[0017] This invention is the first study and report on DEK protein activating dormant neural stem cells to generate a large number of new neurons, and it is also the first time that DEK protein treatment has been used to treat brain aging and stroke in mice. This invention has significant clinical application value in the treatment of various human neurodegenerative and injury diseases. (iv) Description of the attached drawings

[0018] Figure 1 This image shows the expression and distribution characteristics of DEK protein in neural stem and progenitor cells in the subependymal zone (SVZ) of the lateral ventricle of the brain, along with the results of immunofluorescence staining. A represents a schematic diagram of the sagittal and coronal planes of the mouse brain in the SVZ; B represents DEK expression in proliferating neural stem cells (Sox2+Nestin+Ki67+), with the right inset showing a magnified view of the area within the dashed box on the left; C represents DEK expression in hypermagnified cells (ASCL1+), with the right inset showing a magnified view of the area within the dashed box on the left; D represents DEK expression in neuroblastoma cells (DCX+), with the right inset showing a magnified view of the area within the dashed box on the left; E represents DEK expression in BrdU-incorporated dormant neural stem cells (Sox2+GFAP+BrdU-), with the right inset showing a magnified view of the area within the dashed box on the left; F represents a statistical graph showing the expression and distribution characteristics of DEK protein in neural stem and progenitor cells in the subependymal zone of the lateral ventricle of the brain. In the magnified image above, the dashed circle indicates the location of the cell nucleus in a single cell.

[0019] Figure 2 This image shows the expression and distribution characteristics of DEK protein in the subgranular cell layer (SGZ region) of the dentate gyrus in the brain, along with the results of immunofluorescence staining. A represents a schematic diagram of the sagittal and coronal planes of the mouse brain's SGZ region; B represents DEK expression in proliferating neural stem cells (GFAP+Nestin+Ki67+), with the right inset being a magnified view of the area within the dashed box on the left; C represents DEK expression in activated neural stem cells (ASCL1+), with the right inset being a magnified view of the area within the dashed box on the left; D represents DEK expression in neural progenitor cells (GFAP+TBR2+), with the right inset being a magnified view of the area within the dashed box on the left; E represents DEK expression in neuroblastoma cells (DCX+), with the right inset being a magnified view of the area within the dashed box on the left; F represents DEK expression in BrdU-incorporated dormant neural stem cells (Sox2+GFAP+BrdU-), with the right inset being a magnified view of the area within the dashed box on the left; G represents a statistical graph showing the distribution characteristics of DEK protein expression in neural stem progenitor cells of the dentate gyrus in the hippocampus. In the magnified images above, the dashed circles indicate the location of the nucleus in a single cell.

[0020] Figure 3 This image shows the changes in DEK protein in the neural stem cell region during brain aging. SVZ represents the subependymal region of the lateral ventricle, DG-SGZ represents the dentate gyrus of the hippocampus, LV represents the lateral ventricle, A1 represents the immunofluorescence image of DEK expression in the SVZ region of 2-month-old mice in neural stem cells (GFAP+Sox2+), A2 represents the immunofluorescence image of DEK expression in the SVZ region of 6-month-old mice in neural stem cells (GFAP+Sox2+), A3 represents the immunofluorescence image of DEK expression in the SVZ region of 12-month-old mice in neural stem cells (GFAP+Sox2+), A4 represents the statistical graph of the number of DEK-positive neural stem cells in the SVZ region of mice of different ages, and A5 represents the expression of DEK in the DG-SGZ region of 2-month-old mice. Immunofluorescence images of DEK expression characteristics in neural stem cells (GFAP+Sox2+) in the Z region; A6 represents the immunofluorescence image of DEK expression characteristics in the DG-SGZ region of 6-month-old mice in neural stem cells (GFAP+Sox2+); A7 represents the immunofluorescence image of DEK expression characteristics in the DG-SGZ region of 12-month-old mice in neural stem cells (GFAP+Sox2+); A8 represents the statistical graph of the number of DEK-positive neural stem cells in the DG-SGZ region of mice of different ages; B1 is a schematic diagram of the mouse brain, where SVZ represents the subgranular cell layer of the dentate gyrus of the hippocampus, LV represents the lateral ventricle, DG represents the dentate gyrus, and Hippocampus represents the hippocampus. Protein extraction was performed from the SVZ and DG regions; B2 represents the Western blot image of DEK expression levels in total protein extracted from the SVZ region; B3 represents the Western blot image of DEK expression levels in total protein extracted from the Hippocampus region; B4 is a statistical graph of the relative protein expression levels of DEK protein in the SVZ and Hippocampus regions.

[0021] Figure 4 This diagram illustrates and validates the construction principle of Nestin-driven DEK conditional overexpression mice; A represents the schematic diagram of the construction principle of conditional DEK overexpression mice. B represents the gel electrophoresis image for genotype identification of transgenic mouse double-positive progeny; lane 1 represents DNA standard molecular weight (100 to 2000 base pairs) markers, lane 2 represents Luc-EGFP positive genotype, lane 3 represents Luc-EGFP negative genotype, lane 4 represents DNA standard molecular weight (100 to 2000 base pairs) markers, lane 5 represents DEK-EGFP positive genotype, lane 6 represents DEK-EGFP negative genotype, lane 7 represents DNA standard molecular weight (100 to 2000 base pairs) markers, and lane 8 represents CreER... T2 Positive genotype, lane 9 represents CreER T2Negative genotype. C represents the DEK overexpression strategy in adult neural stem cells. D represents the expression of DEK protein in neural stem cells (GFP+) in the SVZ region of the brain of fluorescent control mice and conditionally DEK-overexpressing mice; the inset is a magnified image of the area within the dashed box. E represents a Western blot map showing the expression level of DEK protein in the total protein of the SVZ region of the brain of fluorescent control mice and conditionally DEK-overexpressing mice.

[0022] Figure 5 DEK overexpression activates dormant neural stem cells in the subependymal region of the lateral ventricle of the brain. A1 represents the immunofluorescence pattern of Ki67 expression, a cell proliferation marker, in the SVZ region of the brain of control and conditionally DEK-overexpressing mice after tamoxifen induction; yellow arrows indicate Ki67-positive neural stem cells. A2 represents a statistical graph of the number of proliferating neural stem cells (GFP+Ki67+) in the SVZ region of control and DEK-overexpressing mice. B1 represents a diagram of the BrdU incorporation retention experiment strategy. B2 represents the immunofluorescence pattern of BrdU, which remains labeled after dilution, in the SVZ region of the brain of control and conditionally DEK-overexpressing mice; the SVZ region is marked by a dashed box. B3 represents a statistical graph of the number of dormant neural stem cells (GFP+Sox2+BrdU+) in the SVZ region of control and DEK-overexpressing mice.

[0023] Figure 6 This diagram illustrates the activation of dormant neural stem cells in the subgranular region of the dentate gyrus of the hippocampus by DEK overexpression. A1 represents the characteristic immunofluorescence image of Ki67 expression (a cell proliferation marker) in the SGZ region of the brains of control and conditionally DEK-overexpressing mice after tamoxifen induction. Yellow arrows indicate Ki67-positive neural stem cells. A2 represents a statistical graph showing the number of proliferating neural stem cells (GFP+Ki67+) in the SGZ region of control and DEK-overexpressing mice. B1 represents the characteristic immunofluorescence image of BrdU incorporation retention assay (after dilution, the labeled BrdU remained) in the SGZ region of the brains of control and conditionally DEK-overexpressing mice. Dashed boxes mark the SVZ region. B2 represents a statistical graph showing the number of dormant neural stem cells (GFP+Sox2+BrdU+) in the SGZ region of control and DEK-overexpressing mice.

[0024] Figure 7This image shows the activation of neural stem cells in the subgranular layer of the dentate gyrus of the hippocampus in aged mice by AAV9-GFAP-DEK-GFP. A1 represents the viral injection strategy; A2 represents the injection site indicated by the green arrow on the mouse skull surface; A3 is a schematic diagram of the coronal plane viral injection; A4 is an immunofluorescence image of the coronal section of the brain after viral injection; A5 represents the immunofluorescence results of DEK protein expression in neural stem cells (GFP+GFAP+) in the SGZ region of the brain of mice in the control and DEK overexpression groups after viral infection, with yellow arrows indicating single cells. B1 represents the immunofluorescence image of the expression characteristics of the cell proliferation marker Ki67 in the SGZ region of the brain of mice in the control and DEK overexpression groups after viral infection, with the dashed box representing the dentate gyrus region; B2 represents the statistical graph of the number of proliferating neural stem cells (GFP+Ki67+) in the SGZ region of mice in the control and DEK overexpression groups.

[0025] Figure 8DEK overexpression promotes sustained neurogenesis in the subependymal region of the lateral ventricle, resulting in a large number of neuroblasts and new neurons. A represents the experimental strategy diagram. B1 represents the immunofluorescence map of DCX-positive neuroblast expression distribution in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 1 month of tamoxifen induction; B2 represents the immunofluorescence map of DCX-positive neuroblast expression distribution in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 4 months of tamoxifen induction; B3 represents the immunofluorescence map of DCX-positive neuroblast expression distribution in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 8 months of tamoxifen induction; C1 represents the immunofluorescence map of newly generated mature neurons (GFP+NeuN+) in the olfactory bulb region of the brains of control and conditionally overexpressing DEK mice after 1 month of tamoxifen induction; C2 represents the immunofluorescence map of 4 months of tamoxifen induction... Immunofluorescence images showing the expression and distribution of newly generated mature neurons in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 8 months of tamoxifen induction. C3 represents the immunofluorescence image showing the expression and distribution of newly generated mature neurons in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 8 months of tamoxifen induction. D1 represents the statistical graph showing the number of DCX-positive neuroblasts in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 1, 4, and 8 months of tamoxifen induction. D2 represents the statistical graph showing the number of newly generated mature neurons in the olfactory bulb region of the brains of control and conditionally overexpressing DEK mice after 1, 4, and 8 months of tamoxifen induction. D3 represents the statistical graph showing the number of GFAP+Sox2+ total neural stem cells in the SVZ region of the brains of control and conditionally overexpressing DEK mice after 1, 4, and 8 months of tamoxifen induction.

[0026] Figure 9DEK overexpression promotes sustained neurogenesis and the generation of numerous neuroblasts and new neurons in the subgranular region of the dentate gyrus of the hippocampus. A1 represents the immunofluorescence image of DCX-positive neuroblast expression distribution in the SGZ region of the brains of control and conditionally DEK-overexpressing mice after 1 month of tamoxifen induction; A2 represents the immunofluorescence image of DCX-positive neuroblast expression distribution in the SGZ region of the brains of control and conditionally DEK-overexpressing mice after 4 months of tamoxifen induction; A3 represents the immunofluorescence image of DCX-positive neuroblast expression distribution in the SGZ region of the brains of control and conditionally DEK-overexpressing mice after 8 months of tamoxifen induction; B1 represents the immunofluorescence image of newly generated mature neurons (BrdU+NeuN+) in the dentate gyrus region of the brains of control and conditionally DEK-overexpressing mice after 1 month of tamoxifen induction; B2 represents the immunofluorescence image of neurons (BrdU+NeuN+) in the dentate gyrus region of the brains of control and conditionally DEK-overexpressing mice after 4 months of tamoxifen induction. Immunofluorescence images showing the expression and distribution of newly generated mature neurons in the dentate gyrus of the brains of control and conditionally overexpressing DEK mice after 8 months of tamoxifen induction; B3 represents the immunofluorescence image showing the expression and distribution of newly generated mature neurons in the dentate gyrus of the brains of control and conditionally overexpressing DEK mice after 8 months of tamoxifen induction; C1 represents the statistical graph showing the number of DCX-positive neuroblasts in the SGZ region of the brains of control and conditionally overexpressing DEK mice after 1, 4, and 8 months of tamoxifen induction; C2 represents the statistical graph showing the number of newly generated mature neurons in the dentate gyrus of the brains of control and conditionally overexpressing DEK mice after 1, 4, and 8 months of tamoxifen induction; D3 represents the statistical graph showing the number of GFAP+Sox2+ total neural stem cells in the SGZ region of the brains of control and conditionally overexpressing DEK mice after 1, 4, and 8 months of tamoxifen induction.

[0027] Figure 10This section presents a heatmap showing the genes upregulated and downregulated by DEK overexpression in activating dormant neural stem cells. A represents a heatmap of differentially expressed genes from the transcriptome sequencing results of neural stem cells extracted from the control and conditionally DEK-overexpressing mouse groups. B represents a volcano plot of differentially expressed genes from the transcriptome sequencing results of neural stem cells extracted from the control and conditionally DEK-overexpressing groups. C represents a heatmap of expression profiles of genes related to cell dormancy activation regulation among the top 30 differentially expressed genes from the transcriptome sequencing results of neural stem cells extracted from the control and conditionally DEK-overexpressing groups. D represents a statistical graph of signaling pathways upregulated in the DEK-overexpressing group compared to the control group. E represents a heatmap of expression profiles of near-upstream regulatory genes directly related to cell dormancy activation regulation among the differentially expressed genes from the transcriptome sequencing results of neural stem cells extracted from the control and conditionally DEK-overexpressing groups.

[0028] Figure 11 DEK overexpression activates dormant neural stem cells and inhibits Notch signaling pathways. A1 represents the levels of DEK protein, internal control protein H3, β-actin, and signaling pathway proteins Notch1, Notch2, NICD, Hes1, Hes5, CSL, ASCL1, FoxO3a, P-FoxO3a, P53, P21, and MYC after tamoxifen induction. Western blot images showing changes in total protein expression in the SVZ region of the brains of mice in the control and conditionally DEK-overexpressing groups, as presented by Wnt3a et al. A2 represents the statistical graph of quantitative analysis of the Western blot results; B1 represents the immunofluorescence image showing characteristic expression of Notch1 in the SVZ region of the brains of mice in the control and conditionally DEK-overexpressing groups after tamoxifen induction; B2 represents the immunofluorescence image showing characteristic expression of Notch2 in the SVZ region of the brains of mice in the control and conditionally DEK-overexpressing groups after tamoxifen induction; and B3 represents the immunofluorescence image showing characteristic expression of Notch1 in the SGZ region of the brains of mice in the control and conditionally DEK-overexpressing groups after tamoxifen induction. The images are as follows: B4 represents the immunofluorescence image showing the characteristic expression of Notch2 in the SGZ region of the brain of mice in the control group and the conditionally overexpressing DEK group after tamoxifen induction; C1 represents the immunofluorescence image showing the characteristic expression of ASCL1 in the SVZ region of the brain of mice in the control group and the conditionally overexpressing DEK group after tamoxifen induction, with yellow arrows indicating ASCL1 positive cells; the small image is a magnified view of the dashed box in the large image. C2 represents the immunofluorescence image showing the characteristic expression of ASCL1 in the SGZ region of the brain of mice in the control group and the conditionally overexpressing DEK group after tamoxifen induction, with yellow arrows indicating ASCL1 positive cells; the small image is a magnified view of the dashed box in the large image.

[0029] Figure 12This study aims to activate dormant neural stem cells in the subependymal region of the lateral ventricle of aged mice by DEK overexpression. A represents the experimental strategy: 12-month-old aged control and conditionally DEK-overexpressing mice were induced with tamoxifen, labeled with BrdU for 7 days, and fed for another month before sampling and analysis. B1 represents the immunofluorescence images of GFP+Sox2+Ki67+ proliferating active neural stem cells in the SVZ region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction; the smaller image is a magnified view of the area within the dashed box in the larger image. B2 represents the statistical chart of the number of GFP+Sox2+Ki67+ proliferating active neural stem cells in the SVZ region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction.

[0030] Figure 13 This image shows the activation of dormant neural stem cells in the subgranular region of the dentate gyrus of the hippocampus in aged mice by DEK overexpression. Image A represents the immunofluorescence of GFP+Sox2+Ki67+ proliferating active neural stem cells in the SGZ region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction. Image B represents the statistical count of GFP+Sox2+Ki67+ proliferating active neural stem cells in the SGZ region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction.

[0031] Figure 14 This study demonstrates how DEK overexpression promotes sustained neurogenesis and the generation of numerous neuroblasts and new neurons in the subependymal region of the lateral ventricle of aged mice. A1 represents the immunofluorescence image of GFP+DCX+ neuroblasts migrating to the SVZ region and rostral migration flow in the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction. RMS represents rostral migration flow. The smaller image on the right is a magnified view of the area within the dashed box in the larger image. A2 represents the statistical graph of the number of GFP+DCX+ neuroblasts and RMS in the SVZ region and conditionally DEK-overexpressing mice one month after tamoxifen induction. B1 represents the immunofluorescence image of newly generated mature neurons (GFP+NeuN+) in the olfactory bulb region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction. B2 represents the statistical graph of the number of newly generated mature neurons (GFP+NeuN+) in the olfactory bulb region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction. C1 represents the immunofluorescence image of newly generated mature neurons in the olfactory bulb region of the brain of 2-month-old, 5-month-old, 9-month-old, and 12-month-old control and 12-month-old conditionally overexpressing DEK mice after 1 month of tamoxifen induction. C2 represents the statistical graph of the number of newly generated mature neurons in the olfactory bulb region of the brain of 2-month-old, 5-month-old, 9-month-old, and 12-month-old control and 12-month-old conditionally overexpressing DEK mice after 1 month of tamoxifen induction.

[0032] Figure 15 This study demonstrates how DEK overexpression promotes sustained neurogenesis and the generation of numerous neuroblasts and new neurons in the subgranular region of the dentate gyrus of the hippocampus in aged mice. A1 represents the immunofluorescence image of GFP+DCX+ neuroblasts in the SGZ region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction; A2 represents the statistical graph of the number of GFP+DCX+ neuroblasts in the SGZ region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction; B1 represents the immunofluorescence image of GFP+ new cells in the hippocampus region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction; B2 represents the statistical graph of the number of GFP+ new cells in the hippocampus region of the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction; C1 represents the newly generated mature neurons of GFP+NeuN+ in the brains of control and conditionally DEK-overexpressing mice one month after tamoxifen induction. Immunofluorescence images of the dentate gyrus region: C2 represents the number of newly generated mature neurons (GFP+NeuN+) in the dentate gyrus region of the brains of control and conditionally overexpressing DEK mice after 1 month of tamoxifen induction; D1 represents the immunofluorescence images of newly generated mature neurons (BrdU+NeuN+) in the dentate gyrus region of the brains of 12-month-old control and 12-month-old conditionally overexpressing DEK mice after 1 month of tamoxifen induction; D2 represents the number of newly generated mature neurons (BrdU+NeuN+) in the dentate gyrus region of the brains of 12-month-old control and 12-month-old conditionally overexpressing DEK mice after 1 month of tamoxifen induction.

[0033] Figure 16 This study aimed to establish a mouse model of striatal hemorrhagic stroke. A1 represents the experimental strategy: adult wild-type C57BL / 6 mice were stereotactically injected with type IV collagenase, and samples were collected for analysis 24 hours later. A2 represents a photograph of the lesion induced by collagenase IV-induced intracranial hemorrhage. B represents immunofluorescence staining images of Casp3, a marker of apoptosis, in the lesion and undamaged areas. C represents immunofluorescence staining images of NeuN in mature neurons in the lesion and undamaged areas.

[0034] Figure 17To investigate the effect of DEK overexpression on sustained neurogenesis in the subependymal region of the lateral ventricle in stroke-inducing mice. A represents the experimental strategy: adult control mice and conditionally DEK-overexpressing mice underwent stereotactic injection of type IV collagenase to induce hemorrhagic stroke. After 7 days of continuous EdU drinking water and a total 30-day injury repair cycle, samples were collected for analysis. B represents immunofluorescence staining of the brain lesion area in control and conditionally DEK-overexpressing mice one month after hemorrhagic stroke induced by tamoxifen, with green dashed boxes marking the area around the lesion. C1 represents immunofluorescence staining of GFP+DCX+ neuronal blasts in the brain lesion area of ​​control and conditionally DEK-overexpressing mice one month after hemorrhagic stroke induced by tamoxifen, with white dashed lines indicating newly formed neuronal blasts. C2 represents the number of GFP+DCX+ neuronal blasts in the brain lesion area of ​​control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen; D1 represents the immunofluorescence staining image of newly formed GFP+GFAP+ astrocytes in the brain lesion area of ​​control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen, with a magnified image of the area within the dashed box on the right; D2 represents the number of GFP+GFAP+ astrocytes in the brain lesion area of ​​control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen.

[0035] Figure 18This study demonstrates how DEK overexpression promotes the generation of numerous mature neurons in the subependymal region and striatum of the lateral ventricle in stroke-inducing mice. A1 represents the immunofluorescence staining of GFP+EdU+ neonatal cells in the brain lesion region of control and conditionally DEK-overexpressing mice one month after hemorrhagic stroke induced by tamoxifen. White dashed lines indicate newly generated neuronal mother cells. A2 represents the statistical count of GFP+EdU+ neonatal cells in the brain lesion region of control and conditionally DEK-overexpressing mice one month after hemorrhagic stroke induced by tamoxifen. B1 represents the immunofluorescence staining of GFP+NeuN+ newly generated mature neurons in the brain lesion region of control and conditionally DEK-overexpressing mice one month after hemorrhagic stroke induced by tamoxifen. White dashed lines indicate newly generated neurons. The graph shows the number of newly formed mature neurons (GFP+NeuN+) in the brain lesion region of control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen. B2 represents the number of newly formed mature neurons (GFP+NeuN+) in the brain lesion region of control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen. C1 represents the immunofluorescence staining image of newly formed mature neurons (GFP+NeuN+) in the brain lesion region of control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen. White dashed lines indicate newly formed neuronal mother cells. C2 represents the number of newly formed mature neurons (GFP+NeuN+) in the brain lesion region of control and conditionally overexpressing DEK mice one month after hemorrhagic stroke induced by tamoxifen.

[0036] Figure 19 A schematic diagram illustrating how DEK activates dormant neural stem cells in mice to regress, proliferate, differentiate, contribute to neurogenesis, and participate in maintaining brain function and repairing nerve damage. (V) Detailed Implementation Methods

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Unless otherwise specified, all percentage concentrations in the embodiments of this invention are volume concentrations.

[0038] The final concentration composition of the phosphate buffer (PBS) used in this invention is: 137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate dodecahydrate and 1.76 mM potassium dihydrogen phosphate, with water as the solvent and pH adjusted to 7.4.

[0039] All mice in this invention were housed in a sterile environment with a continuous supply of water and food, and their bedding was changed regularly. All mouse experiments underwent ethical review, and the experimenters adhered to animal welfare principles during the experiments.

[0040] Table 1. Reagents and antibodies used in the embodiments of the present invention:

[0041] Example 1: Expression and distribution of DEK in adult neural stem cell lineages

[0042] 1. Mouse strains

[0043] Eight-week-old adult wild-type C57BL / 6 mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were fed BrdU drinking water (1 mg / mL) for 14 consecutive days, and brain tissue samples were directly obtained. Immunofluorescence was used to detect the co-localization of a series of neural stem / progenitor cell markers with DEK to study the expression and distribution characteristics of DEK in the neural stem cell region in vivo.

[0044] 2. Immunofluorescence detection of DEK, Sox2, Nestin, GFAP, Ki67, BrdU, ASCL1, DCX, and TBR2

[0045] a. Mouse cardiac perfusion and brain tissue fixation

[0046] (1) Preparation of 4% paraformaldehyde fixative: Weigh a certain amount of paraformaldehyde into PBS, heat it in a 60°C water bath to dissolve it, and mix it occasionally during the process. After preparing the 4% paraformaldehyde solution, cool it to room temperature and store it at 4°C for later use. At the same time, pre-cool physiological saline.

[0047] (2) Anesthetize the mice with isoflurane using a small animal anesthesia system until the limbs of the experimental mice are touched and there is no nerve reflex.

[0048] (3) After the mice are fully anesthetized, they are placed in a supine position and fixed with a stainless steel syringe needle on a foam board covered with an absorbent pad. The chest is disinfected with 70% ethanol and the fur is wetted. The chest skin is gently grasped with tissue forceps and lifted up about 0.5 cm. The skin of the chest cavity and the left and right ribs are cut open with dissecting scissors to fully expose the heart and liver.

[0049] (4) Using a syringe filled with pre-cooled 0.9% saline, attach an indwelling intravenous catheter, insert the needle tip into the left ventricle of the mouse heart, cut a small opening in the right atrial valve with dissecting scissors, and after blood flowed out, slowly push the syringe to infuse saline. The infusion volume is about 20 mL, and the liver will appear white and bloodless.

[0050] (5) After the physiological saline perfusion is completed, the syringe containing 4% paraformaldehyde solution is replaced and the perfusion continues. The perfusion volume is about 40 mL. After the mice are fixed with paraformaldehyde, the body is stiff and the tail is rigid, indicating that the perfusion is complete.

[0051] (6) Cut the mouse from the neck and shoulder with tissue scissors, cut open the head skin, repeatedly and lightly scratch the skull with a blade, then peel off the skull with tissue forceps, remove the brain, cut it into brain tissue blocks 2 to 5 mm thick in the coronal or sagittal plane, and immerse it in 4% paraformaldehyde solution overnight in a refrigerator at 4°C.

[0052] b. Frozen sections of brain tissue

[0053] (1) The fixed brain tissue was transferred to a PBS solution containing 30% sucrose for dehydration at 4°C. The brain tissue was dehydrated when it floated in the sucrose solution until it sank to the bottom. The processed brain tissue was completely wrapped in an appropriately sized mold box with OCT embedding agent, equilibrated for 15 min, transferred to dry ice for cryo-embedding, and stored in an ultra-low temperature freezer at -80°C.

[0054] (2) Pre-cool the microtome to -20°C and place the sample inside the microtome to equalize the temperature.

[0055] (3) Apply OCT to the base of the microtome, attach the embedded brain tissue sample to the base, place it in the quick-freezing stage of the cryostat to freeze the newly applied OCT, and load the sample onto the base.

[0056] (4) Adjust the machine parameters to trim the slides to a thickness of 25 μm, discarding slides from non-target areas until the target area is found. Readjust the machine parameters to trim the slides to a thickness of 10 μm, and prepare slides for brain sections of the subependymal zone (SVZ) of the lateral ventricle and the subgranular cell zone (SGZ) of the dentate gyrus of the hippocampus. Slice the SVZ and SGZ tissues to a thickness of 10 μm. The resulting frozen brain sections adhere to the slides. The frozen brain sections can be stored in an ultra-low temperature freezer for long-term use.

[0057] c. Immunohistofluorescence staining

[0058] (1) Equilibrium: The slides of the SVZ and SGZ brain slices prepared in step b were air-dried at room temperature for 30 min and then rinsed with PBS for 5 min to remove residual OCT.

[0059] (2) Secondary fixation: Draw a hydrophobic zone near the tissue with an immunohistochemical pen, add an appropriate amount of 2% paraformaldehyde into the hydrophobic zone, treat at room temperature for 5 min, rinse with PBS 3 times for 5 min each time, and preheat the hot water bath to 68℃.

[0060] (3) Penetration: Use a pipette to add an appropriate amount of PBS containing 0.25% Triton X-100 to the hydrophobic zone for penetration treatment, incubate at room temperature for 15 min, and rinse with PBS 3 times, 5 min each time.

[0061] (4) Antigen retrieval: Immerse the slides in a staining box containing pH 9 antigen retrieval solution, incubate at 68°C for 20 min, then transfer to cold water to cool for 10 min, and rinse the slides with PBS 3 times for 5 minutes each time.

[0062] (5) Blocking: Add an appropriate amount of blocking solution (i.e., PBS containing 10% donkey serum for blocking and 0.1% Triton X-100) to the hydrophobic zone using a pipette, and incubate at room temperature for 1 h.

[0063] (6) Primary antibody incubation: Prepare the primary antibody dilution buffer (i.e., dilute the antibody stock solution in the blocking buffer at a certain ratio). Use a pipette to aspirate and discard the blocking buffer in the hydrophobic zone, then add the prepared primary antibody dilution buffer to it using a pipette, and incubate overnight at 4°C. The antibody dilution ratios are as follows: DEK antibody, 1:200; GFAP antibody, 1:1000; Sox2 antibody, 1:500; Nestin antibody, 1:500; Ki67 antibody, 1:200; TBR2 antibody, 1:100; DCX antibody, 1:200; BrdU antibody, 1:100; ASCL1 antibody, 1:100. After the primary antibody incubation, wash three times with PBS, 5 min each time.

[0064] (7) Secondary antibody incubation: Prepare the fluorescent secondary antibody dilution solution (i.e., dilute the fluorescein-conjugated IgG / Y stock solution (Jackson, USA) in the blocking solution at a volume ratio of 1:200), and then add the prepared secondary antibody dilution solution to the hydrophobic zone in step (6) using a pipette, and incubate at room temperature for 2 h. After the secondary antibody incubation is completed, rinse 3 times with PBS for 5 min each time.

[0065] (8) EdU staining (optional): Tissue samples labeled with EdU must be stained with EdU to detect its signal. The specific method is to prepare the EdU staining working solution according to the EdU Click kit formula, and then use a pipette to add the hydrophobic zone after the secondary antibody incubation in step (7) to fully cover the tissue section. Incubate at room temperature for 45 min. Rinse 3 times with PBS, 5 min each time.

[0066] (9) Nuclear staining: Add an appropriate amount of DAPI staining solution to the hydrophobic zone after EdU staining in step (7) or (8) using a pipette, and incubate at room temperature for 15 min to stain the cell nuclei. Rinse with PBS 3 times, 5 min each time.

[0067] (10) Mounting: Add an appropriate amount of anti-fluorescence quenching mounting solution to the hydrophobic zone using a pipette. Use tweezers to pick up the slide and slowly cover it with a coverslip along the edge of the droplet, avoiding the formation of air bubbles. Store the mounted sample slices away from light for later use, to detect fluorescence signals using a laser confocal scanning microscope.

[0068] We used adult wild-type mice and performed a BrdU serial incorporation experiment, and immunofluorescence detection of a series of neural stem / progenitor cell markers and DEK co-localization. The results showed that in the SVZ region ( Figure 1 DEK protein is abundantly expressed in proliferating neural stem cells (Sox2+Nestin+Ki67+), over-amplified cells (ASCL1+), and neuroblastoma cells (DCX+), but is almost not expressed in dormant neural stem cells (Sox2+GFAP+BrdU-).

[0069] In the SGZ area ( Figure 2 DEK protein is also abundantly expressed in proliferating neural stem cells (GFAP+Nestin+Ki67+), activated neural stem cells (ASCL1+), neural progenitor cells (GFAP+TBR2+), and neuronal blasts (DCX+), while in dormant neural stem cells (Sox2+GFAP+BrdU-), less than 50% of cells express DEK at low levels.

[0070] Conclusion: These results indicate that DEK protein is almost unexpressed or poorly expressed in dormant neural stem cells, but is abundantly expressed in proliferating neural stem cells, hyperamplified cells, and neuronal blasts. It may be an essential factor for neural stem cell activation and participates in the regulation of neural stem cell dormancy and activation.

[0071] Example 2: Changes in DEK during brain aging

[0072] 1. Mouse strains

[0073] Adult wild-type C57BL / 6 mice aged 2 months, 6 months, and 12 months (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were used to study the changes in DEK expression characteristics during brain aging.

[0074] 2. Immunofluorescence detection of DEK expression in neural stem cells of mice at different ages

[0075] DEK immunofluorescence analysis (GFAP, DEK) was performed on mice of different ages (same as step 2 in Example 1). The results are shown below. Figure 3 During the aging process from 2 months to 12 months after birth, the number of DEK-positive neural stem cells in the SVZ and SGZ regions decreased continuously from A1 to A8, decreasing by about 75%.

[0076] 3. Detection of DEK protein level changes by Western blotting assay

[0077] Microscopic anatomy and protein extraction of a.SVZ and hippocampal stem cell region

[0078] The experimental mice were sacrificed by dislocation, and fresh brain tissue was removed using dissecting scissors and surgical forceps. In PBS, a scalpel was used to make a vertical incision along the middle of the lateral ventricle of the forebrain, exposing the bilateral ventricular cavities. The SVZ region of the lateral ventricle wall and the hippocampus were cut using the tip of curved, sharp dissecting forceps. Figure 3 (B1). After removing excess water from the dissected tissues, they were quickly transferred to centrifuge tubes and temporarily stored in liquid nitrogen. This study used TRIzol reagent to extract total protein from the tissues; the specific method is as follows: (1) Grind 50-100 mg of tissue thoroughly with 1 mL of TRIzol reagent using a hand-held homogenizer. After mixing, centrifuge at 12000 g for 10 min at 4°C, transfer the supernatant to a new tube, and let stand at room temperature for 5 min.

[0079] (2) Add 0.2 mL of chloroform, shake well for 15 s, let stand at room temperature for 5 min, then centrifuge at 12000 g for 15 min at 4℃. The upper transparent aqueous phase can be used to extract total RNA, and the lower organic phase can be used for the following protein extraction steps.

[0080] (3) Add 0.3 mL of anhydrous ethanol, mix well until the solution is clear, let stand at room temperature for 5 min, then centrifuge at 2000 g for 10 min at 4℃ and discard the supernatant.

[0081] (4) Add 1.5 mL of isopropanol, let stand at room temperature for 10 min, then centrifuge at 12000 g for 10 min at 4℃ and collect the precipitate.

[0082] (5) Add 2 mL of 0.3M guanidine hydrochloride (dissolved in anhydrous ethanol), crush the precipitate with a pipette tip, rinse on a shaker for 20 min, centrifuge at 7500 g for 5 min at 4℃, and collect the precipitate. This step needs to be repeated twice.

[0083] (6) Resuspend the precipitate with 1 mL of anhydrous ethanol, place at room temperature for 10 min, centrifuge at 7500 g for 5 min at 4℃, remove the anhydrous ethanol, collect the precipitate, and open the centrifuge tube cap until the precipitate is completely dry.

[0084] (7) After drying, the precipitate is incubated in a 55°C water bath overnight with an appropriate amount (about 50-100 μL) of 2% SDS aqueous solution, depending on the amount of precipitate, until the protein is completely dissolved. Then, 5X loading buffer (DTT) is added, and the mixture is thoroughly mixed. The mixture is then incubated in a boiling water bath for 10 min to denature the protein. The protein is then centrifuged at 12000 g for 10 min at room temperature. The supernatant is collected as the protein extract. The protein concentration is determined by the Bradford method and stored at -30°C for later use.

[0085] b. Western blot detection of DEK proteins

[0086] (1) Gel preparation: SDS-polyacrylamide gel was prepared according to the molecular weight of the target protein. The gel concentration used for detecting target proteins of different molecular weights in this experiment was as follows: 12.5% ​​for protein molecular weight ≤70 kDa; 10% for protein molecular weight ≤100 kDa; and 8% for protein molecular weight ≥100 kDa.

[0087] (2) Electrophoresis: Using 1X SDS electrophoresis buffer, load the prepared gel into the ATTO electrophoresis tank, gently blow the buffer in the gel well with a pipette to ensure that the gel well is clean, add the protein extract and standard molecular weight protein sample into the gel well, and perform electrophoresis separation at a constant current of 15 mA per gel.

[0088] (3) Transfer: After electrophoresis, remove the upper stacking gel with a gel release plate, and temporarily store the lower separating gel in the prepared transfer buffer (0.01 M glycine, 0.01 M tris(hydroxymethyl)aminomethane, 10% methanol, solvent: water). Wet the transfer filter paper and the methanol-activated PVDF membrane with the transfer buffer. Using a semi-dry transfer apparatus, place the filter paper, PVDF membrane, gel, and filter paper in that order to form a transfer "sandwich". Moisten each component with a small amount of transfer buffer and use the pusher to make the "sandwich" flat and free of air bubbles. Transfer each transfer "sandwich" at a constant voltage of 7V for 1 hour.

[0089] (4) Antibody incubation: After transfer, the PVDF membrane was rinsed with 1X TBS for 30 s and incubated with 1% blocking buffer at room temperature for 1 h; after blocking, the PVDF membrane was incubated with the prepared primary antibody dilution buffer at 4℃ for 24 h; after primary antibody incubation, the PVDF membrane was rinsed three times with TBS containing 0.5% Tween 20 for 5 min each time and incubated with 0.5% blocking buffer at room temperature for 20 min; after blocking, the secondary antibody dilution buffer corresponding to the primary antibody species was incubated at room temperature for 40 min; after secondary antibody incubation, the PVDF membrane was rinsed three times with TBS containing 0.5% Tween 20 for 5 min each time and set aside. Thanks to the high analytical sensitivity of Western blotting, the primary antibody used in the experiment was diluted at a ratio of 1:1000-1:5000 according to the antibody instructions, and the HRP-conjugated secondary antibody was diluted at a ratio of 1:2500.

[0090] (5) Development: The results of this experiment are based on the detection of horseradish peroxidase coupled to the secondary antibody molecule. The reaction solution was prepared using a chemiluminescence immunoassay kit (ECL). After the reaction solution was dropped onto the PVDF membrane, it was placed in a chemiluminescence imaging instrument, and the experimental results were recorded. The results are shown in […]. Figure 3From B2 to B4, the DEK protein level also decreases with age, decreasing to about 40% of that in older mice (12 months old) compared to younger mice (2 months old).

[0091] Conclusion: Immunofluorescence staining and Western blotting experiments revealed that DEK may be associated with brain aging, especially the aging of neural stem cells.

[0092] Example 3: DEK overexpression activates dormant neural stem cells in the SVZ and SGZ regions.

[0093] 1. Obtaining double-positive offspring from transgenic mice

[0094] a. Construction of transgenic mice

[0095] The R26-DEK-EGFP mouse model was constructed by Shanghai Southern Model Biotechnology Co., Ltd. The construction principle involved targeting ES cells (mouse embryonic stem cells) using homologous recombination, with a CAG promoter-loxp-Neo-loxp-Dek-IRES-EGFP-polyA expression cassette inserted at the Rosa26 gene locus. The brief procedure is as follows: An ES cell targeting vector was constructed using seamless cloning. This vector contained a 1.1 kb 5' homologous arm, a CAG promoter, loxp-PGK-Neo-polyA-loxp, a Dek-IRES-EGFP coding region, a 4.3 kb 3' homologous arm, and an MC1-DTA-polyA negative selection marker. After linearization, the vector was electroporated into ES cells. After screening with neomycin (G418) and guanosine propionate (Ganc), 144 resistant ES cell clones were obtained; long-fragment PCR identification revealed 15 positive ES cell clones with correct homologous recombination. Positive ES cell clones were expanded and injected into the blastocysts of C57BL / 6J mice to obtain chimeric mice. High-proportion chimeric mice were mated with C57BL / 6J mice to obtain 8 positive F1 generation Neo mice, which were then used to obtain R26-DEK-EGFP mice. The R26-DEK-EGFP heterozygous mice showed no significant abnormalities in development or reproduction.

[0096] b. Double-positive offspring of transgenic mice

[0097] Reference Figure 4The diagram illustrates the construction principle of conditionally overexpressing DEK mice in the A-type gene. The DEK-iresEGFP sequence is inserted post-progenitor of the Rosa26 promoter using transgenic technology and remains in an inactive state due to the presence of a preceding termination sequence. In the double-positive offspring obtained by crossbreeding these mice with neural stem cell-specific tool mice (Nestin-CreERT2), the termination sequence in the neural stem cell genome is removed upon tamoxifen induction, initiating DEK overexpression. Similarly, neural stem cell fluorescent reporter mice are obtained through crossbreeding using Luc-EGFP fluorescent control mice, serving as the control group for all experiments.

[0098] The presence of the loxp-PGK-Neo-polyA-loxp expression cassette prevents the transcription of the downstream target gene Dek-IRES-EGFP. The expression site and efficiency of the target gene Dek-IRES-EGFP depend on the tissue type and efficiency of Cre expression. After mating R26-DEK-EGFP mice with Cre mice, in their offspring double-positive mice, the loxp-PGK-Neo-polyA-loxp expression cassette in cells expressing Cre recombinase is knocked out, and the target gene is highly expressed under the drive of the CAG promoter.

[0099] The double-positive experimental mice used in this experiment were obtained by crossing 2-month-old Nestin-CreERT2 (purchased from Jackson Laboratory, catalog number 016261) tool mice with 2-month-old R26-DEK-EGFP mice (constructed in step a), and were denoted as double-positive experimental mice. Nestin-CreER T2 ::R26-DEK-EGFP .

[0100] Double-positive fluorescent control mice were obtained by crossing 2-month-old Nestin-CreERT2 tool mice with 2-month-old R26-Luc-EGFP mice (purchased from Shanghai Southern Model Organisms, catalog number NM-KI-00087), and were designated as double-positive fluorescent control mice. Nestin- CreER T2 ::R26-Luc-EGFP .

[0101] All experiments used genotype-identified double-positive mice.

[0102] c. Genotyping

[0103] Methods for identifying the genotype of newborn mice: (1) Cut off the tails or toes of appropriate length from the double-positive experimental mice and double-positive fluorescent control mice in step b and place them in microcentrifuge tubes pre-filled with 50 μL DNA Extraction Buffer. Centrifuge with a mini centrifuge to fully immerse the mouse tissues in the solution.

[0104] (2) Place the microcentrifuge tube after centrifugation in step (1) into a PCR instrument and digest it at 55℃ for 15 min; 95℃ for 5 min; 16℃ for 10 min. Then add 50 μL Stop Solution to the centrifuge tube to stop the digestion. No centrifugation is required. Take a small amount of supernatant for subsequent detection. The resulting genomic template is stored in a 4℃ refrigerator for a short period of time.

[0105] (3) Genotyping

[0106] Take 1 μL of the supernatant from the double-positive experimental mice and the double-positive fluorescent control mice obtained in step (2) for genomic identification PCR. The supernatant from both the double-positive experimental mice and the double-positive fluorescent control mice was prepared using the primers Cre / Globin in Table 2. The PCR reaction was performed according to the reaction system and procedure in Table 3. Cre genotype( Figure 4 (lanes 2 and 3 corresponding to B in the middle lane); the supernatant of double-positive mice was used with primers R26-DEK / P1, P2, and P3 in Table 2, and PCR reaction was performed according to the reaction system and procedure in Table 4 for identification. R26-DEK-EGFP genotype( Figure 4 Lanes 5 and 6 (corresponding to B in the middle lane) were used. The supernatant from the fluorescent control mice was analyzed using primers R26-Luc / P1, P2, P3, and P4 from Table 2, and PCR was performed according to the reaction system and procedure in Table 5. R26-Luc- EGFP genotype( Figure 4 Lanes 8 and 9 correspond to lane B in the middle section.

[0107] The results are as follows Figure 4 As shown in Figure B, we successfully obtained double-positive experimental mice for subsequent experiments.

[0108] Table 2. Primer sequences for genotype identification

[0109] Table 3 Cre Genotypic PCR reaction system and procedure

[0110] Table 4 R26-DEK-EGFP Genotypic PCR reaction system and procedure

[0111] Table 5 R26-Luc-EGFP Genotypic PCR reaction system and procedure

[0112] 2. Obtaining DEK-overexpressing mice and fluorescent control mice

[0113] Weigh out tamoxifen and use a corn oil:anhydrous ethanol mixture (9:1 by volume) as a solvent. First, resuspend the tamoxifen powder in anhydrous ethanol to disperse it, then add corn oil. Heat in a 37°C water bath to aid dissolution, stirring occasionally to accelerate the process, until a clear and transparent 20 mg / mL tamoxifen solution is obtained. Use freshly prepared tamoxifen solution for each injection.

[0114] Reference Figure 4 The adult neural stem cell overexpression strategy of C involved weighing 2-month-old experimental mice (double-positive experimental mice and double-positive fluorescent control mice) constructed in step 1, and then injecting them intraperitoneally with tamoxifen solution at a dose of 10 μL / g once a day for 5 consecutive days to obtain conditionally overexpressing DEK mice and fluorescent control mice. Three days after the last injection, samples (SVZ region) were taken for immunofluorescence analysis (same as step 2 in Example 1) to analyze the expression of DEK protein in neural stem cells (GFP+) in the SVZ region of the brain of the two groups of mice. Figure 4 (D), the expression level of total protein in the SVZ region of the brain of the two groups of mice was analyzed by immunoblotting. Figure 4 (E).

[0115] 3. Immunofluorescence analysis of DEK activation of dormant neural stem cells

[0116] Three days after the last injection, tamoxifen-induced conditionally overexpressing DEK mice and fluorescent control mice were sampled from the subependymal region of the lateral ventricle and the subgranular cell layer of the dentate gyrus of the hippocampus, respectively, for GFP / Ki67 and GFP / Sox2 / BrdU immunofluorescence analysis (same as step 2 in Example 1). The antibody dilution ratios were as follows: GFP antibody, 1:1000; GFAP antibody, 1:1000; Sox2 antibody, 1:500; Ki67 antibody, 1:200; BrdU antibody, 1:100.

[0117] Immunofluorescence staining analysis of the cell proliferation marker Ki67 revealed that, compared to the control group, overexpression of DEK directly activated dormant neural stem cells, and proliferating neural stem cells (GFP+Ki67+) were observed in the SVZ region (…). Figure 5 (A1 and A2) and SGZ area ( Figure 6 The number of A1 and A2 in the middle increased significantly.

[0118] 4. BrdU incorporation retention experiment

[0119] Reference Figure 5 In step B1, we simultaneously conducted a BrdU incorporation retention experiment. The double-positive experimental mice and double-positive fluorescent control mice from step 1 were fed BrdU-treated drinking water for 14 consecutive days, then induced with tamoxifen for 5 days as in step 2, and continued to be fed normal drinking water for 12 days. Samples were then taken from the SVZ and SGZ regions, respectively, and fluorescence staining analysis was performed using the method in step 2 of Example 1. The results are shown in [Figure 1]. Figure 5 Chinese B2, B3 and Figure 6 B1 and B2.

[0120] The results showed that after DEK overexpression, the number of neural stem cells with BrdU retention markers (GFP+Sox2+BrdU+) in the SVZ and SGZ regions was significantly reduced, indicating that DEK overexpression activated dormant neural stem cells, promoted their proliferation and division, and diluted the BrdU markers.

[0121] 5. DEK overexpression injection

[0122] The method for activating neural stem cells by stereotactic injection of DEK-overexpressing virus and fluorescent control virus into the brains of wild-type mice is as follows: (1) The aged mice C57BL / 6J (12 months old) were anesthetized with 1.5% isoflurane gas using a small animal anesthesia machine.

[0123] (2) After removing the hair from the surface of the mouse’s head with scissors, fix the head on the stereotaxic instrument. Use anatomical forceps to open the mouse’s mouth and attach the upper jaw teeth to the buckle of the stereotaxic instrument. Rotate the fixing screw on the instrument so that it can just fix the mouse’s nose. Finally, insert the fixing rods on both sides into the mouse’s ears to anchor its head.

[0124] (3) Wipe the scalp with iodine to disinfect the surface of the skin, make a small incision with a scalpel, and cut the incision open with tissue scissors.

[0125] (4) Use tissue forceps to pull the cut skin to both sides, use a cotton swab to apply saline to remove the meninges, find the anterior fontanelle on the skull, and mark it as the origin of the coordinate system with a marker.

[0126] (7) Fixed point coordinates: The former brine is taken as the origin ( Figure 7 Mark the coordinates (A2, A3) 2.0 mm backward and 1.5 mm to the right with a marker, drill the hole with a cranial drill, and maintain the drilling force while holding the drill to avoid damaging the brain tissue.

[0127] (8) Draw 2 μL of virus solution (AAV9-GFAP-GFP or AAV9GFAP-DEK-GFP, purchased from Shandong Weizhen Biotechnology), set the flow rate of the microinjection pump to 1 μL / 10 min, fix the microinjector on the brain stereotaxic instrument, lower the needle to the coordinate hole on the skull, reset the longitudinal coordinate and lower the syringe so that the needle tip enters to a depth of 2.3 mm, start the microinjection pump program and start the injection.

[0128] (9) After the injection, leave the syringe for 10 minutes to allow for full absorption. Slowly lift the syringe and clean the blood off the needle with a cotton swab soaked in saline solution to prevent the needle from bending.

[0129] (10) Use tissue forceps to pull the skin on both sides of the wound together, suture the wound with absorbable sutures, disinfect with povidone-iodine, and transfer the mouse to a 37°C heating pad to facilitate its recovery.

[0130] (11) Clean all surgical instruments with alcohol, sterilize them in a high-temperature and high-pressure autoclave, disinfect the operating table, and irradiate with ultraviolet light for 30 minutes.

[0131] Reference Figure 7 In A1, aged wild-type C57BL / 6 mice were injected into the striatum with fluorescent control adeno-associated virus (AAV-Ctrl: AAV9-GFAP-GFP) and DEK-overexpressing adeno-associated virus (AAV-OE: AAV9-GFAP-DEK-GFP). Seven days later, immunofluorescence images of coronal sections of the brain were analyzed in the SVZ and SGZ regions. Immunofluorescence results of DEK protein expression in neural stem cells (GFP+GFAP+) in the SGZ region of the brain of mice in the control virus injection group and the DEK-overexpressing virus injection group are shown in the figure. Figure 7 Immunofluorescence images (A4, A5) showing the characteristic expression of the cell proliferation marker Ki67 in the SGZ region of the brain of mice in the control virus injection group and the DEK overexpression virus injection group. Figure 7 Statistical chart of the number of B1) and proliferating neural stem cells (GFP+Ki67+) Figure 7 (B2), observe whether hippocampal neural stem cells are activated.

[0132] We also significantly increased the number of proliferating neural stem cells in the hippocampus by stereotactic overexpression of DEK virus. Figure 7 These results indicate that DEK can directly activate dormant neural stem cells in the SVZ and SGZ regions, enabling them to enter the cell cycle.

[0133] Example 4: DEK activates dormant neural stem cells to promote neurogenesis

[0134] Immunofluorescence assays showed that DEK promotes the generation of neurogens (DCX+) and mature neurons (NeuN) from neural stem cells.

[0135] To investigate whether DEK overexpression can promote neurogenesis after activating dormant neural stem cells, we conducted a series of lineage tracing experiments using Nestin conditionally overexpressing mice and a control group at 1, 4, and 8 months after tamoxifen induction. We detected and statistically analyzed changes in the number of various cell types during neurogenesis. The specific procedures are as follows: Reference Figure 8 In Example A, double-positive experimental mice and double-positive fluorescent control mice were induced with tamoxifen for 5 days, and three days after the last injection, they were fed BrdU drinking water (1 mg / mL) for 7 consecutive days. After that, they were fed normal drinking water for 1 month, 4 months, and 8 months. Samples (SVZ and SGZ regions) were taken for brain tissue sections, and immunofluorescence analysis was performed on DCX-positive neuronal blasts and GFP. + NeuN + or BrdU + NeuN + The expression distribution of newly generated mature neurons (same as step 2 in Example 1) is shown in the following results. Figure 8 B1, B2, B3, C1, C2, C3, D1, D2 and Figure 9 The antibodies used were A1, A2, A3, B1, B2, B3, C1, and C2, with the following antibody dilution ratios: GFP antibody, 1:1000; DCX antibody, 1:200; NeuN antibody, 1:1000; and BrdU antibody, 1:100.

[0136] Immunofluorescence staining results showed that, compared with the control group, the number of neuronal blasts (DCX+) in the SVZ and SGZ regions increased continuously after DEK overexpression. Figure 8 , Figure 9 The number of newborn neurons (GFP+NeuN+ or BrdU+NeuN+) detected in the olfactory bulb and dentate gyrus regions also continued to increase. Figure 8 , Figure 9 This indicates that overexpression of DEK promotes neurogenesis.

[0137] Example 5: DEK does not disrupt the homeostasis of the neural stem cell bank.

[0138] DEK can promote the entry of dormant neural stem cells into the cell cycle, generating a large number of proliferating neural stem cells and promoting neurogenesis for a prolonged period. Whether the homeostasis of the dormant neural stem cell bank is affected during this process is of concern. Therefore, the effect of DEK on the total number of neural stem cells was detected using immunofluorescence. The specific steps are as follows: Three days after the last injection, the conditionally overexpressing mice induced by tamoxifen in step 2 of Example 3 and the fluorescent control mice were fed with normal drinking water for 1 month, 4 months, and 8 months. Brain tissue samples (SVZ and SGZ regions) were then taken for sectioning. The total number of neural stem cells (GFAP) in the SVZ and SGZ regions of mice overexpressing DEK for 1 month, 4 months, and 8 months was calculated. + Sox2 + Statistical analysis was performed (same as step 2 in Example 1), and the results are shown below. Figure 8 Medium D3 and Figure 9 In C3, the antibody dilution ratios used were: GFAP antibody, 1:1000; Sox2 antibody, 1:500.

[0139] The results showed that, compared with the control group, overexpression of DEK in neural stem cells for a short period of time (1 month, 4 months) increased the number of neural stem cells (GFAP+Sox2+), and then returned to a level comparable to that of normal mice over a long period of time.

[0140] The above results indicate that overexpression of DEK in adult dormant neural stem cells does not disrupt the homeostasis of the neural stem cell pool and does not induce the depletion of the neural stem cell pool over a period of up to 8 months.

[0141] Example 6: Mechanism of DEK-regulated activation of dormant neural stem cells

[0142] a. Overexpression of DEK protein induces changes in gene expression in neural stem cells.

[0143] To determine the molecular mechanism by which DEK regulates the activation of dormant neural stem cells, we first performed transcriptome sequencing analysis on primary adult neural stem cells 24 hours after DEK overexpression to obtain changes in gene expression at the transcriptional level (NCBI database GEO number: GSE200000).

[0144] High-throughput RNA sequencing of the transcriptome (RNA-Seq): SVZ region tissue from step 3a of Example 2 was collected, and primary neural stem cells were obtained using a neural tissue digestion kit (Mitteni, catalog number: 130-092-628). 200,000 primary neural stem cells were resuspended in 1 ml of Neuralbasal maintenance medium containing 2% B27, and 5 μL of 10... 13Cells were incubated at 37°C for 24 hours with vg / mL AAV9-GFAP-GFP (control group) or AAV9-GFAP-DEK-GFP (experimental group), followed by centrifugation. The precipitate was then used as the cell sample. Using the SMARTer® Stranded RNA-Seq Kit (Clontech, catalog number: 634836), 1 mL of TRIzol reagent (Thermo Fisher) was added to the cell samples to extract total RNA. PolyA-tailed mRNA was enriched using Oligo(dT) magnetic beads. Transcriptome libraries were constructed using the Illumina NEBNext® Ultra™ RNA LibraryPrep Kit and sequenced. The sequences were aligned to the mouse reference genome mm39 using HISAT2 software. Based on the alignment location on the reference genome, the number of signals covered by each gene from start to end was counted, and gene expression levels were quantitatively analyzed. Further statistical analysis of the gene expression data was performed to screen for genes with significantly different expression levels among different samples.

[0145] Transcriptome analysis revealed that DEK overexpression in primary neural stem cells induced differential expression of 1730 genes, of which 636 genes were upregulated and 1094 genes were downregulated. Figure 10 (B); GO function query of the top 30 differentially expressed genes revealed that they are all directly involved in cell cycle regulation. Figure 10 KEGG functional enrichment analysis revealed a significant upregulation of signaling pathways regulating neural stem cell activation and cell cycle (such as PI3K-Akt, MAPK, TGF-β, etc.). Figure 10 (D). Numerous reported studies have shown that the negative regulation of dormant maintenance factors is crucial in the activation of dormant neural stem cells. Through differential expression analysis of dormant maintenance genes (such as Notch1, Notch2, Hes5, Foxo3, Apoe, Tp53, etc.) in transcriptome data, we found that overexpression of DEK significantly inhibited the expression of dormant maintenance genes, leading to upregulated expression of neural stem cell division-related genes (such as Mki67, Pcna). Figure 10 (E).

[0146] The above results indicate that overexpression of DEK in neural stem cells inhibits the expression of dormancy maintenance genes, leading to a significant upregulation of cell division-related regulatory molecules and signaling pathways.

[0147] b. Validation of DEK regulatory mechanisms using immunofluorescence and Western blotting

[0148] To verify the above findings, three days after the last injection, the SVZ region tissues of the tamoxifen-induced conditionally overexpressing mice and the fluorescent control mice in step 2 of Example 3 were obtained by microscopic dissection, and the total protein was extracted. The molecular characteristics of the relevant signaling pathways were detected by Western blot. The specific steps were as follows: (1) Immunofluorescence analysis of in situ brain tissue sections (same as step 2 of Example 1), wherein the antibody dilution ratios used were: GFP antibody, 1:1000; Notch1 antibody, 1:400; Notch2 antibody, 1:400; ASCL1 antibody, 1:100. (2) Western blot analysis of tissue proteins in the SVZ region (same as step 3 in Example 2), wherein the antibody dilution ratios used are: DEK antibody, 1:1000; Notch1 antibody, 1:1000; Notch2 antibody, 1:1000; NICD antibody, 1:1000; Hes1 antibody, 1:1000; Hes5 antibody, 1:1000; CSL antibody, 1:1000; ASCL1 antibody, 1:1000; FoxO3 antibody, 1:2000; phosphorylated FoxO3 antibody, 1:2000; P53 antibody, 1:500; P21 antibody, 1:500; MYC antibody, 1:500; Wnt3a antibody, 1:2000; H3 antibody, 1:5000; β-actin antibody, 1:5000.

[0149] The results showed that after DEK overexpression, the protein expression levels of key molecules (Notch1, Notch2, NICD, Hes1, Hes5) in the Notch signaling pathway, a core signaling pathway for maintaining dormancy, were significantly reduced in neural stem cells. Conversely, the expression of ASCL1, a downstream protein in the Notch signaling pathway, was significantly increased. Furthermore, the expression levels of dormancy-related factors FoxO3, P53, and P21 were also significantly reduced, while the expression of activation-related factors MYC and Wnt3a was significantly increased. Figure 11 (A1, A2)

[0150] Based on these results, we used immunofluorescence staining at the brain tissue section level to detect the levels of Notch1 and Notch2 in neural stem cells after DEK overexpression in the SVZ and SGZ regions. Figure 11 (B1, B2, B3, B4) and ASCL1 ( Figure 11 The expression changes of C1 and C2 in DEK revealed a consistent pattern. This indicates that DEK promotes the activation of dormant neural stem cells and neurogenesis by directly inhibiting the Notch signaling pathway, and epigenetically regulates dormancy-activation related factors.

[0151] Example 7: DEK can activate dormant neural stem cells in aged mice.

[0152] 1. Mouse strains

[0153] like Figure 12 In this embodiment, aged (12 months old) double-positive experimental mice were constructed using the method in step 1 of Example 3. Nestin-CreER T2 ::R26-DEK-EGFP and double-positive fluorescent control mice Nestin-CreER T2 ::R26-Luc-EGFP The following experiment was conducted.

[0154] 2. Immunofluorescence detection of DEK-activated dormant neural stem cells in aged mice

[0155] To investigate whether DEK can directly activate dormant neural stem cells in the brains of aged mice, we used Nestin-CreERT2::R26-DEK-EGFP aged (12 months) double-positive experimental mice and Nestin-CreER double-positive fluorescent control mice. T2 ::R26-Luc-EGFP was induced with tamoxifen for 5 days using the method in step 2 of Example 3. Three days after the last injection, the cells were fed with BrdU drinking water (1 mg / mL) for 7 consecutive days, followed by feeding with normal drinking water for 1 month. Samples (SGZ and SVZ regions) were then taken for immunofluorescence analysis of brain tissue sections (same as step 2 of Example 1). The antibody dilution ratios used were: GFP antibody, 1:1000; Sox2 antibody, 1:500; Ki67 antibody, 1:200; DCX antibody, 1:200; NeuN antibody, 1:1000; BrdU antibody, 1:100.

[0156] Fluorescence spectroscopy and immunofluorescence staining revealed that, compared with the control group, overexpression of DEK in neural stem cells of aged mice activated dormant neural stem cells in the SVZ and SGZ regions, significantly increasing the number of proliferating neural stem cells (GFP+Sox2+Ki67+). Figure 12 B1 and B2 Figure 13 (A, B); One month after DEK overexpression in neural stem cells of aged mice, the number of neuronal blasts (GFP+DCX+) in the SVZ and SGZ regions also increased significantly. Figure 14 A1 and A2, Figure 15 (A1 and A2). Under normal physiological conditions, only one SGZ region was present or undetectable in tissue sections of aged mice, while after DEK overexpression, approximately 40 proliferating neural stem cells could be detected. Figure 12 B1 and B2 Figure 13 In A and B, the same phenomenon also exists in the distribution of neuronal blasts. Figure 14 A1 and A2, Figure 15 The data (A1 and A2) are consistent with the pattern of reduction in neural stem cells and their progeny cells in the SVZ and SGZ regions during brain aging as described by previous studies.

[0157] The results above indicate that DEK can activate dormant neural stem cells in the brains of aged mice and significantly enhance neurogenesis.

[0158] Example 8: DEK activates dormant neural stem cells to "rejuvenate" the aged brain.

[0159] To further evaluate the effect of DEK on activating dormant neural stem cells in the brains of aged mice, we compared and studied neurogenesis levels. Considering the varying total number of neural stem cells in the brains of mice of different ages, and the difficulty in using GFP-labeled recombinant cells for analysis and comparison, we performed a BrdU incorporation retention assay during tamoxifen induction in aged mice to label newly generated mature neurons indicating neurogenesis levels. Immunofluorescence staining was used to detect the number of mature neurons generated after DEK overexpression in aged mice, and the level of newly generated mature neurons labeled with BrdU was compared with that of control groups of different ages.

[0160] The double-positive experimental mice (2 months, 5 months, 9 months and 12 months old) and fluorescent control mice constructed by the method in step 1 of Example 3 above were fed with BrdU drinking water (1 mg / mL) for 7 days. Then, they were induced with tamoxifen for 5 days by the method in step 2 of Example 2. Three days after the last injection, they were fed with normal drinking water for 1 month. Brain tissue sections were taken and BrdU+NeuN+ immunofluorescence staining was performed for comparative analysis (same as step 2 of Example 1). The antibody dilution ratio used was: NeuN antibody, 1:1000; BrdU antibody, 1:100.

[0161] The results showed that, compared with the control group, overexpression of DEK in the olfactory bulb and hippocampus regions activated neural stem cells, enhanced neurogenesis, and produced more mature neuronal progeny (GFP+NeuN+). Figure 14 B1 and B2 Figure 15 (C1, C2); As mice age, the number of newly generated mature neurons (BrdU+NeuN+) labeled with BrdU in the olfactory bulb and dentate gyrus of the hippocampus decreases compared to when they are younger. However, after one month of DEK overexpression, this number significantly increases, reaching the neurogenesis level of 5-month-old mice. Figure 14 C1, C2, Figure 15 (D1, D2). The above results indicate that DEK reactivates neurogenesis, thus "rejuvenating" the elderly brain.

[0162] Example 9: DEK activates dormant neural stem cells to promote nerve damage repair in stroke-affected mice.

[0163] a. Construction of a mouse model of stroke

[0164] The method for inducing hemorrhagic stroke by stereotactic injection of collagenase into the brain of wild-type mice is as follows: (1) Anesthetize aged mice (20 months old) with 1.5% isoflurane gas using a small animal anesthesia machine.

[0165] (2) After removing the hair from the surface of the mouse’s head with scissors, fix the head on the stereotaxic instrument. Use anatomical forceps to open the mouse’s mouth and attach the upper jaw teeth to the buckle of the stereotaxic instrument. Rotate the fixing screw on the instrument so that it can just fix the mouse’s nose. Finally, insert the fixing rods on both sides into the mouse’s ears to anchor its head.

[0166] (3) Wipe the scalp with iodine to disinfect the surface of the skin, make a small incision with a scalpel, and cut the incision open with tissue scissors.

[0167] (4) Use tissue forceps to pull the cut skin to both sides, use a cotton swab to apply saline to remove the meninges, find the anterior fontanelle on the skull, and mark it as the origin of the coordinate system with a marker.

[0168] (7) Fixed coordinates: With the front end as the origin, mark the coordinates 0.5 mm forward and 1.5 mm to the right with a marker pen, drill a hole with a skull drill, and maintain the drilling force during the process to avoid damage to the brain tissue.

[0169] (8) Draw 10 μL of collagenase IV solution (150 U / mL), set the flow rate of the microinjection pump to 0.5 μL / 10 min, fix the microinjector on the stereotaxic instrument, lower the needle to the coordinate hole on the skull, reset the longitudinal coordinate and lower the syringe so that the needle tip enters to a depth of 3.5 mm, start the microinjection pump program and start the injection, inject 0.5 μL into each mouse.

[0170] (9) After the injection, leave the syringe for 10 minutes to allow for full absorption. Slowly lift the syringe and clean the blood off the needle with a cotton swab soaked in saline solution to prevent the needle from bending.

[0171] (10) Use tissue forceps to pull the skin on both sides of the wound together, suture the wound with absorbable sutures, disinfect with povidone-iodine, and transfer the mouse to a 37°C heating pad to facilitate its recovery.

[0172] (11) Clean all surgical instruments with alcohol, sterilize them in a high-temperature and high-pressure autoclave, disinfect the operating table, and irradiate with ultraviolet light for 30 minutes.

[0173] Hemorrhagic stroke was induced by injecting 0.5 μL of collagenase IV solution (150 U / mL) into the striatum of adult wild-type mice. After 24 hours, samples were taken to analyze the lesion area and observe for post-hemorrhagic congestion, thus evaluating the establishment of the hemorrhagic stroke model. This model was subsequently applied to the transgenic mouse double-positive progeny constructed in Example 3.

[0174] b. Immunofluorescence analysis of the effect of DEK overexpression on injury repair in stroke-affected mice

[0175] Stroke is a common and sudden neurological injury with a high mortality rate. To investigate whether DEK, as an activating regulator, can play a role in neurological injury repair, we used collagenase IV injection to induce vascular rupture and established a mouse model of hemorrhagic stroke injury. We then used tamoxifen-induced stroke injury repair lineage tracing in the transgenic mouse double-positive progeny constructed in Example 3.

[0176] Adults (2 months old) were injected with tamoxifen three days after the procedure in step a of Example 3, step 2. Nestin- CreER T2 ::R26-Luc-EGFP and Nestin-CreER T2 ::R26-DEK-EGFP Transgenic mice were injected with 0.5 μL of collagenase IV solution (150 U / mL) into the striatum to induce hemorrhagic stroke. After surgery, the mice were fed EdU-containing drinking water (0.25 mg / mL) for 7 consecutive days, followed by feeding them normal drinking water for 23 days, allowing for a one-month post-operative repair period. Brain tissue sections were then sampled for immunofluorescence analysis (same as step 2 in Example 1). The antibody dilution ratios used were: GFP antibody, 1:1000; GFAP antibody, 1:1000; DCX antibody, 1:200; NeuN antibody, 1:1000. EdU signal detection was performed using the EDU-Click kit (Sigma, catalog number: BCK-EDU594), with staining solution prepared and incubated at room temperature for 30 minutes before DAPI staining in the immunofluorescence step.

[0177] Pathological observation, apoptosis detection, and immunofluorescence staining revealed that 24 hours after injecting collagenase IV into the striatum region of mouse brains, we observed extensive congestion in the striatum region. Figure 16 (A2), and this region contains a large number of apoptotic cells (Casp3+) ( Figure 16 In the middle B), there was a significant loss of mature striatal neurons (NeuN+) due to damage. Figure 16 The results (C) indicate that we have successfully constructed a mouse model of hemorrhagic stroke.

[0178] Existing research indicates that during the repair of nerve injury in hemorrhagic stroke, endogenous dormant neural stem cells can only be activated in a limited number of cases in response to this injury stimulus. Figure 19 DEK overexpression significantly enhances the endogenous neural stem cell response to injury repair by activating dormant neural stem cells. Neuroblasts generated from neural stem cells have the ability to migrate to the lesion area and differentiate into mature neurons. Furthermore, astrocytes also play an important role in filling and supporting the brain during injury repair, and inhibiting the inflammatory response. In a hemorrhagic stroke model applied to DEK-overexpressing and fluorescent control mice, after a one-month injury repair phase (…), Figure 17 In the control group (A), compared with the normal physiological damage repair, overexpression of DEK significantly promoted the activation of endogenous neural stem cells, and the number of GFP-labeled neural stem cells and their progeny (GFP+) increased, migrating to the damaged area. Figure 17 In the middle B group, the number of neuroblasts (GFP+DCX+), astrocytes (GFP+GFAP+), and EdU-labeled new total cells (GFP+EdU+) generated from neural stem cells were significantly increased. These results indicate that DEK can promote the activation of endogenous neural stem cells and promote the repair of nerve damage in stroke mice. Figure 17 C1, C2, D1, D2 Figure 18 (A1, A2). Furthermore, density analysis of mature neurons (NeuN+) in the damaged area revealed a significant recovery in the density of mature neurons in the lesion region of DEK-overexpressing mice. Figure 18 (B1, B2, C1, C2) Although only a small portion of these neurons are lineage descendants produced after DEK-activated neural stem cells, we speculate that the repair process of mature neurons may be promoted by the overexpression of DEK to activate neural stem cells and generate a large number of new neuronal progenitors.

Claims

1. The application of a DEK protein in the preparation of a drug for activating dormant neural stem cells to treat brain aging.

2. The application as described in claim 1, characterized in that, The DEK protein has the conserved nucleic acid sequence shown in SEQ ID NO.

1.

3. The application as described in claim 1, characterized in that, The DEK protein has more than 95% similarity to the amino acid sequence shown in SEQ ID NO.

2.

4. The application as described in claim 1, characterized in that, The DEK protein has the amino acid sequence shown in SEQ ID NO.

2.

5. The application as described in claim 1, characterized in that, The application involves overexpressing endogenous DEK protein in dormant neural stem cells or delivering exogenous DEK protein into dormant neural stem cells to activate them and treat brain aging.

6. The application as described in claim 1, characterized in that, The brain aging mentioned includes Alzheimer's disease and Parkinson's disease.

7. The application as described in claim 1, characterized in that, The drug is prepared by expressing and purifying DEK protein in vitro and then loading it onto exosomes, or by loading DEK onto a genetically engineered virus.

8. Application of a DEK protein in the preparation of a reagent to activate dormant neural stem cells.

9. The application as described in claim 9, characterized in that, The reagents include endogenous or exogenous DEK proteins.

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