Application of NeuroD1 in repairing Alzheimer's disease

Abstract

The invention discloses an application of NeuroD1 (NeuroD1) in repairing Alzheimer's disease (Alzheimer's disease). The invention also discloses an application of the carrier for coding NeuroD1 in any one of the following aspects: preventing neuron damage and apoptosis; hippocampus atrophy is inhibited; the neuroinflammation is relieved; repairing blood vessel / blood brain barrier injury; the AD biomarker level of the cerebrospinal fluid is recovered to be normal; the removal of pathological toxic proteins in brain tissues is promoted; glucose metabolism is improved; the space working memory ability is enhanced; and regenerating neurons.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of NeuroD1 in repairing Alzheimer's disease. Background Art

[0002] Neurons are the structural basis and functional units for humans to receive, transmit, store, integrate, and process information. A variety of central nervous system degenerative diseases are usually accompanied by a large number of neuron injuries and even deaths. However, terminally differentiated neurons in the human central nervous system do not have the ability to divide and regenerate. Once the number of functional neurons lost due to central nervous system degenerative diseases reaches a certain threshold, it will cause irreversible damage to the function of the human central nervous system.

[0003] Alzheimer's disease (AD) is the main cause of dementia and the most common neurodegenerative disease. With the aging of the global population, its prevalence has been steadily increasing. This devastating disease has far-reaching social consequences, including a decline in quality of life, an extended lifespan of patients, an increased burden and stress on caregivers, and a large amount of healthcare costs for families and society. The damage and loss of neurons are the main causes of brain dysfunction in Alzheimer's disease. Effective disease-improving treatments must ultimately have the ability to regenerate functional neurons to replace damaged and dead neurons.

[0004] Currently, the treatments for AD mainly include drug therapy and immunotherapy. The main prescribed drugs are cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) and memantine (an NMDA receptor antagonist), aiming to improve cognitive function, memory, and behavioral neurotransmitters through regulation. However, these drugs can only provide moderate symptom relief and cannot prevent the disease from progressing, and their effects will weaken over time. In addition, the side effects of these drugs will affect the quality of life of patients, or the effects are limited. Immunotherapy includes active immunotherapy using vaccines such as AN1792 and passive immunotherapy using CAD106 and monoclonal antibodies such as Aducanumab, lecanemab, and donanemab. Although these immunotherapies have shown varying degrees of success in reducing β-amyloid protein and slowing cognitive decline, serious adverse reactions (such as meningitis) limit their application. In terms of improving cognition, the results are mixed, and the impact on disease progression is uncertain.

[0005] Generally speaking, although the current treatment methods can temporarily relieve the symptoms of some patients, their limitations lead to an urgent need for more effective treatment methods to directly address the underlying pathogenic mechanisms of Alzheimer's disease. Summary of the Invention

[0006] The object of the present invention is to provide a technical solution capable of effectively regenerating neurons and effectively treating Alzheimer's disease for the above technical problems to be solved.

[0007] In order to achieve the above object of the invention, the present invention provides the use of NeuroD1 in repairing Alzheimer's disease.

[0008] On the other hand, the present invention also provides the use of a vector encoding NeuroD1 in any of the following aspects:

[0009] (1) Preventing neuronal damage and apoptosis;

[0010] (2) Inhibiting hippocampal atrophy;

[0011] (3) Reducing neuroinflammation;

[0012] (4) Repairing vascular / blood-brain barrier damage;

[0013] (5) Restoring the level of cerebrospinal fluid AD biomarkers to normal;

[0014] (6) Promoting the clearance of pathological toxic proteins in the brain tissue (such as β-amyloid protein);

[0015] (7) Improving glucose metabolism;

[0016] (8) Enhancing spatial working memory ability;

[0017] (9) Regenerating neurons.

[0018] Preferably, the vector is a viral vector.

[0019] Preferably, the vector is an adeno-associated virus.

[0020] Preferably, the vector overexpresses NeuroD1.

[0021] Preferably, the AD biomarkers include total tau protein, phosphorylated tau 181 protein and phosphorylated tau 231 protein.

[0022] Preferably, the regenerated neurons are: converting hippocampal astrocytes into neurons.

[0023] On the other hand, the present invention also provides the use of a vector encoding NeuroD1 in the preparation of a drug having any of the following functions:

[0024] (1) Preventing neuronal damage and apoptosis;

[0025] (2) Inhibiting hippocampal atrophy;

[0026] (3) Reducing neuroinflammation;

[0027] (4) Repair vascular / blood-brain barrier damage;

[0028] (5) Restore the normal level of cerebrospinal fluid AD biomarkers;

[0029] (6) Promote the clearance of pathological toxic proteins in the brain tissue;

[0030] (7) Improve glucose metabolism;

[0031] (8) Enhance spatial working memory ability;

[0032] (9) Regenerate neurons.

[0033] In the present invention, through the overexpression of the neural transcription factor NeuroD1 mediated by adeno-associated virus (AAV), a large number of functional neurons are regenerated in a non-human primate Alzheimer's disease-like animal model with overexpression of human tau protein in the hippocampus, thereby repairing the structural and functional damage of the brain tissue of the Alzheimer's disease model monkeys. The AAV encoding the neural transcription factor NeuroD1 is delivered into the hippocampus of NHP, and extensive expression of NeuroD1 in the hippocampal region of macaques is observed. Gene therapy based on NeuroD1-AAV can not only repair neuronal damage and loss, but also resist continuous nerve damage and prevent the progression of hippocampal atrophy. In addition, this gene therapy reduces neuroinflammation, restores vascular integrity, and normalizes the level of AD biomarkers in cerebrospinal fluid. In addition, this gene therapy improves glucose metabolism and alleviates the deficits shown by the cognitive behavioral assessment of working memory. Transcriptome analysis shows that NeuroD1 AAV-based gene therapy upregulates genes involved in neuronal and synaptic functions while downregulating those related to neuroinflammation and apoptosis. Overall, the research results indicate that NeuroD1 AAV-based gene therapy has the potential to become an effective treatment strategy for AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Shows a schematic diagram of constructing a non-human primate model with AD-like pathology. (A) Schematic of the engineered AAV (Syn::FLPo and CAG::FRT-hTau) that expresses hTau after activation of the FLPo recombinase. (B) Representative images of tau immunostaining showing elevated tau protein expression, overexpression in the hippocampus of AD-like monkeys compared to normal monkeys after AAV-induced tau protein; scale bars, 1 mm and 20 μm. (C) Quantification of the fluorescence intensity of tau staining in the monkey brain shows that the hTau expression level in AD monkeys is significantly higher than that in normal monkeys; ****p < 0.0001; Student's t-test, N = 5. (D) Representative images of phosphorylated-tau immunostaining showing overexpression of phosphorylated tau (Ser202 / Thr205) in the hippocampus of AD-like monkeys after hTau overexpression; scale bars, 1 mm and 50 μm. (E) Representative images of NeuN immunostaining showing significant neuronal loss and hippocampal atrophy after hTau overexpression. The dotted line demarcates the boundary of the hippocampus in normal monkeys; scale bar, 1 mm. (F) Quantification of NeuN-positive cells in the hippocampus shows their numbers in the DG-CA4 and CA1-CA3 regions after hTau overexpression; ***P < 0.001, **P < 0.01, Student's t-test, N = 5. (G) Representative sagittal plane of T1-weighted MRI images showing the hippocampal volume after hTau overexpression, the area marked by the yellow line; scale bar, 1 cm. (H) Quantification of the normalized hippocampal volume shows the hippocampus after hTau overexpression, each color represents a different animal; ***P < 0.001, Student's t-test, N = 5. (I) Schematic diagram summarizes and illustrates various AD-like pathological phenotypes observed in the NHP AD model.

[0035] Figure 2Shows AAV-based gene therapy for overexpression of NeuroD1 in the monkey hippocampus. A) Schematic illustration of the process for creating the NHPAD model, the gene therapy process based on NeuroD1-AAV, and subsequent detection of the expression level of its transcription factor NeuroD1. (B) Viruses for the treatment group and the inverted NeuroD1 structure diagram, GFAP::GFP were used for both the control group and the NeuroD1 treatment group. (C) Representative images of GFP immunostaining show that AAV can be effectively transduced into most regions of the hippocampus; most GFP+ cells in the control group are astrocytes, while many GFP+ cells in the NeuroD1 treatment group are neurons; scale bar, 1 mm. (D) Quantification of GFP+ coverage of the hippocampal area, indicating that the genes (GFP and NeuroD1) are delivered throughout the monkey body by AAV and are effectively expressed in the hippocampus; N = 4 in the control group, N = 6 in the NeuroD1 group. (E) Representative images of NeuroD1 immunostaining show effective NeuroD1 at 6 weeks and 36 weeks after AAV injection, and a decrease in NeuroD1 expression was observed at the 36-week time point; scale bar, 1 mm and 100 μm. (F) Representative images of NeuroD1 / GFAP / NeuN immunostaining show that NeuroD1 is specifically expressed in astrocytes at 6 weeks after injection, and GFP+ neurons at 36 weeks; scale bar, 20 μm. (G) Quantification of the normalized NeuroD1 fluorescence intensity shows high expression at 6 weeks and a decrease at 36 weeks.

[0036] Figure 3AAV-based NeuroD1 gene therapy regenerates neurons. (A) Schematic diagram depicts the process of developing the NHPAD model, the NeuroD1 AAV-based gene therapy process, and subsequent evaluation of astrocyte-to-neuron conversion and changes in neuron density. (B) Representative images of GFP / GFAP immunostaining show that most AAV-transduced GFP+ cells are GFAP+, in the hippocampus of control monkeys; scale bars, 1 mm and 20 μm. (C) Representative images of GFP / NeuN immunostaining show that many AAV-transduced GFP+ cells in the hippocampus of NeuroD1 monkeys are NeuN+; scale bars, 1 mm and 20 μm. (D) Quantification of changes in cell characteristics between the control group and NeuroD1, as the percentage of cells co-expressing GFP with GFAP or NeuN; ****P<0.0001. (E) Representative confocal images of GFP / GFAP / NeuN immunostaining show potential intermediate state cells during the astrocyte-to-neuron conversion process; scale bar, 20 μm. (F) Representative images of NeuN immunostaining show a significant increase in the number of NeuN+ at 48 weeks after hTau expression, and a significant increase in the number of NeuN+ in the NeuroD1 treatment group; scale bar, 1 mm. (G) Quantification of NeuN+ represents neuron density, evaluates neuron loss in the AD-like macaque model, and evaluates NeuroD1 AAV gene-based neuron regeneration / protection therapy; ****P<0.0001, *****P<0.001, P<0.01, P<0.05.

[0037] Figure 4NeuroD1 AAV-based gene therapy inhibits hippocampal atrophy is shown. (A) Schematic illustration of the development of the NHP AD model; the gene therapy process of administering NeuroD1 AAV, and longitudinal monitoring of hippocampal volume by magnetic resonance imaging and brain sectioning. (B) Representative sagittal plane of T1-weighted MRI scans showing progressive hippocampal atrophy, continuous atrophy of the hippocampus in the control group, and cessation of atrophy in the NeuroD1-treated group; the yellow line outlines the boundary of the hippocampus; scale bar, 1 cm. (C) Representative three-dimensional reconstruction of the monkey hippocampus using the Brainsight neuronavigation system, which can automatically calculate the volume of the hippocampus after three-dimensional reconstruction; scale bar, 5 mm. (D, E) Quantitative MRI scans of the change in hippocampal volume in the control or NeuroD1-treated group showing progressive hippocampal atrophy (D) or cessation of hippocampal atrophy after NeuroD1 AAV-based gene therapy (E). (F) Representative images of DAPI-labeled coronal brain sections showing that NeuroD1 AAV-based gene therapy can prevent the progression of hippocampal atrophy; the yellow line outlines the boundary of the hippocampus; scale bar, 1 mm. (G) Quantitative evaluation of hippocampal atrophy by the area of the hippocampus in coronal brain sections. ****P < 0.0001, *P < 0.05. (H) Quantitative ventricular area, measured from coronal brain sections to evaluate hippocampal atrophy; ****P < 0.0001, 5 cases in the normal group, 4 cases in the control group, and 5 cases in the NeuroD1 group.

[0038] Figure 5It is shown that NeuroD1 AAV-based gene therapy can reduce neuroinflammation. A) Schematic diagram depicting the process of developing an NHP AD model, administering NeuroD1 AAV-based gene therapy, and subsequently evaluating neuroinflammation and leukocyte infiltration. (B) Representative images of GFAP immunostaining show astrocyte activation after hTau overexpression, and significantly reduced astrocyte activation after NeuroD1 overexpression. Scale bar, 1 mm. (C) Representative high-magnification images of GFAP immunostaining show the morphology of resting and activated astrocytes in the NHP hippocampus. Scale bar, 20 μm. (D) Quantification of the cell density of GFAP+ in the hippocampus of the NHP AD model. ****P<0.0001, **P<0.01. (E) Representative images of Iba1 immunostaining show microglial activation, significant microglial activation after hTau overexpression, and reduced activation in the NeuroD1 group. Scale bar, 1 mm. (F) Representative high-magnification images of Iba1 immunostaining show the morphology of resting and activated microglia in the NHP hippocampus. Scale bar, 20 μm. (G) Quantification of the cell density of Iba1+ in the hippocampus of the NHP AD model. ****P<0.0001, N = 5 in the normal group, N = 6 in the control and NeuroD1 groups. (H) Representative images of CD45 immunostaining show leukocyte infiltration after hTau overexpression, and significantly reduced leukocyte infiltration after NeuroD1 overexpression. Scale bar, 1 mm. (I) Representative high-magnification images of CD45 immunostaining show the morphology of brain-infiltrating leukocytes in the NHP hippocampus. Scale bar, 20 μm. (J) Quantification of the density of CD45+ cells in the hippocampus of the NHP AD model. ****P<0.0001, N = 5 in the normal group, N = 6 in the control and NeuroD1 groups.

[0039] Figure 6It is shown that NeuroD1 AAV-based gene therapy can repair vascular and blood-brain barrier damage in AD-like macaques. (A) Schematic illustration depicts the development of the NHP AD model, followed by NeuroD1 AAV-based gene therapy, and the assessment of monkey vascular / blood-brain barrier integrity using Simoa. (B & C) Longitudinal monitoring of AD biomarkers in cerebrospinal fluid, and representative images of laminin immunostaining show higher magnifications of basement membrane thickening, vascular damage (B), and abnormal vascular morphology (C). The treatment group is similar to that observed in healthy monkeys. Arrow, chord vessel; open arrow, vascular occlusion; closed arrow, distorted / bulging vessel. Scale bars, 1 mm, 200 μm, 20 μm. (D) Representative image at high magnification of PECAM-1 immunostaining shows vascular damage and degeneration in the control group. The NeuroD1 treatment group is similar to that observed in healthy monkeys. Arrow, vascular rupture; arrowhead, vascular disintegration. Scale bar, 20 μm. (E & F) Statistics show the quantitative thickening of the percentage of vessels in the basement membrane, abnormal morphology (E), and vascular degeneration (F). ****P < 0.0001, normal group N = 5, control group N = 4, NeuroD1 group N = 6. (G) Representative image of AQP4 immunostaining shows the distribution pattern of AQP4. In the normal group and the NeuroD1 treatment group, there is a polarized distribution of AQP4 around blood vessels, and in the control group, it is a dispersed distribution. Arrow, dispersed signal; arrowhead, polarized signal. Scale bar, 100 μm. (H) Representative line scan intensity distribution graph (pink line) measured along a line segment perpendicular to a blood vessel (green square) represents the width of a specific blood vessel. Line segment, 40 μm.

[0040] Figure 7 It is shown that NeuroD1 AAV-based gene therapy can improve glucose metabolism and spatial working memory. (A) Schematic illustration demonstrates the development process of the NHP AD model and the gene therapy with NeuroD1 AAV, and longitudinal monitoring of hippocampal glucose metabolism and NHP behavioral tests for spatial working memory research. (B) Representative 18 F-FDG PET / MRI fusion images show decreased signal in the hippocampus 8 weeks after hTau overexpression and increased glucose metabolism 32 weeks after NeuroD1 expression. The boundary of the hippocampus is outlined by a dashed line. Scale bar, 1 cm. (C & D) Normalized 18Quantitative analysis of F-FDG signal intensity in the control group (C) and NeuroD1 treatment group (D). Each color represents a different animal. *P < 0.05. (E) Schematic diagram depicting the "delayed response" task, which uses the WGTA to evaluate the "memory retention interval" and thus assess the ability of spatial working memory before and after hTau overexpression and after NeuroD1 AAV gene therapy. (F&G) Recording in the control group (F) and NeuroD1 treatment group. (G) Quantification of the "memory retention interval" in monkeys. Each line represents a different animal. **P < 0.01, *P < 0.05, repeated measures ANOVA with Tukey's post hoc test, N = 3 (F), n = 5 (G).

[0041] Figure 8 Transcriptome analysis showed the effects of NeuroD1 on neuroinflammation, apoptosis, neuronal function, and synaptic transmission. (A) Schematic diagram illustrating the development process of the NHP AD model; gene therapy based on NeuroD1 AAV was administered, followed by bulk RNA sequencing for transcriptome analysis. (B) PCA plot of bulk RNA sequencing data. Intra-group consistency of transcriptome data from normal (green), hTau overexpression control (blue), and NeuroD1 treatment (yellow) monkeys. (C) Heatmap of normal (green) and htau-overexpressing control (blue); NeuroD1 treatment group (yellow) showed hierarchical clustering. The color scale (lower right) indicates the degree of expression: red, high expression; blue, low expression. The selection criteria for DEGs were Q value ≤ 0.05 and |log2FC| ≥ 1. (D) Volcano plot of DEGs. The x-axis represents the fold change after log2 transformation. The y-axis represents -log10-transformed significance. Red dots, upregulated genes; blue dots, downregulated genes. The upper panel is the comparison between the htau overexpression control group and the normal group; the bottom is the comparison between the NeuroD1 treatment group and the htau overexpression control group. (E) Bubble plot of GO enrichment analysis of DEGs. The enrichment factor represents the enrichment degree of GO. The node size represents the number of genes selected for the node, and the color represents the Q value of the enrichment analysis. The Q value is the p value after multiple hypothesis correction. (F) Bubble plot of KEGG pathway analysis of DEGs. The top 10 KEGG pathways were enriched. The node size represents the enrichment factor, and the color represents the p value of the enrichment analysis. The enrichment factor is the ratio of the number of DEGs annotated in the pathway (as shown on the y-axis) to the total number of genes annotated in the pathway.

[0042] Figure 9It is shown that NeuroD1 AAV-based gene therapy can prevent neuronal damage and loss in AD-like monkeys. (A) Representative images of Nissl staining showed a significant decrease in the number of Nissl-stained cells in the control group, while a significant increase in the number of Nissl-stained cells in the NeuroD1 treatment group. Scale bars: 1 mm and 200 μm (insets). (B) Quantification of the density of nsll-stained cells to evaluate neuronal loss in AD-like monkeys and neuronal regeneration / protection after NeuroD1 overexpression. ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA with Tukey's post hoc test, normal group N = 5, control group N = 4, NeuroD1 group N = 6 groups.

[0043] Figure 10 It is shown that in brain slices of AD-like monkeys, CD45-positive cells are unlikely to be microglia. (A) Representative images of CD45 immunostaining (20X) showed leukocyte infiltration and macrophage proliferation in the monkey hippocampus after hTau overexpression. Note that most CD45+ cells are Iba1-negative. Scale bar, 50 μm. (B) Representative images of CD45 immunostaining (63X) showed leukocyte infiltration and macrophage proliferation in the monkey hippocampus after hTau overexpression. Note that most CD45+ cells are Iba1-negative. Scale bar, 10 μm.

[0044] Figure 11 It is shown that NeuroD1 AAV-based gene therapy can partially restore the levels of AD biomarkers in the cerebrospinal fluid of AD-like monkeys. (A) Normalized cerebrospinal fluid total tau levels were quantitatively detected by Simoa before and after 12 weeks of hTau expression and after 32 weeks of NeuroD1 expression. **P < 0.01, one-way ANOVA with Tukey's post hoc test, N = 5. (B) Normalized cerebrospinal fluid levels of phosphorylated tau 181 and phosphorylated tau 231 were quantitatively detected by Simoa before and after 12 weeks of hTau expression and after 32 weeks of NeuroD1 expression. ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA with Tukey's post hoc test, N = 5. (C) Normalized cerebrospinal fluid levels of Aβ42 and Aβ40 were quantitatively detected by Simoa before and after 12 weeks of hTau expression and after 32 weeks of NeuroD1 expression. **P < 0.01, *P < 0.05, one-way ANOVA plus Tukey's post hoc test, N = 5. (D) Quantification of the Aβ42 / Aβ40 ratio in cerebrospinal fluid by Simoa before and after 12 weeks of hTau expression and after 32 weeks of NeuroD1 expression. *P < 0.05, one-way ANOVA with Tukey's post hoc test, N = 5.

[0045] Figure 12 Transcriptome analysis revealed upregulation of AD-like monkey neuroinflammation and apoptosis and downregulation of neurodevelopment and synaptic transmission. (A) Bubble plot of GO enrichment analysis of DEGs. The top 10 GO pathways. The rich factor represents the enrichment degree of GO. The node size represents the number of selected genes, and the color represents the Q value of the enrichment analysis. The Q value is a multiple hypothesis corrected p value. (B) Bubble plot of KEGG pathway analysis of DEGs. The top 10 KEGG pathways. The node size represents the rich factor, and the color represents the p value of the enrichment analysis. The rich factor is the ratio of the number of DEGs annotated in a pathway (shown on the y-axis) to the number of all genes annotated in that pathway. Detailed implementation mode

[0046] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0047] 1. Establishing an NHP model with AD-like pathology through the hippocampus and overexpressing hTau

[0048] Although many successes have been achieved in rodent AD models, they also have great limitations, such as short lifespan, difficulty in cognitive testing, and different brain structures and tau pathology from patients. On the other hand, non-human primates (NHPs) have genetic, anatomical, physiological, and pathological similarities to humans, and thus have become important tools in studying the pathogenesis and treatment interventions of AD. In recent decades, NHP models of Alzheimer's disease, especially transgenic primates, have made significant progress, providing valuable insights into Alzheimer's disease pathology and treatment trials. However, factors such as high cost, ethical issues, long lifespan, and limited translational relevance have hindered their widespread use.

[0049] To evaluate the safety and efficacy of NeuroD1 AAV-based gene therapy, an NHP model with AD-like pathology was generated by overexpressing the tau protein (hTau). Briefly, two AAV vectors (AAV Syn::FLPo + AAV CAG::FRT-hTau) were injected (45 μL unilaterally, 90 μL bilaterally) into the bilateral hippocampi of adult rhesus monkeys. Under the Syn promoter, AAV Syn::FLPo will specifically express the recombinase FLPo in neurons, which will induce strong expression of hTau in neurons ( Figure 1 , A). Approximately 10 weeks after AAV injection, it was found that the expression level of hTau protein in the hippocampus of the mice was significantly higher than that of the control group ( Figure 1 , B). High-magnification imaging found that tau protein accumulated excessively in the somata and apical dendrites of pyramidal neurons ( Figure 1, B, illustration). Quantitatively, the tau levels in the hippocampus of AD-like monkeys were 3.5 - 4 times that of control monkeys ( Figure 1 , C). In the AD-affected brain, hyperphosphorylation of tau at specific sites (such as S202 or T205) leads to the formation of neurofibrillary tangles (NFTs). Consistently, many hippocampal neurons were found to show strong overexpression of phosphorylated tau (S202 / T205) in their somata and dendrites at 10 weeks ( Figure 1 , D).

[0050] Neurodegeneration is a common pathological feature of Alzheimer's disease and is associated with the decline in cognitive ability of AD patients. After hTau overexpression, the immunoreactivity of NeuN in the hippocampus of macaques was significantly reduced compared to the control group without hTau protein expression ( Figure 1 , E). In terms of the number of NeuN-positive cells, after hTau overexpression, the reduction in the hippocampal DG / CA4 and CA1 / CA2 / CA3 regions was approximately 55% and 65%, respectively, indicating a significant loss of hippocampal neurons in this NHP AD model ( Figure 1 , F). Hippocampal atrophy usually leads to severe amnesia, which is a major feature of Alzheimer's disease and is also closely related to the progression of mild dementia. Obvious hippocampal atrophy was observed after hTau overexpression in NHP ( Figure 1 , G, yellow contour). Data from quantitative MRI scans showed that the measured hippocampal volume decreased by 12 - 23% 9 weeks after hTau overexpression, indicating hippocampal atrophy in the NHP AD model ( Figure 1 , H). Notably, the observed AD-like pathology is highly unlikely to be attributed to mechanical damage associated with stereotaxic injection or GFP overexpression. This conclusion is supported by the fact that it can be explained by control macaques. In addition to pathological features such as tau hyperphosphorylation, neuronal loss, and hippocampal atrophy, the AD-like NHP model also exhibits many other characteristics of pathological AD. These include 3R / 4R tau accumulation, tau propagation, neuroinflammation, lack of Aβ clearance, vascular abnormalities, and cognitive impairment ( Figure 1 , I), indicating that a potentially suitable NHP model has been developed to further evaluate therapeutic interventions for AD.

[0051] 2. Widespread expression of the neural transcription factor NeuroD1 in the hippocampus of AAV-injected monkeys

[0052] To investigate the efficacy of NeuroD1 AAV-based gene therapy in the NHP AD model, an AAV9 vector encoding NeuroD1 (NCBI Gene ID: 4760) and GFP (GFAP(CMVe)::NeuroD1+GFAP::GFP) was injected (45 μL unilaterally, 90 μL bilaterally) after 12 weeks of overexpression of hTau in the monkey hippocampus. The control virus was an inverted NeuroD1 and GFP (GFAP(CMVe)::Inverted NeuroD1+GFAP::GFP))( Figure 2 , A-B). AAV9 GFAP::GFP was used to label astrocytes by initiating GFP fluorescence in the monkey hippocampus using the astrocyte promoter GFAP. The use of a strong CMV enhancer (CMVe) can cause high expression levels of NeuroD1, which is crucial for astrocyte transformation.

[0053] AAV was injected at 6 evenly distributed injection sites as determined by T1-weighted magnetic resonance imaging scans. Thirty-six weeks later, GFP expression was still widely detected throughout the hippocampal region( Figure 2 , C), and approximately 70% of the hippocampal region was covered by GFP+ cells( Figure 2 , D). NeuroD1 immunostaining clearly showed hippocampal expression at 6 weeks and 36 weeks after injection of AAV, and the total expression level was significantly lower at 36 weeks than at 6 weeks after injection( Figure 2 , E, quantified in Figure 2 , G). High-magnification images showed that NeuroD1 was mainly expressed in astrocytic nuclei at 6 weeks, while at 36 weeks, NeuroD1 expression was mainly observed in neuronal nuclei( Figure 2 , F), suggesting that hippocampal astrocytes expressing NeuroD1 had become neurons. Over time, the expression of NeuroD1 may be due to the downregulation of astrocyte-specific GFAP promoter activity after astrocyte-to-neuron transformation. Taken together, these results demonstrate that the AAV-based gene delivery method is effective and that widespread expression of NeuroD1 can be effectively achieved in the monkey hippocampus in this gene therapy.

[0054] 3. NeuroD1 AAV-based gene therapy prevents neuronal degeneration

[0055] Neurodegeneration is a defining feature of Alzheimer's disease, and significant neuronal loss can be observed in many rodent and NHP AD models, as previously demonstrated, and the neuroregenerative potential of NeuroD1AAV-based gene therapy in the NHP AD model was further explored( Figure 3, A). AAV9 GFAP::GFP was injected to label monkey hippocampal astrocytes, and AAV encoding inverted NeuroD1 (as a control for non-functional protein expression) and AAV encoding functional NeuroD1 were co-injected. In the control group with AAV encoding inverted NeuroD1, almost all GFP+ cells maintained an astrocytic morphology after 36 weeks and were co-labeled with the astrocyte marker GFAP ( Figure 3 , B). In contrast, in the treatment group, many GFP+ cells were infected with the neuronal marker NeuN 36 weeks after NeuroD expression ( Figure 3 , C). Quantitatively, the control group was dominated by GFAP+ astrocytes (~80%) among GFP+ cells, with less than 5% of NeuN+ neurons. The NeuroD1 treatment group showed a significant cell identity shift, with GFAP+ astrocytes reduced to ~15% and NeuN+ neurons becoming the majority (~65%) among GFP+ cells, indicating that a substantial portion of astrocytes expressing NeuroD1 may have been converted to neurons 36 weeks after injection ( Figure 3 , D). In NHPs without an astrocyte lineage tracing tool, capturing the intermediate states of direct reprogramming may help confirm the astrocyte-to-neuron conversion in the monkey hippocampus. Six weeks after NeuroD1 expression, some intermediate state cells were observed to be co-labeled with astrocyte and neuronal markers (GFAP and NeuN) ( Figure 3 , E), indicating that astrocytes can be successfully converted to neurons.

[0056] Subsequently, the neuronal density in the rat brain was measured by NeuN immunostaining, comparing the monkey hippocampus with or without NeuroD1 gene therapy. Compared with the normal monkey hippocampus ( Figure 3 , F, left), after overexpression of hTau followed by overexpression of GFP in the control group, NeuN+ cells were significantly reduced in the CA2 / CA3 / CA4 regions and moderately reduced in the CA1 / DG regions as well ( Figure 3 , F). In contrast, NeuroD1 treatment led to a substantial recovery of NeuN+ cells throughout the monkey hippocampus ( Figure 3 , F, right). Quantitatively, 48 weeks after hTau, in the DG / CA1 / CA2 / CA3 / CA4 regions within the hippocampus, the neuronal density of NeuN+ in the control group was significantly reduced, by 45 - 75% ( Figure 3 , G). In contrast, compared with the control group, the NeuroD1 treatment group showed a significant repair effect ( Figure 3, G). Considering that the reduction of NeuN immunoreactivity can be caused not only by neuronal loss but also by the depletion of nerve cells, resulting in the loss of protein or antigenicity, neurons were further quantified by Nissl. Consistent with the NeuN staining results, the cell density was significantly restored after Nissl staining in the NeuroD1 treatment group. Compared with normal monkeys in the control group, the cell density of Nissl staining decreased ( Figure 9 ). In summary, these results indicate that NeuroD1 AAV-based gene therapy can effectively prevent AD-like hippocampal neuronal damage and degeneration in monkeys.

[0057] 4. Prevention of hippocampal atrophy progression by NeuroD1 AAV gene therapy

[0058] Hippocampal atrophy is closely related to cognitive dysfunction. Since NeuroD1 treatment can prevent hippocampal neuronal damage and degeneration in the NHP-AD model, it was further investigated whether this treatment might affect hippocampal atrophy. To track changes in hippocampal volume, T1-weighted MRI brain scans were performed before and after tau-induced lesions and after NeuroD1 repair ( Figure 4 . The results showed that the volume of the hippocampus in the control group of macaques decreased steadily, with a significant reduction in volume 8 weeks after tau overexpression and a further decrease 12 weeks after injection of control AAV. However, hippocampal atrophy in the NeuroD1 treatment group was successfully inhibited ( Figure 4 , B, C). Quantitatively, the standardized hippocampal volume obtained from the data measured by MRI scans showed that the atrophy rate in the control group increased from ~15% to ~25% within 16 weeks. In contrast, in the NeuroD1 treatment group, the decrease in hippocampal volume stopped after NeuroD1 treatment, and there was no further progression after a reduction of approximately 15% in hippocampal volume ( Figure 4 , D, E). For MRI analysis, additional comparisons of the average cross-sectional area of the monkey hippocampus were made. These average cross-sectional areas were calculated by averaging 10 evenly spaced coronal brain slices, comprehensively representing the hippocampus from its anterior to middle and posterior parts. The results were consistent with those of the MRI scans. The monkey hippocampal region in the NeuroD1 treatment group showed less atrophy, approximately 17%, while the control group showed approximately 30% atrophy ( Figure 4 , F, G). Along with hippocampal atrophy, the lateral ventricles of AD monkeys in the control group expanded by approximately 140%, while in the NeuroD1 treatment group, the lateral ventricles only expanded by approximately 60% ( Figure 4 , H). Collectively, these findings suggest that NeuroD1 AAV-based gene therapy can inhibit persistent hippocampal atrophy.

[0059] 5. Relief of neuroinflammation by NeuroD1 AAV-based gene therapy

[0060] Neuroinflammation is a key mechanism in the development of AD mediated by cells such as microglia and astrocytes, ultimately leading to neuronal death and dysfunction. GFAP is a recognized marker of astrocyte activation and its expression changes, and its level is often associated with the neuroinflammatory process. GFAP immunostaining was performed ( Figure 5 , A), and it was found that hyperactivated astrocytes in the hippocampus were from AD-like monkeys in the control group, rather than from the NeuroD1 treatment group ( Figure 5 , B). Magnified images showed that GFAP+ cells exhibited typical reactive astrocyte morphology in the control group, such as cytoskeletal hypertrophy and abnormally long processes. In contrast, astrocytes in the NeuroD1 treatment group were similar in morphology to resting astrocytes in the brain ( Figure 5 , C). Quantitatively representing the number of GFAP+ cells, the hippocampus of monkeys in the control group showed a significant increase, ranging from 3 to 4 times the normal level. However, in the NeuroD1 treatment group, the number of GFAP+ cells was significantly reduced compared to the control group, suggesting that NeuroD1 AAV-based gene therapy can effectively reduce the reactivity of astrocytes in the hippocampus of the NHP AD model ( Figure 5 , D).

[0061] Microglial activation is another feature of neuroinflammation. The activation of microglia was evaluated by Iba1 immunostaining ( Figure 5 , A), and it was found that the number of Iba1+ cells in the control group increased significantly compared to the normal level, while the number of Iba1+ in the NeuroD1 treatment group decreased substantially ( Figure 5 , E). More interestingly, in the hippocampus of monkeys in the NeuroD1 treatment group, most Iba1+ cells restored the ramification of resting microglia and were star-shaped. In contrast, in the control group, a considerable proportion of Iba1+ cells underwent morphological changes and showed the morphology of activated microglia, which were spherical, rod-shaped, or amoeboid-like ( Figure 5 , F). Quantitatively, the cell density of Iba1+ in the hippocampus of the control group showed a significant increase in all regions of the brain, 3 to 4 times higher than the baseline. In contrast, the number of cells in the NeuroD1 treatment group decreased significantly, with a 15 - 30% decrease compared to the baseline increase, indicating that NeuroD1 AAV-based gene therapy can effectively reduce microglial activation in the hippocampus of AD-like monkeys ( Figure 5 , G).

[0062] Brain-infiltrating leukocytes and macrophages are considered to be the cause of this disease. The progression of AD promotes blood-brain barrier (BBB) damage, enhances chronic inflammatory responses, and exacerbates nerve injury. CD45 is a leukocyte marker. CD45 staining was performed on the hippocampus of a 36-week NHP AD model after injection of control or NeuroD1-expressing AAV (Figure 5 , A). Although CD45 labels leukocytes, macrophages, and microglia in the central nervous system, the results of the study showed that most CD45+ cells did not express Iba1, indicating that most of these CD45+ cells were unlikely to be microglia ( Figure 10 ). In fact, almost no CD45+ leukocytes and macrophages were detected in the hippocampus of the normal group, while the AD-like monkeys in the control group showed the presence of a large number of CD45+ leukocytes and macrophages. However, the NeuroD1 treatment group showed a significant decrease in the CD45 signal level ( Figure 5 , H, I, J). Taken together, these findings suggest that AAV-based NeuroD1 gene therapy can successfully alleviate neuroinflammation in the hippocampus of AD-like monkeys.

[0063] 6. Gene therapy based on NeuroD1 AAV repairs blood vessels and blood-brain barrier integrity

[0064] Vascular problems, including chronic cerebral hypoperfusion, blood-brain barrier leakage, and persistent vascular inflammation, play an important role in the progression of sporadic AD. The thickening of the basement membrane in vascular neurodegenerative diseases may impede blood circulation to the brain and accelerate disease development. Laminin staining was performed. Laminin is a basement membrane marker ( Figure 6 A). Thickening of the vascular basement membrane and vascular abnormalities ( Figure 6 , B, C) including string vessels (open arrows), vascular occlusion (open arrows), and vascular distortion / bulging (closed arrows, Figure 6 in C) were observed in the control group. In contrast, there was less thickening of the blood vessels in the hippocampus of the NeuroD1 treatment group and fewer obvious abnormalities ( Figure 6 , B, C right). In addition, staining for the PECAM-1 / CD31 endothelial marker ( Figure 6 , A) showed obvious vascular damage / degeneration in the control group, marked by vascular rupture (arrow) and disintegration (arrow) in the control group ( Figure 6 , D). In contrast, vascular damage / degeneration was significantly reduced in the treatment group ( Figure 6 , D right). Quantitatively, the thickening of the basement membrane, vascular damage, and degeneration in the control group increased by 8 - 12 times, while the treatment group only increased by 2 - 3 times, suggesting that this gene therapy has the potential to repair vascular damage in the hippocampus of the NHP AD model ( Figure 6 , E, F)

[0065] AQP4 is mainly expressed in the end-feet of astrocytes, and the end-feet are crucial for astrocyte apoptosis. Disruption of AQP4 function can lead to disruption of the blood-brain barrier. In the lymphatic system, AQP4 enables the effective exchange of water and solutes between cells, thus promoting the clearance of waste from the brain. AQP4 immunostaining showed diffuse staining in the control groupFigure 6 In contrast, the distribution of AQP4 signal in the treatment group was basically restored (arrow). AQP4 was observed to be diffusely distributed in the control group and polarized in the normal group and the NeuroD1 treatment group. Figure 6 (H), indicating that overexpression of hTau protein disrupts the integrity of the blood-brain barrier, which can be partially restored by NeuroD1 AAV-based gene therapy.

[0066] Total tau protein, phosphorylated tau 181 protein, and phosphorylated tau 231 protein in cerebrospinal fluid were positively correlated, while Aβ42 peptide and Aβ42 / Aβ40 ratio in cerebrospinal fluid were negatively correlated, making them specific biomarkers for Alzheimer's disease. In addition, astrocytic AQP4 ultimately disrupts perivascular drainage and the lymphatic system, leading to defective clearance of Aβ / tau. Using Simoa technology to measure total tau protein, phosphorylated tau 181 protein, and phosphorylated tau 231 protein in monkey cerebrospinal fluid samples collected before and after repair, significant increases were found 12 weeks after hTau overexpression, but significant decreases were observed 32 weeks after NeuroD1 expression. Figure 11 (A, B). Similarly, the ratios of Aβ42, Aβ40, and Aβ42 / Aβ40 were evaluated in monkey cerebrospinal fluid samples, and significant decreases in Aβ42 levels and Aβ42 / Aβ40 ratios were found 12 weeks after hTau overexpression. However, 32 weeks later, both Aβ42 levels and Aβ42 / Aβ40 ratios of NeuroD1 were significantly increased, indicating enhanced Aβ clearance after NeuroD1 treatment. Figure 11 (C, D). In summary, NeuroD1 AAV-based gene therapy can effectively repair vascular and blood-brain barrier damage and may help AD monkeys clear brain waste.

[0067] 7. NeuroD1 AAV-based gene therapy can improve hippocampal glucose metabolism and spatial working memory.

[0068] Fluorine-18 fluorodeoxyglucose 18 (18F-FDG) PET scan is a sensitive method for differentiating changes in glucose metabolism in the brain. Notably, in specific brain regions closely related to the pathological diagnosis of AD, effective Alzheimer's disease treatments often improve the metabolic activity of the hippocampus. Therefore, longitudinal 18 18F-FDG PET brain imaging was performed on the same group of monkeys and evaluated before and after hTau overexpression and after NeuroD1 AAV-based gene therapy. Figure 7 (A). Consistent with Figure 3 the neuronal loss shown, glucose metabolism in the monkey hippocampus decreased significantly 8 weeks after hTau overexpression, and further decreased 32 weeks after injection of AAV in the control group of monkeys. Figure 7, B, C). In contrast, after 32 weeks of NeuroD1 overexpression, glucose metabolism in the hippocampus of AD-like monkeys in the treatment group was significantly improved, and neuronal activity was restored ( Figure 7 , B, D) Glucose metabolism was significantly reduced 8 weeks after hTau overexpression, indicating substantial neuronal damage and degeneration in the monkey hippocampus. Subsequently, 32 weeks after injection of AAV, glucose metabolism continued to decline in the control group ( Figure 7 , C), suggesting further neuronal damage and loss. In contrast, after 32 weeks of NeuroD1 AAV-based gene therapy, there was a substantial recovery of glucose metabolism ( Figure 7 , D), suggesting possible restoration of neuronal function in the NeuroD1 treatment group.

[0069] Spatial working memory is a key assessment for understanding the pathological progression of Alzheimer's disease and a potential therapeutic intervention for AD. Although working memory has traditionally been thought to be related to the prefrontal cortex in many studies, evidence has been provided for the involvement of the hippocampus in spatial working memory. To assess spatial working memory in rhesus monkeys, the "delayed response" (DR) task operated using the Wisconsin General Test Apparatus (WGTA) has been widely adopted, which is a test of the cognitive ability of non-human primates. In the DR task, the monkey is required to simply remember the location of a food reward hidden in a box. The monkey is considered to be able to reliably remember the location of the object food reward, and after a specified delay period, its line of sight is blocked by an opaque cover, and it accurately selects the bait well in at least 26 out of 30 attempts on the first try for 3 consecutive days of testing. ( Figure 7 , E). The results showed that 8 weeks after hTau expression, the "memory retention time" of the monkeys decreased significantly, indicating a significant impairment in spatial working memory ability (Figure 7F, G). Up to 21 weeks after injection of AAV, the monkeys in the control group remained at a low level ( Figure 7 , F). In contrast, the monkeys treated with NeuroD1 had a significant improvement in "memory retention time" in the same time window as the control group ( Figure 7 , G). These results provide convincing evidence that NeuroD1 AAV-based gene therapy can effectively improve spatial working memory deficits in rats.

[0070] 8. Transcriptome analysis shows the effects of NeuroD1 on neuroinflammation and apoptosis, as well as the upregulation and downregulation of neuronal function and synaptic transmission

[0071] Transcriptome analysis is an effective tool that can identify gene expression changes, clarify molecular mechanisms and pathways related to diseases or biological functions; reveal treatment effects, discover new genes, and guide the potential contributions of treatment strategies. Therefore, RNA sequencing and transcriptome analysis were performed to identify differentially expressed genes (DEGs) and signaling pathways regulated by NeuroD1 overexpression in an attempt to reveal the molecular mechanism of NeuroD1 AAV-based gene therapy ( Figure 8 , A). The results of principal component analysis (PCA) showed that the transcriptome data of 3 clusters of normal monkeys and 3 AD-like monkeys overexpressing tau protein, and 5 monkeys treated with NeuroD1 AAV-based gene therapy were divided into three discrete regions in the PCA, indicating the consistency of the transcriptome data within the group ( Figure 8 , B). The heatmap of DEGs from hierarchical clustering not only demonstrated the consistency within the data population but also showed significant differences between different groups. Many DEGs were upregulated in the control group, but downregulated in the treatment group, and vice versa, indicating that NeuroD1 AAV-based gene therapy systematically restored the brains of AD-like monkeys to a healthier state ( Figure 8 , C). The volcano plot showed that 1,579 genes were upregulated and 1,353 genes were upregulated, with statistically significant differences between the control group and the normal group (P<0.05) ( Figure 8 , upper panel of D), and 974 genes were upregulated and 745 genes were downregulated in the NeuroD1 treatment group and the htau overexpression control group ( Figure 8 , lower panel of D).

[0072] Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identify the components related to biological processes, molecular functions, and cellular functions associated with a set of genes and map the genes to known signaling and metabolic pathways related to diseases or biological processes. Therefore, GO analysis was performed, and the results showed that in the hippocampus of AD-like monkeys, biological processes related to neuroinflammation, apoptosis, autophagy, synaptic pruning, cytotoxicity, and amyloid fiber formation were significantly upregulated; while the functions of ion channels and transporters, presynaptic neurotransmitter release, nuclear RNA surveillance, and postsynaptic neurotransmitter receptor trafficking were significantly downregulated ( Figure 12 , A). Similarly, KEGG pathway analysis of DEGs showed that in the hippocampus of AD-like monkeys, Alzheimer's disease, many other neurodegenerative diseases, neuroinflammation, phagocytosis, and innate immune responses were significantly upregulated; while the major neurodevelopmental signaling pathways, neuroactive ligand-receptor interactions, and metabolic regulatory signaling pathways were significantly downregulated ( Figure 12 , B).

[0073] In contrast to the transcriptomic changes induced by hTau overexpression, in the hippocampus of the NeuroD1 treatment group, the content of NHPs, GO enrichment analysis of DEGs revealed that the biological processes and cellular component assembly related to presynaptic and postsynaptic, apical dendrites, mitochondrial energy metabolism, resting membrane potential maintenance, and synaptic transmission were significantly upregulated; while the processes related to biological and neuroinflammatory responses, apoptosis, and phagocytosis were significantly downregulated ( Figure 8 , E). Similarly, the results of KEGG pathway analysis of DEGs showed that in the hippocampus of the NeuroD1 treatment group, the signals of NHPs were significantly regulated in the pathways related to neurodevelopment, calcium signaling, mitochondrial energy metabolism, long-term potentiation, axon guidance, and neuroactive ligand-receptor interaction; while the signaling pathways related to pathology, neuroinflammation, innate immune response, phagocytosis, and the formation of new blood vessels were significantly downregulated ( Figure 8 , F). Overall, transcriptomic analysis indicated that NeuroD1 AAV-based gene therapy could systematically restore the AD monkey-like brain to a healthier state, mainly by upregulating neuronal function and synaptic transmission, while downregulating neuroinflammation and apoptosis.

[0074] In summary, in the present invention, AAV encoding NeuroD1 was delivered to AD-like hippocampal monkeys. Widespread expression of NeuroD1 in the hippocampus prevented apoptotic neurodegeneration of neurons, stopped hippocampal atrophy, reduced neuroinflammation, and repaired vascular BBB damage. In addition, CSF AD biomarkers, hippocampal metabolism, and spatial working memory were all partially restored. Transcriptomic analysis indicated that NeuroD1 AAV-based gene therapy upregulated neuronal development and synaptic transmission and downregulated neuroinflammation and apoptosis, presenting a promising multifaceted approach for treating AD. The research results showed that NeuroD1 AAV-based gene therapy could prevent neuronal loss, stop hippocampal atrophy, reduce neuroinflammation, repair vascular / blood-brain barrier damage, restore CSF AD biomarker levels, improve glucose metabolism, and enhance spatial memory in the NHP AD model, thus highlighting its therapeutic potential.

Claims

1. Use of NeuroD1 in the repair of Alzheimer's disease.

2. Use of a vector encoding NeuroD1 in any of the following aspects: (1) Preventing neuronal damage and apoptosis; (2) Inhibiting hippocampal atrophy; (3) Alleviating neuroinflammation; (4) Repairing vascular / blood-brain barrier damage; (5) Restoring the levels of cerebrospinal fluid AD biomarkers to normal; (6) Promoting the clearance of pathological toxic proteins in the brain tissue; (7) Improving glucose metabolism; (8) Enhancing spatial working memory ability; (9) Regenerating neurons.

3. The application according to claim 2, characterized in that The vector is a viral vector.

4. The application according to claim 2, wherein The vector is an adeno-associated virus.

5. The application according to claim 2, wherein The vector overexpresses NeuroD1.

6. The application according to claim 2, wherein The AD biomarkers include total tau protein, phosphorylated tau 181 protein, and phosphorylated tau 231 protein.

7. The application according to claim 2, characterized in that The regenerated neurons include: converting hippocampal astrocytes into neurons.

8. Use of a vector encoding NeuroD1 in the preparation of a drug having any of the following functions: (1) Preventing neuronal damage and apoptosis; (2) Inhibiting hippocampal atrophy; (3) Alleviating neuroinflammation; (4) Repairing vascular / blood-brain barrier damage; (5) Restoring the levels of cerebrospinal fluid AD biomarkers to normal; (6) Promoting the clearance of pathological toxic proteins in the brain tissue; (7) Improving glucose metabolism; (8) Enhancing spatial working memory ability; (9) Regenerating neurons.

9. The application according to claim 8, wherein The vector is an adeno-associated virus.

10. The application according to claim 8, characterized in that, The vector overexpresses NeuroD1.

Images