Application of Gpnmb gene in related tau protein lesion

Abstract

The invention relates to the technical field of treatment of neurodegenerative diseases, in particular to application of a Gpnmb gene in related tau protein diseases. The invention relates to a pharmaceutical composition, which comprises a small molecule compound capable of activating a v-ATP enzyme subunit ATP6V0D1 and / or ATP6V0D2, and is used for recovering lysosomal acidification and tau protein degradation capability of Gpnmb-deficient microglial cells, the small molecule compound is hypericin, and the pharmaceutical composition is used for treating neurodegenerative diseases related to tau protein accumulation. The application of the Gpnmb gene in related tau protein lesions reveals that deletion of the Gpnmb gene can aggravate pathological changes, neurodegenerative lesions and cognitive impairment of tau protein, clarifies the key effect of the Gpnmb gene in neuroprotection, and explains that Gpnmb regulates assembling of a v-ATP enzyme subunit ATP6V0D1 / D2 and maintains lysosomal acidification so as to promote degradation of the tau protein from the molecular level, so that the application of the Gpnmb gene in the related tau protein lesions is realized, and the application of the Gpnmb gene in the related tau protein lesions is realized. And a new target spot is provided for treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of neurodegenerative disease treatment, and particularly relates to a method for treating tauopathy Gpnmb application of the gene in related tauopathy. BACKGROUND

[0002] Aging is the most significant risk factor for Alzheimer's disease (AD) and related tauopathies, but how age-related cellular decline accelerates tau-driven neurodegeneration remains a key question. One of the most prominent features of aging is the gradual decline of lysosomal function, which disrupts the clearance mechanism of misfolded and aggregated proteins. As the core of protein homeostasis, lysosomes rely on the acidic environment maintained by vacuolar ATPase (V-ATPase) to degrade toxic proteins such as tau. With age, the lysosomal acidification capacity decreases, leading to the accumulation of protein aggregates, which in turn triggers synaptic dysfunction, neuronal loss, and glial activation. This makes lysosomal dysfunction a nexus of aging and neurodegeneration.

[0003] Tau aggregation, as a hallmark of Alzheimer's disease (AD), triggers synaptic dysfunction, neurofibrillary tangle formation, and progressive neurodegeneration. In the PS19 mouse model, overexpression of tau recapitulates key features of human tauopathy, including hippocampal synaptic damage, glial activation, and neuronal death. Notably, microglia not only respond to tau aggregates but also promote their spread, forming a vicious cycle: lysosomal dysfunction exacerbates inflammatory responses, while accelerating the spread of tau.

[0004] Recent research has found that glycoprotein non-metastatic melanoma protein B (GPNMB) is a key player in regulating lysosomal integrity. With age and the development of neurodegenerative diseases, the expression of GPNMB significantly increases, indicating that it is a "firefighter" for cells to cope with stress. From a molecular mechanism perspective, GPNMB, by binding to CD44 and V-ATPase subunits, can both stabilize the lysosomal acidification environment and maintain its protein degradation function. Once macrophages lose GPNMB, lysosomal clearance dysfunction occurs - but as long as the "parts" of V-ATPase are supplemented, this problem can be easily solved. In the aging brain, GPNMB is enriched in microglia, which may have a dual role: protecting lysosomal function and inhibiting the release of inflammatory cytokines. Despite these findings, whether GPNMB has a protective effect on tau pathology remains completely unexplored.

[0005] GPNMB is a previously unidentified molecular brake that maintains lysosomal function and inhibits age-related neurodegeneration induced by tauopathy. In PS19 mouse models, Gpnmb gene deletion impairs V-ATPase activity, leading to lysosomal alkalinization, tau clearance inhibition, and excessive microglial inflammatory responses. These pathological cascades accelerate hippocampal neurodegeneration, manifested as thinning of the CA1 pyramidal layer and synaptic loss. Notably, a small molecule compound, hyperforin, discovered through structure-guided screening, restores lysosomal acidification and microglial degradation capacity in the Gpnmb deficient background. Comprehensive research results show that GPNMB is at the intersection of protein aggregation and neuroinflammation, suggesting that GPNMB-targeted therapeutic strategies may be a promising treatment for age-related tauopathy and neurodegenerative diseases.

[0006] To this end, a kind of Gpnmb gene in related tauopathy is designed to provide another technical solution to the above technical problems. SUMMARY

[0007] Therefore, it is necessary to provide a kind of Gpnmb gene in related tauopathy for solving the technical problems proposed in the background art.

[0008] To solve the above technical problems, the present application adopts the following technical solutions: A pharmaceutical composition comprising a small molecule compound capable of activating v-ATPase subunits ATP6V0D1 and / or ATP6V0D2 for restoring Gpnmb lysosomal acidification and tau degradation capacity of deficient microglia.

[0009] As a preferred embodiment of the pharmaceutical composition provided by the present application, the small molecule compound is hyperforin.

[0010] As a preferred embodiment of the pharmaceutical composition provided by the present application, the pharmaceutical composition is used for treating neurodegenerative diseases related to tau accumulation.

[0011] A method for screening candidate compounds for treating tauopathy, comprising the following steps: Molecular docking of candidate compounds with ATP6V0D1 and / or ATP6V0D2 subunits; Evaluate the promoting effect of the compound on lysosomal acidification; Select compounds that can restore GpnmbCompounds that enhance the degradation capacity of tau protein in defective microglia.

[0012] The use of the prepared compound in the preparation of a medicament for the prevention or treatment of tau protein lesions, the use including by enhancing... Gpnmb Gene expression or activity improves lysosomal function in microglia and promotes the degradation of tau protein.

[0013] As a preferred embodiment of the application provided by the present invention, the Gpnm The b gene promotes lysosomal acidification by regulating the assembly of the v-ATPase subunits ATP6V0D1 and / or ATP6V0D2.

[0014] As a preferred embodiment of the application provided by the present invention, the tau protein lesions include Alzheimer's disease, tau protein hyperphosphorylation, aggregation, or neurodegenerative lesions manifested in the PS19 transgenic tau mouse model.

[0015] A sort of Gpnmb The application of genes as biomarkers in the diagnosis or prediction of tau protein disease progression involves assessing disease status or treatment response by detecting the level of the extracellular domain of GPNMB in cerebrospinal fluid or tissue.

[0016] It is clear without a doubt that the technical solution described above in this application can solve the technical problem that this application aims to address.

[0017] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects: 1. A method provided by the present invention Gpnmb The application of genes in related tau protein lesions reveals Gpnmb Gene deletion exacerbates pathological changes in tau protein, neurodegenerative diseases, and cognitive impairment, clarifying its crucial role in neuroprotection and elucidating its molecular function. Gpnmb By regulating the assembly of the v-ATPase subunit ATP6V0D1 / D2, maintaining lysosomal acidification, and thereby promoting the degradation of tau protein, a new therapeutic target is provided.

[0018] 2. In the PS19 mouse model, this invention... Gpnmb Gene deletion impairs type V ATPase activity, leading to lysosomal alkalization, hindering tau protein clearance, and triggering excessive microglial inflammatory responses; these pathological cascades accelerate hippocampal neurodegeneration, manifested as thinning of the CA1 pyramidal layer and synapse loss; structure-based screening identified the small molecule compound hypericin, which can... GpnmbRestoring lysosomal acidification function and microglial degradation capacity in the presence of defects; Comprehensive research results show that GPNMB is at the intersection of protein aggregation and neuroinflammation, suggesting that GPNMB-targeted therapeutic strategies may become a promising treatment for age-related tau pathology and neurodegenerative diseases.

[0019] 3、The present application can restore lysosomal function in a defective model by screening for small molecule compounds, such as hypericin, which can penetrate the blood-brain barrier. Gpnmb The extracellular domain (ECD) of GPNMB is significantly elevated in cerebrospinal fluid and can be used as a biomarker for tau pathology to aid early diagnosis and efficacy evaluation of the disease. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed in the embodiment description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 For the Gpnmb gene deletion of the present application Gpnmb Schematic diagram of Gpnmb gene deletion accelerating cognitive decline in PS19 mice; Figure 2 For the Gpnmb gene deletion of the present application Schematic diagram of Gpnmb gene deletion accelerating cognitive decline in PS19 mice; Figure 3 Gpnmb For the Gpnmb gene deletion of the present application Schematic diagram of Gpnmb gene deletion accelerating cognitive decline in PS19 mice; Figure 4 Gpnmb For the Gpnmb gene deletion of the present application Schematic diagram of Gpnmb gene deletion accelerating cognitive decline in PS19 mice; Figure 5 Gpnmb For the Gpnmb gene deletion of the present application Figure 6 For the Gpnmb gene deletion of the present application Figure 7 For the Gpnmb gene deletion of the present application Gpnmb Schematic diagram of Gpnmb gene deletion accelerating cognitive decline in PS19 mice; Figure 8 For the Gpnmb gene deletion of the present application Schematic diagram of Gpnmb gene deletion accelerating cognitive decline in PS19 mice;Figure 9 Fig. 9.5-month-old WT and KO mice hippocampus contrast illustration; Gpnmb Fig. 9.5-month-old WT and KO mice hippocampus contrast illustration; Figure 10 Fig. 10. Open field test results illustration of the present application; Figure 11 Fig. 11. Average expression levels of selected marker genes in different cell types illustration of the present application; Figure 12 Fig. 12. Immunolabeling subcellular localization illustration of the present application; Figure 13 Fig. 13. Gene expression contrast illustration of the present application. DETAILED DESCRIPTION

[0022] In order to make the objects, technical solutions and advantages of the present application clearer, the following will further describe the present application with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0023] In order to make those skilled in the art better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely with reference to the accompanying drawings.

[0024] It should be noted that the embodiments in the present application and the features and technical solutions in the embodiments can be combined with each other without conflict.

[0025] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0026] Embodiment One With reference to Figures 1-13 , a Gpnmb application of the gene in related tauopathy.

[0027] 1. In PS19 mice, Gpnmb Deletion of the gene exacerbates tau pathology A large cerebrospinal fluid (CSF) proteomics dataset from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort was analyzed. This analysis showed that levels of GPNMB cytoplasmic domain (CD) and extracellular domain (ECD) were significantly elevated in A - T - compared to A + T + individuals (p<0.05) (Fig. 1). Figure 1A). To further explore the potential role of GPNMB in tauopathies, its expression was examined in 9.5-month-old wild-type (WT) and PS19 tau transgenic mice. Western blot (WB) analysis showed that GPNMB levels were elevated in the hippocampus of PS19 mice, but no changes were observed in the cortex ( Figure 1 B, Figure 8 A, B). To explore whether GPNMB could modulate tau pathology, PS19; Gpnmb knockout (KO) mice were crossed with PS19 mice to generate PS19; Gpnmb KO (PS19; GKO) mice. At 9.5 months of age, immunohistochemical analysis showed that p-tau202 / 205 + and p-tau217 + were significantly higher in the hippocampus of PS19; GKO mice than in PS19 controls ( Figure 1 C, D). WB further confirmed these elevations ( Figure 1 E, F). Thioflavin-S labeling also showed increased neurofibrillary tangle-like aggregates in the CA1, CA3, and DG subregions ( Figure 1 G, H). These findings suggest that Gpnmb the absence of GPNMB accelerates tau hyperphosphorylation and aggregation in PS19 mice.

[0028] 2、 Gpnmb GKO mice exacerbate neurodegeneration and neuroinflammation in PS19 mice Tau-related neurodegeneration was assessed by Sudan black staining, and it was found that PS19; GKO mice had more severe brain atrophy, with reduced hippocampal volume and increased ventricular volume, in stark contrast to PS19 mice ( Figure 2 A, B). NeuN immunostaining results showed that the density of pyramidal neurons in the CA1 region and the density of granule cells in the dentate gyrus (DG) were significantly reduced in PS19; GKO mice ( Figure 2 C, D). In line with the phenomenon of neuronal loss, the number of PSD95 / SYP colocalization spots in the hippocampal samples of PS19; GKO mice was reduced, and the expression levels of PSD95 and SYP were significantly lower than those in PS19 controls ( Figure 2 E-H). These data suggest that Gpnmb the absence of GPNMB exacerbates neurodegeneration and synaptic dysfunction in tauopathy mice.

[0029] Given that the association between tau pathology and neuroinflammation has been confirmed, the activation of glial cells was further evaluated. Compared with PS19 mice, PS19; GKO mice had more activated microglia (Iba1 + ) and disease-associated microglia (DAM, Clec7A+ ) significantly increased (p < 0.05, t-test) in PS19;GKO mice (Fig. 1 Figure 2 I, J). Activated astrocytes (GFAP + ) and A1 reactive astrocytes (C3+) were also significantly increased (p < 0.05, t-test) in PS19;GKO mice (Fig. 1 Figure 2 K, L). These findings suggest that Gpnmb Gene deletion exacerbates tau-associated neuroinflammation.

[0030] 3、 Gpnmb Gene deletion exacerbates cognitive impairment in PS19 mice To investigate Gpnmb whether gene deletion affects cognitive decline in tauopathy, 9-month-old PS19 and PS19;GKO mice were subjected to a series of behavioral tests. In the novel object recognition (NOR) task (Fig. 2 Figure 3 A), both strains showed similar levels of locomotor activity during the habituation phase (Fig. 2 Figure 10 A and B), but PS19;GKO mice spent significantly more time in the center of the experimental arena than PS19 controls (Fig. 2 Figure 3 B), which is consistent with changes in emotional or anxiety-related behavior. During the test phase, PS19 mice showed a preference for the novel object, while PS19;GKO mice failed to discriminate between the novel and familiar objects (Fig. 2 Figure 3 C), indicating impaired recognition memory.

[0031] Subsequently, the hippocampus-dependent spatial learning and memory abilities were assessed using the Morris water maze (MWM) test. Compared to PS19 mice, PS19;GKO mice showed longer escape latencies during the 6-day acquisition phase (Fig. 2 Figure 3 D, E). In the probe trial, PS19;GKO mice spent less time in the target quadrant and crossed the previous platform location fewer times than PS19 controls (Fig. 2 Figure 3 F, G), despite comparable swimming speeds (Fig. 2 Figure 10 C), suggesting that the deficits stem from impaired spatial memory rather than interference from motor or motivational factors.

[0032] In the contextual fear conditioning test, PS19;GKO mice exhibited significantly reduced freezing responses in the 24-hour contextual memory recall test (Fig. 2 Figure 3 H), while the immediate post-shock freezing response and cued fear memory in a novel environment remained normal, indicating selective impairment of hippocampus-dependent contextual memory. Taken together, these data lead to the conclusion that: Gpnmb gene deletion exacerbates hippocampal dysfunction in tauopathy, leading to recognition impairment, spatial memory deficits, and contextual memory impairment.

[0033] 4. Gpnmb Genetic defects disrupt the microglial lysosomal gene network in mouse Tau proteinosis. To investigate the molecular mechanism of GPNMB regulation in Tau proteinopathy, the following studies were conducted: Gpnmb The effects of gene knockout on cell type-specific transcriptome changes in the hippocampus. Hippocampal tissues from 9.5-month-old PS19 and PS19;GKO mice were analyzed using single-nuclear RNA sequencing (snRNA-seq). Following a rigorous quality control process (including binuclear RNA removal, batch effect correction, and normalization), 24,636 high-quality single-nuclear transcriptome datasets were obtained. These datasets were then classified into six major cell types based on classical markers. Figure 4 A and Figure 11 A).

[0034] Comparative analysis of cell type composition revealed that PS19;GKO mice had a significantly reduced number of excitatory neurons and a significantly increased microglial cell population. Figure 4 B). Differential gene expression (DEG) analysis revealed 3,157 differentially expressed genes in excitatory neurons (2,001 upregulated and 1,156 downregulated; corrected P < 0.05, |log2FC| > 0.25) and 995 differentially expressed genes in inhibitory neurons (659 upregulated and 336 downregulated). Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis indicated that the differentially expressed genes in neurons were mainly enriched in pathways related to neurodegenerative diseases, including mitochondrial dysfunction and apoptosis, suggesting... Gpnmb The absence of neurons disrupts neuronal homeostasis. Figure 4 CE). In microglia, 1,340 differentially expressed genes were identified (835 upregulated and 505 downregulated), and KEGG enrichment analysis showed significant overexpression in the lysosomal and phagosome pathways. Figure 4 F). Notably, these differentially expressed genes in microglia are associated with lysosomal acidification, hydrolytic enzyme activity, and vesicle transport, suggesting that GPNMB may participate in tau protein degradation by regulating lysosomal function in microglia. Figure 4 G). Taken together, these findings indicate Gpnmb Gene deletion can exacerbate neuronal loss, enhance microglia activation, and promote lysosomal dysfunction.

[0035] 5. GPNMB deficiency can impair lysosomal function in microglia, thereby affecting the clearance of Tau protein. Given that PS19 mice GpnmbGene deletion disrupts the lysosomal gene network in microglia. This study investigated whether GPNMB directly participates in the regulation of lysosomal compartments. Immunofluorescence staining showed that GPNMB partially co-localizes with the lysosomal marker CD68 in primary microglia. Figure 5 A). To assess the lysosomal degradation capacity, the DQ-BSA-Red probe was used. This probe is cleaved by proteases in the acidic lysosomal compartment, generating fluorescent fragments and enhancing fluorescence intensity. Live-cell imaging techniques were used to... Gpnmb Primary microglia from knockout mice and wild-type mice were treated with DQ-BSA-Red, and the degradation process was quantified in real time. During the 18-hour observation period, Gpnmb The mean red fluorescence intensity of knockout microglia was significantly lower than that of the wild-type control group. Figure 5 (B, C) indicates impaired lysosomal proteolytic activity. Confocal microscopy further confirmed this. Gpnmb This defect of knocking out microglia ( Figure 5 D, E).

[0036] The potential mechanisms of this lysosomal dysfunction were then investigated. Acridine orange (AO) staining revealed that wild-type microglia maintained intact lysosomal membranes, indicated by orange fluorescence (indicating acidic pH). In contrast, Gpnmb Knockout microglia exhibited green AO fluorescence, indicating lysosomal membrane damage and intraluminal alkalization. Figure 5 F, G). To further quantify lysosomal acidification, ratio analysis was performed using Lysosensor Yellow / BlueDND-160. Compared to wild-type control, Gpnmb Knocking out microglia significantly reduced the yellow / blue emission ratio, confirming... Gpnmb Gene deletion disrupts lysosomal acidification ( Figure 5 H, I).

[0037] Given the important role of microglia in Tau protein clearance, the study evaluated... Gpnmb Does gene knockout affect the phagocytosis and degradation of Tau fibers by microglia? In the experiment, primary microglia were first co-incubated with pHrodo-labeled Tau-PFF for 9 hours (phagocytosis phase), and then cultured in fresh medium for another 9 hours (degradation phase). Although Gpnmb The phagocytic capacity of knockout microglia is comparable to that of wild-type microglia. Figure 5 (J, K), but during the degradation stage Gpnmb The pHrodo fluorescence intensity in knockout microglia was significantly higher than that in wild-type microglia. Figure 5 J, K), indicating that its Tau fiber removal function is defective.

[0038] These findings suggest that GPNMB is crucial for maintaining lysosomal function in microglia, particularly its acidification and degradation capabilities. Gpnmb Gene deletions disrupt lysosomal integrity, leading to defects in tau protein clearance, which may promote the accumulation of tau protein in neurodegenerative diseases.

[0039] 6. GPNMB interacts with the v-ATPase subunit ATP6VOD1 / D2, regulating lysosomal acidification. To investigate how GPNMB regulates lysosomal function in microglia, a subset analysis of the microglia population was performed, identifying 10 unique subsets. Figure 6 A, B). Among them, PS19 mice showed high expression. Gpnmb Microglial subsets (Gpnmb-High, clusters 1 and 6) showed high expression of disease-associated microglial (DAM) markers. Figure 6 C and Figure 13 C). Notably, in PS19;GKO mice Gpnmb Knockout microglia (Low-G, clusters 2-5, 7-10) reduced the number of cells, while expressing high levels of [something]. Gpnmb The number of microglia (High-G) increased significantly. Figure 6 D). For high expression Gpnmb Differential gene expression analysis of microglia showed that the expression of ATP6V0D2 and Colec12 was upregulated in PS19;GKO mice, while Ctss , H2-K1 and Slc11A2 downregulation of expression ( Figure 6 E). KEGG pathway enrichment analysis revealed particularly prominent abnormalities in lysosome and phagosome function, especially in acidification capacity and hydrolytic enzyme activity. Figure 6 E), which indicates that GPNMB plays a crucial role in maintaining lysosomal function.

[0040] Given that ATP6V0D2 is a key v-ATPase subunit regulating proton transport and lysosomal acidification, this study investigated whether GPNMB affects v-ATPase activity. Quantitative proteomics analysis showed that Tau protein treatment increased the expression levels of multiple v-ATPase subunits (ATP6V0D1 / D2) in wild-type microglia, but... Gpnmb This effect disappears completely when cells are knocked out. Figure 6 F). This finding strongly suggests that GPNMB is essential for Tau-induced v-ATPase assembly. Immunoprecipitation-mass spectrometry (IP-MS) showed specific enrichment of ATP6V0D1 and ATP6V0D2 in the microglial GPNMB interactome. Figure 6G). Co-immunoprecipitation experiments further validated the interaction of GPNMB with ATP6V0D1 / ATP6V0D2 Figure 6 H). Taken together, these results suggest that GPNMB can regulate lysosomal acidification by directly interacting with v-ATPase subunits ATP6V0D1 / D2.

[0041] 7. In GPNMB-deficient microglia, activation of ATP6V0D1 / D2 using Hypericin enhances lysosomal acidity and promotes Tau protein degradation From Gpnmb Deficiency-induced v-ATPase dysfunction is the main cause of lysosomal acidity abnormalities in microglia. To functionally validate these findings, lysosomal acidity was measured using Green DNS-189. While overexpression of GPNMB alone had no effect, overexpression of ATP6V0D1 or ATP6V0D2 significantly enhanced lysosomal acidification, with ATP6V0D2 exhibiting stronger potency ( Figure 7 A). Importantly, co-expression of GPNMB with either ATP6V0D1 or ATP6V0D2 synergistically enhanced acidification beyond the individual subunit effects ( Figure 7 A). Therefore, an attempt was made to screen compounds targeting v-ATPase to restore lysosomal pH. Compounds that could promote lysosomal acidification were selected and subjected to molecular docking with ATP6v0D1 and ATP6V0D2 subunits. A total of 572 compounds could dock with ATP6V0D1 and ATP6V0D2 ( Figure 7 B). Among them, 84 compounds had a docking score greater than 19 with the ATP6V0D1 subunit, 53 with the ATP6V0D2 subunit, and 21 were common to both ( Figure 7 C). Next, these compounds were used to treat WT and Gpnmb KO primary microglia at 10 μΜ, and LysoSensor™ Green DND-189 was used to assess changes in lysosomal acidity. The results showed that Boscalid (Boscalid) and Hypericin (Hypericin) specifically restored lysosomal pH in Gpnmb KO microglia ( Figure 7 D, E). Since Boscalid is a major component of pesticides. Subsequently, Hypericin was chosen to treat 293T cells overexpressing ATP6V0D1 and ATP6V0D2 subunits. The experimental results showed that in cells overexpressing ATP6V0D1 and ATP6V0D2, Hypericin further acidified the lysosomes ( Figure 7F), indicating that hyperforin enhances v-ATPase function by targeting these subunits. Since lysosomal acidity is a key factor affecting lysosomal hydrolase activity, it was tested whether hyperforin could restore Gpnmb the ability of primary microglia to degrade Tau protein. Notably, hyperforin treatment partially restored Gpnmb the ability of microglia to degrade Tau protein Figure 7 G, H). Integrating these findings indicates that hyperforin can modulate lysosomal acidity and degradative function by regulating the activity of ATP6v0D1 and ATP6v0D2 subunits.

[0042] 8. Summary In neurodegenerative diseases, GPNMB levels are elevated, but its functional role remains unclear. The present invention found that, Gpnmb gene knockout exacerbates tau pathology, neuron loss and neuroinflammation. Single-nucleus RNA sequencing showed that, Gpnmb deficient microglia in the brains of mutant mice have significant lysosomal dysfunction. Mechanistically, Gpnmb deletion disrupts the assembly of the v-ATPase complex, leading to impaired lysosomal acidification and, in turn, impeding lysosomal degradation of tau. Crucially, this deficiency in the deficient model was successfully repaired by using small molecule drugs that activate v-ATPase subunits ATP6V0D1 and ATP6V0D2. Integrating these findings, GPNMB was identified as a key factor regulating lysosomal function in microglia, and small molecule drugs targeting v-ATPase were considered as a potential therapeutic approach to alleviate age-related tau accumulation and neurodegenerative pathology. Gpnmb

[0043] GPNMB is a multifunctional protein that is widely involved in regulating cell differentiation, migration, inflammatory balance and neuroprotection. Transgenic studies have shown that overexpression of GPNMB in astrocytes and neurons can enhance memory function and increase GluA1 levels in the hippocampus. At the same time, motor neuron-specific GPNMB expression can improve neurodegenerative diseases caused by TDP43 mutations. A large amount of evidence shows that microglia are the main source of GPNMB in the context of neuropathology. In adult rats, GPNMB-positive cells also express the microglial marker OX42; after LPS stimulation, GPNMB + / OX42 + microglia increased in number. In glioma-associated microglia, APP / PS1-derived CD11c + ​GPNMB expression levels are further elevated in microglia and Alzheimer’s disease patients. Disease-associated microglia (DAM / MGnD) found in mouse models of Alzheimer’s disease have characteristic transcriptional signatures, where GPNMB is a core marker of microglial activation, expressed only during neurodegeneration and co-expressed with Trem2 、 ApoE 、 Clec7A and CST7 Recent studies have revealed three key aspects of GPNMB + microglial function: First, the GPNMB-EYA2 + subpopulation, common to AD and PD, exerts neuroprotection by autophagy clearance of Ab and a-synuclein. Second, brain cell dynamic modeling revealed two GPNMB + lipid-associated microglia (LAM) subpopulations: LAM-A drives Ab proteinopathy, and LAM-B mediates pathological transmission of Ab to tau. Third, experimental data from the present invention demonstrate that Gpnmb deletion exacerbates tau pathology, directly validating this mechanistic model. Taken together, these findings establish a core regulatory role of Gpnmb + positive microglia in neurodegenerative proteinopathies.

[0044] Increasing evidence suggests that GPNMB plays a key role in maintaining lysosomal function and mitigating neurodegeneration. In the PS19 mouse model of tauopathy, GPNMB expression is significantly upregulated at 9.5 months of age, at which point it appears to counteract lysosomal dysfunction and promote Tau degradation. Gpnmb gene deletion exacerbates tau pathology in male and female mice. Mechanistically, Gpnmb deficiency interferes with lysosomal acidification by disrupting v-ATPase assembly and reducing Tau degradation, highlighting its key role in maintaining lysosomal integrity. Moreover, ASO-mediated Gpnmb knockdown exacerbates lysosomal dysfunction, MHC-II dysregulation, and proinflammatory secretion in Grn deficient macrophages, further indicating that GPNMB plays an important role in immune regulation and lysosomal homeostasis. Furthermore, GPNMB plays a key role in phagocytosis, and its deficiency impairs both engulfment and degradation processes, thereby exacerbating pathological manifestations. Given that GPNMB upregulation in microglia occurs in synchrony with neurodegeneration, its induction is likely a compensatory mechanism to counteract disease progression. GPNMB, as a key protective factor in neurodegeneration, functions by stabilizing lysosomes and enhancing protein clearance.

[0045] GPNMB is a glycosylated type I transmembrane protein that undergoes extracellular domain cleavage, releasing its extracellular domain (ECD) to the extracellular space. This suggests that the main form of GPNMB in the cerebrospinal fluid can be the secreted ECD. New evidence supports GPNMB as a promising biomarker for neurodegenerative diseases, with its levels elevated in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and aging. However, the functional role of GPNMB ECD remains unclear. Interestingly, while GPNMB-ECD has been shown to promote tissue repair, its role in the central nervous system seems to be divergent. Previous studies suggest that GPNMB-ECD accelerates neuronal aging, which is consistent with the finding that this protein induces endothelial cell senescence in aged tissues. Moreover, Gpnmb-expressing cells have been shown to attenuate the senescence phenomenon in adipose tissue, while GPNMB immunotherapy has been shown to improve aging-related phenotypes. Gpnmb The phenomenon of pathological aggravation in gene knockout mice can be due to the absence of membrane-bound GPNMB, which is essential for lysosomal function. However, whether GPNMB-ECD is directly involved in the pathological process of tau protein, particularly in Alzheimer’s disease, remains an unsolved mystery that requires further research.

[0046] Lysosomal dysfunction, particularly acidification dysfunction due to impaired v-ATPase activity, drives tau protein aggregation and contributes significantly to the pathogenesis of Alzheimer’s disease by promoting the accumulation of neurotoxic proteins. Therefore, restoring lysosomal homeostasis by reducing lysosomal stress, promoting lysosomal repair / replacement, or remodeling, or enhancing lysosomal autophagy, represents a compelling therapeutic strategy that can delay or reverse disease progression. Although TFEB activation has been widely used to enhance lysosomal function, widespread TFEB induction can trigger excessive lysosomal biosynthesis, disrupt cellular homeostasis, and produce off-target effects; in addition, many TFEB-directed small molecule drugs show limited blood-brain barrier (BBB) permeability, limiting their translational potential in central nervous system diseases. While previous studies on lysosomal defects in AD have focused on neurons, new evidence suggests that microglial lysosomal systems are key determinants of the disease. Here, it is shown that Gpnmb Lacking lysosomal acidification by impairing the function of v-ATPase subunits ATP6V0D1 and ATP6V0D2, which are enriched in microglia. Through a targeted small molecule screen, it was identified that hyperforin, a blood-brain barrier-permeable carbocyclic dianthracene derivative from the Hypericum genus of plants, with reported antidepressant, metabolic, and neuroprotective activities, can restore GpnmbLysosomal pH in microglia. Integrated analyses revealed that these data uncovered a GPNMB-dependent regulatory mechanism that modulates microglial v-ATPase activity and lysosomal acidification levels, and placed hyperforin as a preclinical development candidate for Alzheimer's disease microglia-targeted therapy.

[0047] In summary, this study demonstrated that GPNMB in microglia plays a protective role in tauopathy mouse models by facilitating tau degradation. From the molecular mechanism, GPNMB facilitates lysosomal acidification by regulating the assembly process of v-ATPase, especially ATP6V0D1 / D2 subunits. Further studies found that hyperforin, a small-molecule compound targeting ATP6V0D1 / D2, can restore lysosomal acidification function caused by Gpnmb deficiency. These findings suggest that GPNMB is a potential therapeutic target for improving tau clearance efficiency in neurodegenerative diseases, opening up a new way for disease treatment. Gpnmb Lysosomal acidification dysfunction caused by genetic defects. These findings suggest that GPNMB is a potential therapeutic target for improving tau clearance efficiency in neurodegenerative diseases, opening up a new way for disease treatment.

[0048] 9. Figure Description Figure 1 A) Comparison of cerebrospinal fluid GPNMB cytoplasmic domain (CD) and extracellular domain (ECD) levels in A-T- and A+T+ individuals in the ADNI cohort. (B) Western blot analysis of GPNMB, total tau protein, and p-Tau (Thr202 / Ser205) in hippocampal and cortical lysates of wild-type (WT) and PS19 mice. n=3 per group. (C) Representative images of p-Tau202 / 205 and p-Tau217 immunohistochemical staining in the hippocampus of 9.5-month-old PS19 and PS19;GKO mice (scale bar: 500 μm). (D) Quantification of the area covered by p-Tau (Thr202 / Ser205) (left) and p-Tau (Thr217) (right) in the hippocampus. n=10 / 10, 9.5-month-old PS19 male / female; n=9 / 10, 9.5-month-old PS19;GKO male / female. (E) Representative thioflavine-S staining images of the dentate gyrus, CA1, and CA3 regions of the hippocampus of 9.5-month-old PS19 and PS19;GKO mice (scale bar: 100 μm). (F) Quantification of thioflavine-S fluorescence intensity. Fluorescence intensity was normalized to PS19. n=10 / 10, 9.5-month-old PS19 male / female; n=9 / 10, 9.5-month-old PS19;GKO male / female.

[0049] Figure 2(A) Representative images of brain sections from 9.5-month-old PS19 and PS19;GKO mice stained with Sudan black (scale bar: 1 mm). (B) Quantitative analysis of hippocampal volume (left) and lateral ventricle volume (right). n = 10 / 10, 9.5-month-old PS19 male / female; n = 9 / 10, 9.5-month-old PS19;GKO male / female. (C) Representative images of NeuN immunostaining in the hippocampus of 9.5-month-old PS19 and PS19;GKO mice (scale bar: 500 μm). (D) Quantitative analysis of CA1 pyramidal cell layer and dentate gyrus granule cell layer thickness. n = 10 / 10, 9.5-month-old PS19 male / female; n = 9 / 10, 9.5-month-old PS19;GKO male / female. (E) Representative images of PSD95 and SYP colocalization puncta in the CA1 region of the hippocampus of 9.5-month-old PS19 and PS19;GKO mice (scale bar: 5 μm). (F) Quantitative analysis of PSD95 and SYP colocalization puncta in the CA1 region of the hippocampus. n = 10 / 10, 9.5-month-old PS19 male / female; n = 9 / 10, 9.5-month-old PS19;GKO male / female. (G) Representative Western blot images of PSD95 and SYP in the hippocampus. (H) Quantitative analysis of PSD95 and SYP levels. PSD95 and SYP levels were first normalized to actin and then to the level in PS19. Experimental groups included: 9.5-month-old PS19 mice (n = 6) and PS19;GKO mice (n = 6). (I) Representative images of Ibal (left) and Clec7A (right) immunostaining in the hippocampus of 9.5-month-old PS19 and PS19;GKO mice (scale bar: 500 μm). (J) Percentage of Ibal (left) and Clec7A (right) immunostaining coverage area in the hippocampus: n = 10 / 10, 9.5-month-old PS19 male / female; n = 9 / 10, 9.5-month-old PS19;GKO male / female. (K) Representative images of GFAP (left) and C3 (right) immunostaining in the hippocampus of 9.5-month-old PS19 and PS19;GKO mice (scale bar: 500 μm). (L) Percentage of GFAP (left) and C3 (right) immunostaining coverage area in the hippocampus: n = 10 / 10, 9.5-month-old PS19 male / female; n = 9 / 10, 9.5-month-old PS19;GKO male / female.

[0050] Figure 3(A) Open field test and NOR test schematic. (B) Dwell time in the center of the open field test. (C) Exploration time of different objects in the NOR test (familiar objects: Object A [OA] and Object B [OB]; novel object: Object C [OC]). (D) Representative trajectory images of the training phase on day 6 of the MWM test. (E-H) Escape latency (E), dwell time in the target quadrant (F), and the number of platform crossings in the probe trial (G) of the training phase. (H) Freezing levels in the contextual fear conditioning test.

[0051] Figure 4 (A) Uniform Manifold Approximation and Projection (UMAP) visualization of the single-nuclei RNA sequencing (snRNA-seq) data of 24,636 cells from the hippocampus of PS19 and PS19;GKO mice (left panel) and comparative analysis between different genotypes (right panel). Cell type annotations are based on the supplementary Figure 4 (A) Marker gene expression determination. (B) Pie chart showing the relative proportion of cell types in PS19 and PS19;GKO samples. Color coding corresponds to the cell classes annotated in (A). (C-E) Volcano plots presenting the differentially expressed genes (DEGs) of all excitatory neurons, inhibitory neurons, and microglia cells between PS19;GKO and PS19 mice. Bar charts show the top five key pathways (KEGG pathways) enriched in the differentially expressed genes. (F) Functional annotation of the differentially expressed genes in excitatory and inhibitory neurons in Alzheimer’s disease and neurodegenerative disease pathways. (G) Functional annotation of the differentially expressed genes in microglia cells in phagosome and lysosome pathways.

[0052] Figure 5In particular, (A) co-localization of GPNMB with CD68 in primary microglia cells labeled with Ibal (scale bar: 5 μm). (B) Representative live cell images of DQ-BSA (red) signal in wild-type and Gpnmb knockout primary microglia cells. (C) Time course of DQ-BSA (red) mean red intensity. n = 6 / 6, wild-type / Gpnmb knockout. (D) Representative images of DQ-BSA (red) signal in wild-type and Gpnmb knockout primary microglia cells (scale bar: 20 μm). (E) Quantitative analysis of DQ-BSA (red) fluorescence intensity. Fluorescence intensity was normalized to wild-type. n = 5 / 5, wild-type / Gpnmb knockout. (F) Representative images of acridine signal in wild-type and Gpnmb knockout primary microglia cells (scale bar: 20 μm). (G) Quantitative analysis of pyrroloquinolinone (red) density. Density was normalized to wild-type. n = 5 / 5, wild-type / Gpnmb knockout. (H) Representative images of wild-type and Gpnmb knockout primary microglia cells stained with lysosomal sensor yellow / blue (scale bar: 20 μm). (I) Ratio of yellow fluorescence signal (acidic) to blue fluorescence signal (basic). Fluorescence intensity ratio was normalized to wild-type. n = 5 / 5, wild-type / Gpnmb knockout. (J) Light microscope confocal images showing microglia-mediated phagocytosis and lysosomal degradation process of pHrodo red-labeled tau fibril protein fibers (scale bar: 20 μm). (K) Quantitative analysis of tau protein fibril fluorescence intensity. Fluorescence intensity was normalized to wild-type. n = 5 / 5, wild-type / Gpnmb knockout.

[0053] Figure 6Figure 6. Gpnmb deficiency alters the composition of microglial subpopulations. (A) UMAP plot of microglial subpopulations in PS19;GKO mice. (B) Stacked bar plot comparing the composition of microglial subpopulations in PS19;GKO and PS19 mice, bar colors are distinguished by cell classes labeled in (A). (C) UMAP plot of Gpnmb expression in microglial subpopulations in PS19 mice. (D) Proportion of high-G (clusters 1 and 6) and low-G (clusters 2, 3, 4, 5, 7, 8, 9, and 10) microglia in PS19 and PS19;GKO mice. (E) Volcano plot of differentially expressed genes in high-G microglia in PS19;GKO and PS19 mice, differentially expressed genes enriched in phagosome and lysosome pathways are labeled in red. (F) Proteome schematic. Heatmap showing the relative gene expression levels of v-ATPase subunits in wild-type and Gpnmb knockout primary microglia, n = 3 per group. *P < 0.05, **P < 0.01. (G) Mass spectrometry schematic. Scatter plot showing proteins detected by mass spectrometry, wild-type primary microglia lysate was subjected to endogenous GPNMB co-immunoprecipitation using anti-GPNMB antibody, or Gpnmb knockout primary microglia control was subjected to detection, proteins enriched in wild-type IP group are labeled in red. (H) Western blot image showing the results of co-immunoprecipitation experiment of ATP6V0D1 / D2 and GPNMB.

[0054] Figure 7 Figure 7. Gpnmb deficiency alters lysosome acidity and tau clearance. (A) Molecular docking schematic. (B) Scatter plot showing the docking scores of compounds with ATP6v0D1 / D2 and the fold change of acidification relative to DMSO. Compounds with docking scores higher than 19 and Log2FC (fold change) values greater than 1.2 are labeled in red. (C) Venn diagram showing the overlapping area of highlighted compounds in (B). There are 21 compounds that present docking scores higher than 19 in both ATP6v0D1 and ATP6v0D2. (D) Small molecule screening schematic. (E) Scatter plot showing the fold change of lysosome acidity in primary microglia treated with compounds. Lysosome acidity was detected using lysosome sensor Green DND-189, fluorescence intensity was normalized to wild-type DMSO control. (F) Quantitative analysis of lysosome sensor Green DND-189 fluorescence intensity, fluorescence intensity was normalized to solvent control (DMSO). n = 4 per group. (G) Representative confocal microscopy images showing hyposin-mediated lysosomal degradation of pHrodo-labeled tau fibers (scale bar: 20 pm). (H) Quantitative analysis of Tau PFFs fluorescence intensity, fluorescence intensity was normalized to wild-type DMSO control. n = 3 per group.

[0055] Example Two Based on the above-mentioned example one, specific experimental data is provided.

[0056] 1. Mice Wild-type (WT) C57BL / 6J mice were purchased from the Experimental Animal Center of Chongqing Medical University. Transgenic PS19 mice (B6 strain; C3-Tg(Prnp-MAPT*P301S)PS19Vle / J, strain number #008169) were purchased from Jackson Laboratory and were bred in hemizygous form with C57BL / 6 genetic background. Gpnmb knockout (KO) mice were purchased from Sino-British Laboratory Animal Limited Company (catalog number KOCMP-93695-Gpnmb-B6J-VA). All experimental animals were housed in a standard germ-free environment with a temperature controlled at 22 ± 1 °C, humidity of 50 ± 5%, and a 12-hour light / dark cycle. Food and drinking water were freely available.

[0057] 2. Primary microglia culture Primary microglia cultures were prepared from postnatal day 1-3 (P1-P3) mice. Briefly, after careful removal of the meninges, the cortex and hippocampus were dissected, minced and digested with 0.25% trypsin. Dissociated cells were seeded in poly-L-lysine (150 μg / mL; Sigma-Aldrich, P6282)-coated cell culture flasks and cultured in DMEM medium (Gibco, 11965) containing 10% fetal bovine serum (FBS; Sigma, F8318) and 1% penicillin / streptomycin (Gibco, 15140122). Three days after in vitro culture (DIV3), granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng / mL, PeproTech, 315-03) was added to the culture medium to promote microglial proliferation. Microglia were isolated by mechanical dissociation (180 rpm for 15 min) at DIV9 and subsequently used for experiments.

[0058] 3. Experimental procedures 3.1 Brain collection Nine and a half-month-old mice were anesthetized with intraperitoneal injection of Avertin and pre-treated with heart perfusion of PBS. The left hemisphere was fixed in 4% paraformaldehyde (PFA) solution at 4 °C for 36 hours, followed by cryoprotection with sucrose gradient (24 hours at 25% concentration and 72 hours at 30% concentration) and finally embedded with optimal cutting temperature (OCT) embedding medium (Sakura, catalog number 4583). The fixed left hemisphere was cut into 40 pm coronal sections covering the hippocampus using a cryostat (Leica / CM3050S) and stored in a cryoprotective solution containing 30% ethylene glycol, 20% glycerol and 50% double-concentration PBS at -20 °C. The right hemisphere was directly snap-frozen in liquid nitrogen and stored at -80 °C.

[0059] 3.2 Volumetric analysis For each mouse, every sixth coronal section (covering the entire hippocampus, thickness 40 pm, interval 240 pm) was mounted on microscope slides (10 sections per mouse) and air-dried for two hours at room temperature. Sections were stained with Sudan black (Sigma, cat. no. G1691) to enhance visualization of the hippocampus. The procedure was as follows: slides were first rinsed with 70% ethanol for 5 seconds, then incubated with Sudan black solution for 2 minutes at room temperature, rinsed sequentially with 70% ethanol (1 minute) and Milli-Q water (1 minute), and finally mounted with 50% glycerol. Stained sections were imaged at 10x magnification using a Zeiss AxioScan.Zl slide scanner. Regions of interest (ROIs) were manually outlined for all sections using ZEN Blue software (Zeiss) to quantify hippocampal and lateral ventricle volumes. The formula for volume (mm3) was: volume = (average of all ten section areas) x 2.4 mm2.

[0060] 3.3 Immunohistochemistry Brain sections were rinsed three times in PBS (5 minutes each) to remove cryoprotectant and then incubated in 0.3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity. After additional PBS washes, sections were blocked with 10% normal goat serum to reduce non-specific binding and then incubated with primary antibodies (anti-p-tau (Ser202, Thr205; Invitrogen, MN1020B, 1 :300) and anti-p-tau (Thr217)) overnight at 4 °C. The next day, sections were washed in PBS (3 times, 5 minutes each) and incubated with an avidin-biotin complex (ABC; Vector Labs, PK-6100, 1 :400) for 1 hour at room temperature. After further PBS washes, sections were developed and stained using DAB peroxidase substrate (ZSGB-Bio, ZLI-9018) and mounted after washing. After drying, sections were dehydrated through an ethanol gradient, cleared in xylene, and mounted with neutral resin. Stained sections were imaged at 20x magnification using an AxioScan.Zl slide scanner (Zeiss). Images were further processed and quantified using Fiji (ImageJ).

[0061] 3.4 Protein extraction and immunoblotting Frozen brain hemisphere samples were homogenized in ice-cold RIPA lysis buffer (with complete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail) using a mechanical homogenizer. The supernatant was collected after centrifugation at 4°C, 140,000 x g for 15 min and analyzed. Total protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Cat#23225). For immunoblotting, equal amounts of protein (10-30 pg per lane) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 (with 5% bovine serum albumin, abbreviated as BSA, Cat# TBST) for 1 h at room temperature, and then incubated with primary antibodies (diluted in blocking buffer) overnight at 4°C. The antibodies used included: b-actin (1:5,000, Proteintech, Cat# 60008-1-Ig), GAPDH (1:1,000, Zhongshan Golden Bridge, Cat# TA-08), total tau protein (1:5,000, Invitrogen, Cat# AHB0042), phosphorylated tau protein Thr181 (1:1,000, Cell Signaling Technology, Cat# 12885S), phosphorylated tau protein Ser202 / Thr205 (1:1,000, Invitrogen, Cat# MN1020), phosphorylated tau protein Thr217 (1:1,000, mAb 2A7), PSD95 (1:1,000, Merck Millipore, Cat# MAB1596), and synaptophysin (1:200, Abeam, Cat# ab16659). After three 10-min washes with TBST, the membranes were incubated with secondary antibodies (1:10,000) conjugated with luciferase for 1 h at room temperature. Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Cat# WBKLS0500) and imaged by UVP ChemiDoc imaging system.

[0062] 3.5 Sulfur yellow S staining Brain sections were washed with PBS buffer for three times (5 min each), mounted on slides and air-dried at room temperature. Thioflavin S (Thio-S; Sigma-Aldrich, Cat# T1892) working solution was freshly prepared in 50% ethanol and filtered through a 0.22 pm filter. Sections were incubated with Thio-S for 8 min at room temperature in the dark, followed by a quick differentiation with 80% ethanol for 15 s. After washing with ddH2O buffer (5 times, 5 min each), the slides were covered with an anti- fluorescent quenching mounting medium. Images were acquired using a Zeiss LSM980 confocal microscope (excitation wavelength 488 nm, emission wavelength 500-550 nm) with uniform laser power and gain settings for all samples. The mean fluorescence intensity (MFI) of Thio-S positive areas in hippocampal subfields (dentate gyrus DG, CA1, CA3) was quantitatively analyzed after background subtraction using ImageJ software.

[0063] 3.6 Immunofluorescence Brain sections were washed with PBS buffer for three times (5 min each), blocked with 10% normal donkey serum and incubated with primary antibodies overnight at 4°C: anti-NeuN antibody (Abeam, ab104224, 1 :400), anti-GFAP antibody (Cell Signaling Technology, 3670S, 1 :500), anti-IBAl antibody (Wako, 019-19741, 1 :500), anti-PSD95 antibody (Millipore, MAB1596, 1 :200), anti- synaptophysin antibody (Abeam, ab16659, 1 :200), anti-C3 antibody (Abeam, ab11862, 1 : 100), anti-CD68 antibody (Bio-Rad, MCA1957G, 1 :200), and anti-Clec7a antibody (Biolegend, 144303, 1 :50). After completion of primary antibody incubation, sections were washed with PBS buffer for three times (5 min each) and incubated with Alexa Fluor-labeled secondary antibodies (Invitrogen, 1 :500) for 1 h at room temperature. After washing with PBS buffer for three times (5 min each) again, the slides were mounted with ProLong Gold anti-quenching mounting medium (Invitrogen, P36930) and sealed with coverslips using transparent nail polish. Images were acquired using an Olympus / SpinSR microscope (10x or 20x magnification) or a Zeiss LSM980 confocal microscope (63x magnification).

[0064] 4. Mouse testing Mice were subjected to different tests including open field test, novel object recognition test, Morris water maze for assessing spatial learning, fear conditioning, etc.

[0065] 5 Mouse identification 5.1 Immunocytochemistry For immunostaining in vitro, cells were fixed with 4% paraformaldehyde. After washing twice with PBS, cells were blocked with 10% normal donkey serum, then incubated with primary antibodies (anti-IBAl, Oasisbiofarm, OB-PGP049, 1:500; anti-GPNMB, CST, MAB1596, 1:200; anti-CD68, Bio-Rad, MCA1957G, 1:200) at 4°C overnight. After primary antibody incubation, cells were washed three times with PBS (5 min each) and incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:500) for 1 h at room temperature. Images were acquired using a zeiss LSM980 confocal microscope (63x magnification). For DQ-BSA detection, cells were pulsed with 10 pg / ml DQ-red-BSA for 9 h at 37°C, washed to remove excess probe, and imaged after fixation. Lysosomal membrane permeability (LMP) was assessed by pre-treating cells with 1 pg / ml arcidine orange (5 min in culture medium), followed by washing and confocal imaging. For a rough estimation of lysosomal acidity, cells were incubated with 1- pM LysoSensor Yellow / Blue DND-160 or LysoSensor Green DND-189 in 37°C culture medium for 5 min, washed, and imaged by confocal microscopy or Incucyte system. Fluorescence intensity was quantified using Fiji / ImageJ software.

[0066] 5.2 Live cell imaging For assessing lysosomal activity, cells were seeded in 24-well plates and treated with 10 pg / ml DQ-red-BSA. Real-time imaging was performed using the Incucyte system for 18 h.

[0067] 5.3 Tau labeling and fibrillization Human Tau monomer with N-terminal 6His tag was successfully expressed in prokaryotic system and purified by nickel affinity chromatography (Ni-NTA). Tau pre-fibrils (Tau-PFF) were prepared by inducing in vitro polymerization of purified Tau monomer with heparin. Subsequently, Tau-PFF was covalently labeled with pHrodo™ Red SE fluorescent dye following the operation guideline provided by Thermo Fisher Scientific. The final product was aliquoted at 10 mM concentration and stored at -80°C for later use.

[0068] 5.4 Tau phagocytosis and degradation To evaluate the phagocytic ability of microglia on Tau-PFF, researchers treated primary microglia with 20 mM Tau-PFF for 9 hours. Subsequently, the medium containing Tau-PFF was replaced with fresh medium, and the cells were cultured for another 9 hours to detect the degradation efficiency of Tau-PFF. In the rescue experiment, 10 µM compound was added to the fresh medium to observe its effect on the degradation of Tau-PFF. Finally, the cells were fixed with 4% paraformaldehyde for immunocytochemical detection.

[0069] 5.5 Single-cell RNA sequencing and analysis Under anesthesia, 9.5-month-old PS19 and PS19;GKO mice were treated with transcardial perfusion of cold physiological saline. The hippocampal tissue was dissected into 1.5 milliliter RNase-free centrifuge tubes, quickly frozen with liquid nitrogen, and stored at -80°C. Single nuclei suspension was prepared and stained with 0.4% trypan blue to evaluate cell viability. Isolated nuclei with a viability rate >80% were selected and introduced into the Chromium Controller for gene expression module (GEM) construction. After completing GEM fragmentation and cDNA amplification, the library was prepared by fragmentation and adapter ligation, and the quality was verified.

[0070] Quality control, normalization, batch correction, dimensionality reduction, and clustering were performed using Seurat (version 5.1.0)50,51. The quality control filter excluded cells with fewer than 300 or more than 7,000 detected genes, as well as cells with mitochondrial gene expression greater than 10%. Genes expressed in fewer than three cells were also removed. Researchers used scDblFinder (v1.17.1, https: / / github.com / plger / scDblFinder)52 to screen for potential doublet cells. After screening, a total of 24,970 high-quality single-cell transcriptomes were retained, with 11,070 cells in the PS19 group and 13,900 cells in the PS19;GKO group.

[0071] The data processing pipeline included the following steps: first, standardization was performed using the NormalizeData tool combined with UMI count regression, followed by data scaling using ScaleData. Principal component analysis was completed using RunPCA software, and batch effect correction was achieved using Harmony v1.2.053 version. Dimensionality reduction was also completed using RunPCA, and finally, uniform manifold approximation projection (UMAP) technology was used to generate embedded data. Unsupervised clustering analysis was performed using the FindClusters tool at a resolution of 1.2.

[0072] 5.6 Cell type annotation Cell types were classified by detecting the expression of typical marker genes. Six main cell types were ultimately identified: excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, oligodendrocyte precursor cells (OPCs), and astrocytes. Cells that could not be clearly labeled (n=334) were marked as "unknown" and excluded from subsequent analyses.

[0073] 5.7 Difference Analysis and Enrichment Differentially expressed genes (DEGs) between PS19 and GKO were identified using the MAST method implemented in the FindMarkers function of the Seurat software (with the test.use parameter set to "MAST"). The screening criteria were: genes with an adjusted p-value < 0.05 and a mean log2 fold change ≥ 0.25 were considered significantly differentially expressed genes. Based on the two groups of differentially expressed genes, functional enrichment analysis of related signaling pathways (KEGGs) was performed using the richR software package (v0.0.37; development team: https: / / github.com / guokai8 / richR).

[0074] 5.8 Clustering and Differential Analysis of Microglia Subpopulations Microglia were extracted and re-clustered using Seuratv 5.1.0 software at a resolution of 0.8. Based on the average expression level of the Gpnmb gene, subclusters were divided into high-expression and low-expression groups. Subsequently, the MAST method was used to analyze the high and low expression levels in the PS19 and PS19;GKO groups. Gpnmb Differential expression analysis was performed on microglia. Finally, KEGG enrichment analysis was conducted on the selected gene set using the richR software package (v0.0.37).

[0075] 5.9 Co-immunoprecipitation (co-IP) HEK293 cells (approximately 1 × 10⁻⁶ cells per 10 cm culture dish) 7 Cells co-expressing hGPNMB-HA and hATP6V0D1 / 2-Flag or GFP-Flag were washed twice with PBS and then lysed with lysis buffer (#P0013, Beyotime). The lysis buffer was clarified by centrifugation (15,000g, 10 min, 4°C), and the supernatant was incubated with anti-HA or anti-Flag magnetic beads at 4°C for 24 h on a rotary mixer. The magnetic beads were washed three times with washing buffer (50 mM Triss, 150 mM Sodium Chloride, 0.1% Triton X-100, pH 7.5), and the bound proteins were eluted by boiling in SDS loading buffer (95°C, 5 min). Immunoprecipitated proteins were analyzed by Western blotting.

[0076] 5.10 Mass spectrometry analysis Proteins were extracted from wild type (WT) and Gpnmb - / - Primary microglia cells were extracted using cell lysis buffer (#P0013, Beyotime). GPNMB antibody was added to the sample and incubated at 4°C for 24 hours, followed by the addition of Protein A / G magnetic beads (#HY-K0202, MCE) to the complex and continued incubation at 4°C for 24 hours. The magnetic beads were then washed three times with wash buffer (50 mM Tris, 150 mM sodium chloride, 0.1% Triton X-100, pH 7.5) and boiled in loading buffer at 95°C for 5 minutes to elute the proteins bound to the GPNMB antibody.

[0077] The target proteins were stained with Coomassie Brilliant Blue after purification by SDS-PAGE. The gel strip matching the molecular weight of the target protein was cut off and destained with 50% acetonitrile solution of 50 mM ammonium bicarbonate. The destained gel strip was dehydrated with 100% acetonitrile, followed by incubation with 25 mM TCEP in 50 mM ammonium bicarbonate solution at 37°C for 30 minutes to reduce the protein. Next, the gel strip was dehydrated with 100% acetonitrile again and incubated with 25 mM iodoacetamide in 50 mM ammonium bicarbonate solution at room temperature for 30 minutes in the dark to alkylate the protein. Finally, the gel strip was dehydrated with 100% acetonitrile, followed by incubation with 200 ng of modified sequencing grade trypsin (Thermo Fisher Scientific) in 50 mM ammonium bicarbonate solution at 37°C for 6 hours for in-gel digestion of the protein.

[0078] The digestion process was terminated by adding 50 mL of 1% formic acid to the gel pieces. Subsequently, 300 mL of acetonitrile was added and the mixture was mixed well with the gel pieces for 30 minutes at room temperature to elute the digested proteins (i.e. the peptide mixture). The peptide mixture was transferred to a 1.5 mL centrifuge tube and dried using a Thermo Scientific SpeedVac concentrator. The dried peptide mixture was dissolved in 50 mL of 1% formic acid and subjected to LC-MS / MS analysis using a QExactive HF-X mass spectrometer equipped with an EASYnLC-1200 system. The liquid chromatography separation was performed on a 25 cm column (Picofrit 75 mm i.d. packed with Magic C18 resin) pre-equilibrated with 100% mobile phase A (0.1% formic acid in HPLC water). The peptide mixture was eluted using a gradient elution method: from 3% to 35% mobile phase B (0.1% formic acid in 80% acetonitrile) over 35 minutes at a flow rate of 300 nL / min, followed by a 10 minute period at a flow rate of 300 nL / min with mobile phase B at 35-55%, a 5 minute period at a flow rate of 500 nL / min with mobile phase B at 55-100%, and a final 5 minute period at a flow rate of 500 nL / min with mobile phase B at 100%.

[0079] Mass spectrometry data were acquired in data-dependent mode, covering a mass range of 400-1800 m / z. For analysis, the 20 most abundant double- or triple-charged ions from each mass spectrum were selected for analysis by tandem mass spectrometry (MS / MS). The mass spectrometer parameters were set as follows: capillary voltage 2.0 kV, capillary temperature 320 °C, resolution 60,000, target value 3,000,000, collision energy 27 NCE. Mass spectrometry data were processed using Proteome Discoverer software (version 2.4, Thermo Scientific) and compared against a human protein database (UniProt 2022_01 version). The precursor ion mass tolerance was set to 10 ppm and the fragment ion mass tolerance was set to 0.01 Da. The propionamide modification of cysteine and the oxidation modification of methionine were set as fixed and variable modifications, respectively.

[0080] 5.11 Proteomics Primary microglia cells were seeded in 6 cm dishes and treated with Tau-PFF for 24 hours. 1 x 106cells were washed with ice-cold DPBS three times and collected with a pre-chilled spatula. The cells were then lysed in RIPA lysis buffer containing protease and phosphatase inhibitors for 5 minutes on ice. After removing cell debris by centrifugation at 15000g for 10 minutes at 4°C, the supernatant was transferred to a new centrifuge tube. The protein samples were digested with trypsin for peptide preparation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in data-independent acquisition (DIA) mode. DIA data were processed using DIA-NN software with the following parameters: enzyme, trypsin; maximum number of cleavage sites, 1; fixed modification, carboxymethylation (C); dynamic modifications, oxidation (M) and acetylation (protein N-terminus). The identified proteins were required to meet the screening criteria of false discovery rate (FDR) < 1% by database searching.

[0081] 5.12 Molecular docking of acidic small molecule compounds with ATP6V0D1 and ATP6V0D2 The molecular acidity data used was derived from a database titled “SLC7A11 is a non-canonical H+ transporter in lysosomes”. This database screened 4240 small molecule compounds from the Drug Repurposing Hub for their effect on lysosomal acidity in HAP1 cells. We selected 761 small molecule compounds that enhanced lysosomal acidity and performed molecular docking with ATP6v0D1 and ATP6v0D2 proteins. We successfully docked 578 compounds. The chemical structure data of acidic small molecule compounds was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), while the predicted three-dimensional structure of human ATP6V0D1 and ATP6V0D2 proteins was obtained from the AlphaFold protein structure database (https: / / alphafold.ebi.ac.uk / ). To assess the potential interaction of acidic compounds with target proteins, we used the molecular docking method DeepDock46 based on geometric deep learning, which can predict the ligand binding conformation of target proteins. After installing DeepDock through its official GitHub repository, we performed deep docking simulations to evaluate the binding conformation and binding affinity of compounds with ATP6V0D1 and ATP6V0D2. The final docking scores were used to screen candidate molecules for subsequent validation. For each target (ATP6V0D1 and ATP6V0D2), we selected compounds with a DeepDock docking score greater than 19 (indicating stronger predicted binding) and an activity threshold (compound vs. DMSO) greater than 1.2. After double screening, 102 compounds were retained for ATP6V0D1 and 58 for ATP6V0D2.

[0082] 5.13 Screening small molecule compounds affecting lysosomal acidity The top 50 molecules with the highest docking scores were selected from each group as candidate molecules. Wild type (WT) and Gpnmb Knockout primary microglia were seeded in 96-well plates and incubated at 37°C overnight. Microglia were treated with compounds at 10 µM concentration for 60 minutes. Lysosomal acidity was detected using LysoSensor Green DND-189. After washing with HBSS, imaging was performed using the Incucyte imaging system and the mean fluorescence intensity of each cell was calculated. The fluorescence intensity values were normalized to the WT-DMSO group with 0.1% DMSO as the solvent control.

[0083] 6. ADNI study Cerebrospinal fluid proteomic data used were from the ADNI database (https: / / adni.loni.usc.edu). Participants were classified into case and control groups by revised AT classification framework using amyloid-beta (amyloid-12, referred to as Ab) and Tau protein biomarker levels in cerebrospinal fluid samples. The framework was applied to each cohort separately and used independently to assess Ab and Tau biomarkers. Levels of Ab and Tau were log10 transformed to approximate normal distribution, followed by z-score transformation for further standardization, ensuring mean of 0 and standard deviation (SD) of 1. Ab values ranged from -2.81 to 2.98 and Tau values ranged from -2.59 to 2.41. Quantitative Ab and Tau measurements were dichotomized into high (biomarker positive) and low (biomarker negative) categories using Gaussian mixture models in the mclust R package (version 6.1.1). Participants with high Ab levels and high Tau levels in cerebrospinal fluid were classified as amyloid / tau positive (A + T + ), while participants with low Ab and low Tau levels were classified as control (A - T - ).

[0084] 7. Quantification and statistical analysis Data are presented as mean ± standard error (S.E.M.) and are derived from >3 biological replicates. Outliers were identified and removed using ROUT method (Q=1%, GraphPad Prism 9.0). The choice of statistical test was based on the data distribution and homogeneity of variance: D’Agostino-Pearson test for >8 samples, Anderson-Darling test for >5 samples, Shapiro-Wilk test for >3 samples to assess normality; for two-group comparisons, if data were normally distributed and had equal variances, two-tailed unpaired t-test was used; if the normality assumption was not met, Mann-Whitney U test was used. For multiple-group comparisons, if the normality and homogeneity of variance assumptions were met, two-way ANOVA (with Dunnett’s post-test if necessary) was used; if variances were unequal, Brown-Forsythe or Welch ANOVA was used; for non-normal data, Kruskal-Wallis test (with Dunn’s post-test) was used. The sample size was not pre-specified for the statistical method, and the number of biological replicates for each experiment is detailed in the corresponding figure legends. The criteria for statistical significance were P < 0.05 for single comparisons and q < 0.05 for multiple comparisons. Significance is marked as: *P < 0.05, **P < 0.01, ***P < 0.001.

[0085] The preferred embodiments of the application disclosed above are only to help explain the principles of the application. The preferred embodiments do not describe all the details of the application, nor limit the application to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the specification. The specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the application, so that those skilled in the art can well understand and utilize the application. The application is limited only by the claims and their full scope and equivalents.

Claims

1. A pharmaceutical composition, characterized by, Small molecule compounds capable of activating v-ATPase subunits ATP6V0D1 and / or ATP6V0D2 for use in restoring Gpnmb lysosomal acidification and tau protein degradation capacity of defective microglia cells.

2. The pharmaceutical composition according to claim 1, wherein The small molecule compound is hyperforin.

3. A pharmaceutical composition according to either of claims 1 or 2, characterized in that, The pharmaceutical composition is used for treating neurodegenerative diseases associated with tau protein accumulation.

4. A method of screening candidate compounds for the treatment of tauopathies, characterized in that, The steps are as follows: Molecular docking of candidate compounds with ATP6V0D1 and / or ATP6V0D2 subunits is performed; The promoting effect of the compound on lysosome acidification is evaluated; selecting compounds capable of restoring Gpnmb compounds that restore the ability of defective microglia cells to degrade tau protein.

5. Use of the compound produced in the production of claim 4 in the manufacture of a medicament for the prevention or treatment of tauopathy, characterized in that, The applications include improving lysosomal function in microglia cells, promoting degradation of tau protein, by enhancing Gpnmb expression or activity of a gene.

6. Use according to claim 5, characterized in that, The Gpnmb Genes facilitate lysosomal acidification by regulating the assembly of v-ATPase subunits ATP6V0D1 and / or ATP6V0D2.

7. Use according to claim 5, characterized in that, The tauopathy includes Alzheimer's disease, tau protein hyperphosphorylation, aggregation or neurodegenerative pathology exhibited in a tau transgenic mouse model PS19.

8. A method of treating a tauopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof. Gpnmb Use of the gene as a biomarker in diagnosing or predicting progression of a tauopathy, characterized in that, The disease state or treatment response is evaluated by detecting the level of GPNMB extracellular domain in cerebrospinal fluid or tissue.

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