Application of HOPX in improvement / treatment of hippocampal dependent dysmnesia
By upregulating or overexpressing HOPX in the hippocampus, and using the HOPX overexpression vector to improve hippocampus-dependent memory impairment, the problem of age-related reduction in hippocampal neurogenesis was solved, and memory function was improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-14
- Publication Date
- 2026-03-31
AI Technical Summary
Current technologies have failed to effectively reverse age-related reduction in hippocampal neurogenesis and hippocampal-dependent memory impairment, and there is a lack of effective treatment options.
By upregulating or overexpressing HOPX protein, gene therapy can be performed in the hippocampus using HOPX overexpression vectors such as AAV and lentiviral vectors to promote cell proliferation, differentiation, and increase the number of neurons in the hippocampus.
Directly combating the age-related reduction in hippocampal neurogenesis and improving hippocampal-dependent memory disorders provides a new treatment strategy. In vivo experiments show that HOPX overexpression can improve memory function.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to the application of HOPX in improving / treating hippocampal dependent memory disorders. Background Technology
[0002] The cognitive decline associated with aging, particularly hippocampal-dependent learning and memory impairment, is a core issue severely impacting the quality of life for older adults. One of the key pathophysiological bases behind this is the significant decrease in hippocampal neurogenesis with age. Currently, there are no clinical therapies that can effectively reverse this process and significantly improve memory.
[0003] As early as the 1980s, homeotic genes had been extensively studied in Drosophila. The common 180-base-pair DNA sequence contained in these genes was named a homeobox, and the protein encoded by it was called a homeodomain (HD). This domain can both bind DNA and mediate protein-protein interactions. However, Hopx, also known as Hop (homeodomain-only protein homeobox), is a special homeotic gene that lacks DNA-binding ability. It was discovered in 2002 by Chen, Shin, and others. In the nervous system, Hopx is specifically expressed in radial glia-like (RGL) cells of the dentate gyrus (DG) in the adult mouse brain. Its gene sequence (e.g., human HOPX gene, accession number NM_032495) and protein sequence (e.g., human HOPX protein, accession number Q9BPY8) have been indexed in public databases (such as NCBI and UniProt). In existing technologies, HOPX has been reported to be associated with cardiac development, certain tumor progression, and expression in the nervous system. Currently, HOPX is attracting attention in tumor research, with evidence suggesting it may act as a tumor suppressor gene, and its methylation status is related to tumor development and progression; however, the specific mechanisms of action of HOPX in different tumor types are not yet fully understood and require further investigation.
[0004] Previous studies have indicated that HOPX exists in hippocampal neural progenitor cells. However, current technology has not revealed the specific causal relationship between HOPX expression levels and hippocampal neurogenesis during the aging process, nor has it proposed a treatment plan to reverse the age-related reduction in hippocampal neurogenesis, hippocampal atrophy, and memory dysfunction by actively replenishing (or upregulating) HOPX. Summary of the Invention
[0005] The purpose of this invention is to address the above-mentioned problems by providing an application of HOPX in improving / treating hippocampal dependent memory disorders.
[0006] To achieve its objective, the present invention employs the following technical solution:
[0007] The first aspect of the present invention provides the use of HOPX as a target in screening drugs to improve / treat hippocampal dependent memory disorders.
[0008] The drug upregulates HOPX expression.
[0009] The improvements or treatments include: slowing the progression, alleviating symptoms, or restoring function; the hippocampal-dependent memory disorder is an age-related hippocampal-dependent memory disorder.
[0010] The drug promotes cell proliferation, differentiation, and / or increases the number of neurons in the hippocampus.
[0011] A second aspect of the invention provides the use of HOPX protein or a polynucleotide encoding HOPX protein in the preparation of a medicament for improving / treating hippocampal-dependent memory impairment.
[0012] The drug contains a polynucleotide encoding the HOPX protein, and / or contains a substance that can promote the expression of the polynucleotide encoding the HOPX protein; the hippocampus-dependent memory disorder is an age-related hippocampus-dependent memory disorder.
[0013] The drug is an HOPX overexpression vector.
[0014] A third aspect of the invention provides the use of a genetically engineered vector containing the above-described polynucleotide encoding the HOPX protein in the preparation of a medicament for improving / treating hippocampal-dependent memory impairment.
[0015] Preferably, the gene engineering vector is an HOPX overexpression vector.
[0016] Preferably, the HOPX overexpression vector is an adeno-associated virus (AAV) vector, a lentiviral vector, or a retroviral vector.
[0017] The beneficial effects of this invention are:
[0018] (1) It provides a new target and a new drug for improving / treating hippocampal dependent memory disorders. For the first time, it directly links the "replenishment" or "functional upregulation" of HOPX with reversing age-related hippocampal memory decline, opening up a new path and strategy for treating age-related cognitive disorders.
[0019] (2) In vivo experiments of the present invention have demonstrated that HOPX recombinant / overexpression can directly counteract the reduction of hippocampal neurogenesis caused by aging and improve the phenotype at the cellular and molecular levels.
[0020] (3) This invention provides a solid theoretical and experimental foundation for the development of HOPX-based gene therapy, protein drugs or small molecule agonists, and has good prospects for translational application. Attached Figure Description
[0021] Figure 1 The study showed that conditional knockout of HOPX led to hippocampal structural disorder and a reduction in neural stem cells.
[0022] Figure 2 The expression of HOPX and stem cell markers in the hippocampal DG region was shown.
[0023] Figure 3 The results of the novel object recognition test for mice are shown.
[0024] Figure 4 Immunofluorescence staining image for experiments showing that overexpression of HOPX can promote hippocampal neurogenesis and maintain a stem cell pool. Detailed Implementation
[0025] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.
[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.
[0027] Example 1
[0028] 1. Main experimental materials and reagents
[0029] 1.1 Main Reagents
[0030]
[0031] 1.2 Laboratory animal husbandry and modeling
[0032] Male C57BL / 6J mice, weighing approximately 20-22g at 6 weeks of age and 28-30g at 30 weeks of age, were selected for this study and purchased from Jiangsu Jicui Pharmaceutical Co., Ltd. The mice were housed under normal conditions: a temperature of approximately 25℃, humidity of approximately 55%, and a 12-hour light-dark cycle. The mice had free access to water and food. After one week of acclimatization, the mice were randomly and evenly divided into a control group and an experimental group.
[0033] Hopx flox / flox Mouse: loxP sites were inserted on both sides of exon 2 of the Hopx gene; the flux mouse can be mated with tissue-specific Cre tool mice to obtain a mouse model in which the Hopx gene is knocked out in a specific cell type or tissue.
[0034] hGFAP-Cre mice: A transgenic mouse model genetically engineered based on the expression of Cre recombinase driven by the human glial fibrillary acidic protein (GFAP) promoter. This promoter specifically activates Cre recombinase in the central nervous system, thereby mediating gene recombination.
[0035] Hopx flox / flox The mice, including hGFAP-Cre mice, were jointly constructed by our laboratory and Professor Han Jiahuai's research group using conventional homologous recombination methods in embryonic stem cells. Human GFAP-Cre (hGFAP-Cre), Nestin-Cre, and ROSA-Cre were also used. ERT2 The mice were obtained from Jackson Laboratory.
[0036] Hopx hGFAP-cKO Conditional knockout mouse model: Cage matching was performed using the Cre / loxP system principle, Hopx flox / flox Hopx mice, which are neural stem cell-specific conditional knockout mice, are obtained by crossing mice with hGFAP-Cre mice. hGFAP-cKO Conditional knockout mouse model.
[0037] Establishment of a mouse model of HOPX overexpression lentivirus in the hippocampus: Recombinant lentivirus (rLV) was injected into the hippocampus of mice using stereotactic injection. The experimental group was injected with recombinant lentivirus carrying the HOPX gene (rLV-Nestin-mHopx-WPRE) to achieve specific overexpression of HOPX (complementation); the control group was injected with empty vector control virus (rLV-Nestin-WPRE). Ten 30-week-old mice were used in each group. Injection parameters were as follows: injection volume was 1 µL, and viral titer was 2.5 × 10⁻⁶. 12 The injection rate was vg / mL at a rate of 200 nL / min. Stereoscopic localization was performed with the anterior fontanelle as a reference, targeting the hippocampus: 1.9 mm posterior to the anterior fontanelle (AP), 2.3 mm lateral to the midline (ML), and 1.8 mm subsurface of the skull (DV). Mice were routinely housed after viral injection. Beginning at week 4 post-injection (after viral expression), behavioral tests (novel object recognition) were performed to assess learning and memory function. After the behavioral tests, mice were sacrificed and their brains were harvested. Immunofluorescence staining was used to detect the expression and localization of target proteins such as HOPX, SOX2, and GFAP in the hippocampus.
[0038] 2. Experimental methods and results
[0039] Immunohistochemical results showed that Hopx f / f Conditional knockout mice showed a significant reduction in hippocampal volume and a decrease in the number of newly generated neurons.
[0040] Short-term tracking via edu revealed decreased proliferative activity of neural stem cells (NSCs) in the dentate gyrus (DG) region, indicating impaired stem cell niche maintenance.
[0041] 2.1 Conditional knockout of HOPX leads to hippocampal structural disorder and a reduction in neural stem cells.
[0042] Experimental objective: To investigate the effects of HOPX gene dosage on hippocampal development and neural stem cell maintenance, we constructed and analyzed mice with different genotypes to systematically evaluate the changes in hippocampal tissue structure and stem cell pool after HOPX knockout.
[0043] Model and Methods: We constructed the Hopx conditional knockout mouse system using the Cre-loxP system. By using Hopx... flox / flox Mice were crossed with hGFAP-Cre to obtain offspring with varying degrees of Hopx gene knockout, including: wild-type (WT), Hopx... f / f or Hopx f / + ;), Hybrid type (HET, Cre-Hopx) flox / + ) and homozygous knockout (MUT, Cre-Hopx) flox / flox ) Take 8-10 week old mice of the above three genotypes (n≥5 in each group), fix them by cardiac perfusion, and then collect brains to prepare frozen sections.
[0044] We performed the following tests on the brain slices:
[0045] Cell proliferation and stem cell analysis: Proliferating cells and neural stem cells in the hippocampal DG region were labeled using immunofluorescence staining with anti-Ki67 (a cell proliferation marker) and anti-Sox2 (a neural stem cell marker), respectively. Ki67-positive and Sox2-positive cells in the hippocampus (especially the DG subgranular zone) were counted under high magnification.
[0046] Results and conclusions:
[0047] Hippocampal tissue structure destruction: tissue staining ( Figure 1 The results showed that, compared with the complete and well-defined hippocampal structure of the WT group mice, the hippocampal morphology of the HET group was basically normal but slightly smaller, while the MUT group showed severe hippocampal structural disorder, with the dentate gyrus (DG) structure being blurred or even completely disappeared, indicating that the complete absence of HOPX would lead to severe hippocampal malformation.
[0048] The neural stem cell pool showed graded damage: Immunofluorescence staining and quantitative statistical results showed that:
[0049] The number of Ki67-positive cells showed a significant decreasing trend in the WT, HET, and MUT groups. The number of proliferating cells in the MUT group was reduced by more than 70% compared with the WT group (**p < 0.001).
[0050] The number of Sox2-positive neural stem cells also showed a gradient decrease in the order WT > HET > MUT. The number of stem cells in the MUT group was significantly lower than that in the WT group by about 60% (**p < 0.001).
[0051] Summary: The above results demonstrate that HOPX gene deletion impairs hippocampal development in a dose-dependent manner. Heterozygous knockout already leads to a reduction in the number of neural stem cells and proliferating cells, while homozygous knockout causes severe defects ranging from the cellular level (drastic reduction in stem cells / proliferating cells) to the tissue level (hippocampal structural damage and loss of the dentate gyrus). This clearly reveals that HOPX is an indispensable key gene for maintaining normal hippocampal tissue structure and neural stem cell pool homeostasis.
[0052] 2.2 Downregulation of HOPX expression during aging is accompanied by atrophy of the hippocampal neural stem cell pool.
[0053] Experimental Objective: To investigate the expression dynamics of HOPX during aging and its relationship with hippocampal neural stem cell (NSC) homeostasis, we systematically analyzed the expression changes of HOPX and stem cell markers in the hippocampus of wild-type mice from the embryonic stage to old age.
[0054] Model and Methods: Wild-type (C57BL / 6J) mice were used in this study. Samples were collected at six key developmental and senescence time points: embryonic day 15.5 (E15.5), day 10 after birth (P10), day 21 after birth (P21), 3 months of age (3M), 18 months of age (18M), and 20 months of age (20M) (n≥3 at each time point). Coronal frozen sections were prepared from the posterior brain tissue. Multiple immunofluorescence staining was performed on the dentate gyrus (DG) region of the hippocampus. Specific antibodies were used to detect the expression of HOPX, the neural stem cell marker SOX2, and the astrocyte and resting-state stem cell marker GFAP. Confocal microscopy imaging was used, and image analysis software was employed to quantitatively analyze the fluorescence intensity of positive signals and the number of colocalized cells.
[0055] Results and conclusions:
[0056] Immunofluorescence staining results ( Figure 2 The results showed that in the hippocampal DG region, the expression of HOPX and stem cell markers exhibited a regular change with age:
[0057] HOPX expression shows an age-dependent decline: HOPX protein expression levels in the hippocampus (especially the DG region) show a significant gradient decline from the embryonic stage (E15.5) to the senescent stage (20M).
[0058] The number of SOX2+ neural stem cells decreases with aging: Consistent with the expression pattern of HOPX, the number of SOX2-positive cells also decreases significantly in a gradient from the embryonic stage to old age. This indicates that the pool of identifiable neural stem cells in the hippocampus continues to shrink with age.
[0059] Dynamic changes in the GFAP+ cell population: The expression pattern of GFAP differs slightly from the other two. GFAP signaling is widespread and strong from P10 to adulthood (3M); however, from adulthood into old age (18M, 20M), its signal intensity and the number of SOX2+ GFAP+ double-positive resting neural stem cells also show a significant decrease.
[0060] Summary: This study demonstrates that during physiological aging, the expression level of the key regulator HOPX in the hippocampus exhibits a synchronous, age-dependent decline in the expression of neural stem cell markers SOX2 and GFAP, as well as the number of stem cells. This strong positive correlation suggests that downregulation of HOPX expression may be functionally linked to age-related atrophy of the hippocampal neural stem cell pool and decline in neurogenesis, providing crucial molecular phenotypic evidence for further research into replenishing HOPX to combat age-related cognitive decline.
[0061] 2.3 Overexpression of HOPX can improve the memory ability of mice to a certain extent.
[0062] Novel object recognition is an experimental method for studying animal working memory, based on the animal's exploratory psychology towards new things. The experimental method is as follows:
[0063] 1. Before the experiment, two identical cylindrical blocks and one square block need to be prepared. The experiment is conducted in a four-channel open field and consists of three stages.
[0064] 2. First stage: On the first day, let the mice explore freely in the open field for 10 minutes to familiarize themselves with the area.
[0065] 3. Second stage: On the second day, place two identical cylindrical blocks in the open field, ensuring that the blocks are odorless and do not move. The blocks are about 10 cm away from the side wall of the open field. Place the mouse in the open field with its back to the blocks at the same distance from the two blocks, and let it explore freely for 10 minutes.
[0066] 4. Third stage: On the third day, one of the cylindrical blocks was changed to a square block. The mouse was placed in an open field with its back to the block and at the same distance from the block. The experiment lasted for 5 minutes.
[0067] 5. Using the Noldus EthoVision XT small animal behavior recording and analysis system, the time that the mouse's nose tip lingered around the object was recorded.
[0068] In the new object recognition experiment, after the control group mice became familiar with the experimental site and learned and memorized two identical novel objects, the mice did not show any preference for spatial location. However, when another unfamiliar object was introduced, the normal control group mice showed interest in the unfamiliar object, while the experimental group mice that were reintroduced with HOPX showed a greater degree of interest in the novel object than the control group. Figure 3 The experimental group mice showed a significantly higher exploration time ratio (recognition index) for new objects than the control group, indicating that their short-term memory was improved.
[0069] 2.4 Overexpression of HOPX can promote hippocampal neurogenesis and maintain the stem cell pool.
[0070] Experimental objective: To verify whether active HOPX replenishment can reverse neuropathy caused by aging or defects, we used a lentiviral vector to specifically overexpress HOPX in the hippocampus of adult mice and systematically evaluated its effects on cell proliferation, neural differentiation, and stem cell maintenance.
[0071] Model and Methods: The "hippocampal HOPX overexpression lentiviral mouse model" constructed in Section 2.1 was used. Specifically, 6-week-old (1.5-month-old) wild-type C57BL / 6J mice were stereotactically injected into the hippocampus. The experimental group was injected with an overexpressing lentivirus carrying the HOPX gene (rLV-Nestin-mHopx-WPRE), while the control group was injected with an empty lentivirus (rLV-Nestin-WPRE). Approximately 4 weeks after viral injection (i.e., when the mice were about 3 months old), a one-week behavioral test, including new object recognition, was performed. At 6 months of age, the mice were sacrificed and their brains were harvested. After preparing brain sections, the dentate gyrus (DG) region of the hippocampus was subjected to immunofluorescence multiplex staining. Specific antibodies were used to detect HOPX (to verify overexpression efficiency), the cell proliferation marker Ki67, the immature neuron marker Doublecortin (DCX), the neural stem cell marker SOX2, and GFAP, and the images were obtained using confocal microscopy.
[0072] Results and conclusions:
[0073] Immunofluorescence staining ( Figure 4 The results showed that, compared with the control group, the HOPX overexpression group exhibited a variety of regenerative phenotypes in the hippocampus:
[0074] Overexpression validation was successful: the fluorescence signal intensity of HOPX protein in the hippocampal DG region of the HOPX overexpression group was significantly upregulated compared with the control group, confirming the success of virus-mediated gene delivery and expression.
[0075] Improved maintenance of neural stem cell pool: The number of SOX2-positive neural stem cells and GFAP-positive cells in the HOPX-overexpression group were significantly increased compared with the control group, suggesting that HOPX helps maintain the size and homeostasis of the stem cell pool.
[0076] Summary: This experiment demonstrates that active reintroduction (overexpression) of the HOPX gene in the hippocampus of adult mice can effectively reverse age-related neurodegenerative phenotypes. Its mechanism of action involves simultaneously enhancing the proliferative activity of neural stem cells, promoting their differentiation into neurons, and maintaining stem cell pool homeostasis. This provides direct functional evidence for HOPX as a key target for intervention in age-related cognitive impairment or hippocampal damage repair.
Claims
1. Use of HOPX as a target in screening drugs for improving / treating hippocampus-dependent memory impairment.
2. Use according to claim 1, characterized in that: The drugs up-regulate the expression of HOPX.
3. Use according to claim 1, characterized in that: The improvement or treatment includes delaying the progress, alleviating the symptoms, or restoring the function; and the hippocampus-dependent memory impairment is an aging-related hippocampus-dependent memory impairment.
4. Use according to claim 1, characterized in that: The drugs promote hippocampus cell proliferation, differentiation, and / or increase the number of neurons.
5. Use of HOPX protein or polynucleotide encoding HOPX protein in the preparation of drugs for improving / treating hippocampus-dependent memory impairment.
6. Use according to claim 5, characterized in that: The drugs comprise polynucleotide encoding HOPX protein, and / or comprise substances capable of promoting the expression of polynucleotide encoding HOPX protein; and the hippocampus-dependent memory impairment is an aging-related hippocampus-dependent memory impairment.
7. Use according to claim 6, characterized in that: The drugs are HOPX overexpression vectors.
8. Use of a genetic engineering vector comprising polynucleotide encoding HOPX protein as claimed in claim 5 in the preparation of drugs for improving / treating hippocampus-dependent memory impairment.
9. Use according to claim 8, characterized in that: The genetic engineering vector is a HOPX overexpression vector.
10. Use according to claim 7 or 9, characterized in that: The HOPX overexpression vector is an adeno-associated virus (AAV) vector, a lentivirus vector, or a retrovirus vector.