HMGB1 inhibitors for treatment of APOE4-associated tauopathies, including alzheimer's disease

Abstract

Inhibitors of High Mobility Group Frame Protein 1 (HMGB1) can significantly reduce HMGB1 nuclear-cytoplasmic translocation, glial hyperplasia, neurodegeneration, Tau pathologies, and myelin defects, especially in subjects having an APOE4 allele, as described herein. Accordingly, the methods described herein comprise administering to a subject having at least one genomic APOE4 allele one or more inhibitors of High Mobility Group Frame Protein 1 (HMGB1).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing date of U.S. Application No. 63 / 354,728, filed on June 23, 2022, the disclosure of which is incorporated herein by reference.

[0003] Government funding

[0004] This invention was made with government support under R01AG071697 and P01AG073082 awarded by the National Institutes of Health, National Institute on Aging. The government has certain rights in this invention.

[0005] Incorporation by Reference into the Sequence Listing

[0006] The sequence listing is provided herewith as an xml file "2338683.xml" created on May 31, 2023, and its size is 20,464 bytes. The contents of this xml file are incorporated herein by reference in their entirety. Background Art

[0007] Alzheimer's disease is a chronic neurodegenerative disease that is responsible for 60% to 70% of dementia cases. See, e.g., Burns, A., et al. "Alzheimer's Disease," BMJ 338 (2009); World Health Organization, "Dementia Fact Sheet," 2017, available at who.int / en / news-room / fact-sheets / detail / dementia. There are approximately 30 to 35 million people with Alzheimer's disease worldwide. See the World Health Organization above. Alzheimer's disease affects approximately 6% of people aged 65 and older. Burns, A., et al., supra. Alzheimer's disease is one of the most economically expensive diseases in developed countries. See, e.g., Bonin-Guillaume, S., et al. "The Economical Impact of Dementia," PresseMedicate, 34, 35 (2005). However, there is currently no known cure for Alzheimer's disease.

[0008] Therefore, there remains a need for treatments for Alzheimer's disease and other tauopathies. Summary of the invention

[0009] As described herein, inhibitors of high mobility group box protein 1 (HMGB1) can significantly reduce HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, Tau pathology and myelin defects, especially in subjects with APOE4 alleles. Therefore, the methods described herein include administering one or more inhibitors of high mobility group box protein 1 (HMGB1) to subjects with at least one genomic APOE4 allele. In some cases, the subject has two genomic APOE4 alleles. For example, the subject may express a detectable level of APOE4 protein. Some examples of HMGB1 inhibitors that can be used include glycyrrhizic acid, ethyl pyruvate, nicotine, (-) epigallocatechin gallate (EGCG), tanshinone, chlorogenic acid, emodin-6-O-β-D-glucoside, rosmarinic acid, isorhamnetin-3-O-galactoside, polygonum hydropiper, forsythiaside B, chloroquine, acteroside, shikonin, carbenoxolone, quercetin, lycopene, nafamostat mesylate, gabexate mesylate, sivelestat sodium, HMGB1 monoclonal antibody (m2G7 or #10-22), recombinant HMGB1 box A protein, acetylcholine, nicotinic acetylcholine receptor subtype α7 agonist GTS-21, peptide P5779, resveratrol, metformin, or a combination thereof. In some cases, the subject may show the following symptoms: HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, tau pathology (accumulation of tau protein) or myelin defect. For example, the subject may show at least one symptom of tau disease. Such tau disease is a disease characterized by the deposition of abnormal tau protein in the brain. For example, tau disease can be a neurodegenerative disorder, Alzheimer's disease, Pick disease, progressive supranuclear palsy, cortical basal degeneration, argyrophilic grain disease, primary age-related tau disease, chronic traumatic encephalopathy or frontotemporal dementia. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figures 1A to 1N It was shown that the accumulation and spread of Tau pathology was reduced in PS19-fE4 mice after APOE4 was removed from neurons but not astrocytes. Figure 1AThe ApoE levels in the hippocampal lysates of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre measured by ELISA are shown schematically (PS19-fE4: no Cre, n=21; PS19-fE4 / Syn1-Cre, n=18; PS19-fE4 / GFAP-Cre, n=17; PS19-fE3: no Cre, n=17; PS19-fE3 / Syn1-Cre, n=11; PS19-fE3 / GFAP-Cre, n=11). For each individual mouse, the ApoE level was normalized relative to the weight of the dissected hippocampal tissue. Data are expressed as mean ± SEM, using one-way ANOVA and Tukey post hoc multiple comparison test. Figure 1B Representative images of pTau stained with anti-AT8 in the hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre are shown (scale bar, 500 μm). Figure 1C Diagrammatically shown are the percentages of quantified pTau (AT8) coverage in the hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with or without Cre (PS19-fE4: no Cre, n=25; Syn1-Cre, n=17; GFAP-Cre, n=16; PS19-fE3: no Cre, n=20; Syn1-Cre, n=15; GFAP-Cre, n=15). Figure 1D Shown are representative images of anti-AT8 (green) and anti-TUJ1 (red) western blots in the RAB fraction of hippocampal tissue lysates from 10-month-old PS19-fE4 and PS19-fE3 mice with or without Cre. Figure 1E Shown are representative images of anti-AT8 (green) and anti-TUJ1 (red) western blots in RIPA fractions of hippocampal tissue lysates from 10-month-old PS19-fE4 and PS19-fE3 mice with or without Cre. Figure 1F Diagrammatically shown are quantified AT8-positive pTau levels relative to TUJ1 in the RAB fraction of hippocampal lysates of 10-month-old PS19-fE4 and PS19-fE3 mice with or without Cre (PS19-fE4: no Cre, n=21; Syn1-Cre, n=18; GFAP-Cre, n=17; PS19-fE3: no Cre, n=17; Syn1-Cre, n=11; GFAP-Cre, n=11). Figure 1GDiagrammatically shown are quantified AT8-positive pTau levels relative to TUJ1 measured by western blot analysis in RIPA fractions of hippocampal lysates from 10-month-old PS19-fE4 and PS19-fE3 mice with or without Cre (PS19-fE4: no Cre, n=21; Syn1-Cre, n=18; GFAP-Cre, n=17; PS19-fE3: no Cre, n=17; Syn1-Cre, n=11; GFAP-Cre, n=11). Figure 1H Schematic diagram showing the experimental design of the Tau propagation study, which included a 12-week incubation period following unilateral hippocampal injection of AAV2-Tau-P301S (2.10E+13 vg / mL) in fE4 mice with and without Cre or in fE3 mice without Cre. Fig. 1I Representative images of fE4 mouse brains immunostained for human Tau (HT7 antibody) 12 weeks after injection are shown, with black dots indicating injection sites (X=+1.5; Y=-2.1; Z=-2.1) and a notch in the left hemisphere distinguishing the non-injected side (scale bar, 1 mm). Figure 1J Shown are representative images of 10-month-old fE4 mice immunostained for GFP 2 weeks after unilateral injection with AAV2-GFP (1.0E+13 vg / mL) into the right hippocampus, demonstrating the absence of viral spread to the uninjected hippocampal side (scale bar, 900 μm). Figure 1K Shown are representative images of the non-injected hippocampal side of 13-month-old fE mice with and without Cre after immunostaining for human Tau (HT7 antibody) (scale bar, 500 μm). Figure 1L Shown are representative images of the non-injected hippocampal side of 13-month-old fE mice with or without Cre after immunostaining with pTau (AT8 antibody) (scale bar, 500 μm). Figure 1M Graphically depicted are the average number of HT7 (human Tau)-positive cells per hippocampal section on the non-injected hippocampal side of 13-month-old fE mice with or without Cre. Figure 1N Graphically depicted are the average number of AT8 (pTau)-positive cells per hippocampal section on the non-injected hippocampal side of 13-month-old fE mice with or without Cre. Figures 1K to 1M The quantified data are n = 8 mice / genotype group and Figure 1B , 1E The data in , 1F, 1K, and 1M are expressed as mean ± SEM, and one-way ANOVA and Tukey's post hoc multiple comparison test were used.

[0011] Figures 2A to 2BIt was shown that removal of APOE4 from neurons but not astrocytes ameliorated neuronal hyperexcitability in the hippocampus of PS19-fE4 mice. Figure 2A The mean normalized fPSP slope of the CA1 stratum radiatum in response to Schaffer collateral stimulation is graphically shown.APOE4 expression in neurons renders the CA3-CA1 network hyperexcitable as evidenced by the enhancement of responses to synaptic stimulation. Figure 2B The diagram shows the calculation Figure 2A Individual fPSP slope gain values ​​for all experiments in. PS19-fE3: n = 1 3, N = 2; PS19-fE4: n = 20, N = 4;

[0012] PS 1 9-fE4 / Syn 1-Cre: n = 25, N = 4; PS 1 9-fE4 / GFAP-Cre: n = 1 3, N = 2. Data are expressed as mean ± SEM, and one-way ANOVA and Tukey's post hoc multiple comparison test were used.

[0013] Figures 3A to 3J It was shown that whether APOE4 was removed from neurons or astrocytes, neurodegeneration was reduced. Figure 3A Shown are representative images of the ventral hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre after staining with Sudan Black to enhance hippocampal visualization (scale bar, 1 mm). Figure 3B Quantification of hippocampal volume in 10 month old PS19-fE4 and PS19-fE3 mice with and without Cre is graphically shown. Figure 3C Quantification of posterior lateral ventricle volume in 10 month old PS19-fE4 and PS19-fE3 mice with and without Cre is graphically shown. Figure 3D Shown are representative images of the CA1 hippocampal subfield of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre after immunostaining for the neuronal marker NeuN (scale bar, 50 μm). Figure 3E Diagrammatically shows the thickness of the CA1 neuronal cell layer of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 3F Representative images of the hippocampal dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre after immunostaining for the neuronal marker NeuN are shown (scale bar, 100 μm). Figure 3GThe thickness of the dentate gyrus granule cell layer of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre is shown schematically. Figure 3B , 3C All quantifications in 3E, and 3G, PS19-fE4: no Cre, n = 25; Syn1-Cre, n = 17; GFAP-Cre, n = 16, and PS19-fE3: no Cre, n = 20; Syn1-Cre, n = 15; GFAP-Cre, n = 15 mice. All data are expressed as mean ± SEM, using one-way ANOVA and Tukey's post hoc multiple comparison test. Figure 3H Figure 3 shows the hippocampal volume (mm) in PS19-fE4 mice (n=25). 3 ) and AT8 coverage area (%). Pearson correlation analysis (bilateral). LV, lateral ventricle; DG, dentate gyrus. Fig. 3I Figure 3 shows the hippocampal volume (mm) in PS19-fE4 mice (n=25). 3 ) and posterior ventricular volume (mm 3 ) Pearson correlation analysis (bilateral). LV, lateral ventricle; DG, dentate gyrus. Figure 3J Figure 3 shows the hippocampal volume (mm) in PS19-fE4 mice (n=25). 3 ) and the thickness of the cell layer of CA1 neurons (μm) (j). Pearson correlation analysis (bilateral). LV, lateral ventricle; DG, dentate gyrus.

[0014] Figures 4A to 4O It was shown that whether APOE4 was removed from neurons or astrocytes, microgliosis and astrogliosis were reduced. Figure 4A Diagrammatically depicted is the percent Iba1 coverage area in the hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 4B Graphically shown is the correlation between percent Iba1 coverage area and hippocampal volume in PS19-fE4 mice (n=25). Figure 4C Diagrammatically depicted is the percent CD68 coverage area in the hippocampus of 10 month old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 4D The correlation between percent CD68 coverage and hippocampal volume in PS19-fE4 mice (n=25) is shown graphically. Figure 4D n = 8 mice / genotype group, and all data are presented as mean ± SEM using one-way ANOVA and Tukey's post hoc multiple comparison test. Figure 4EDiagrammatically depicted is the percent GFAP coverage area in the hippocampus of 10 month old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 4F Diagrammatically depicted is the percent S100β coverage area in the hippocampus of 10 month old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 4G Graphically shown is the correlation between percent GFAP coverage and hippocampal volume in PS19-fE4 mice (n=25). Figure 4H The correlation between the percentage S100β coverage area and hippocampal volume of PS19-fE4 mice (n=25) is shown graphically. For quantification, PS19-fE4: no Cre, n=25; Syn1-Cre, n=17; GFAP-Cre, n=16, and PS19-fE3: no Cre, n=20; Syn1-Cre, n=15; GFAP-Cre, n=15 mice. All data are expressed as mean ± SEM, using one-way ANOVA and Tukey post hoc multiple comparison test. Pearson correlation analysis (bilateral). Fig. 4I Graphically depicted are the percent Iba1 coverage area in the non-injected hippocampal side of 13 month old fE mice with and without Cre 12 weeks after unilateral injection of AAV2-Tau-P301S. Figure 4J The percentage GFAP coverage in the uninjected hippocampus of 13-month-old fE mice with and without Cre 12 weeks after unilateral injection of AAV2-Tau-P301S is shown graphically. The AAV2 virus does not directly penetrate the uninjected hippocampus, and microgliosis is reduced after neuronal APOE4 or astroglial APOE4 is removed from the uninjected hippocampus of fE4 mice that received unilateral injection of AAV2-Tau-P301S. For quantification, Figure 4K Diagrammatically depicted is the percent MBP coverage area in the hippocampal CA1 subfield of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 4L Diagrammatically depicted is the percent NG2 coverage area in the hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 4M The APOE protein levels in the cell lysates of primary neurons cultured for 14 days from the hippocampus and cortex of PS19-fE4 and PS19-fE4 / GFAP-Cre mice measured by ELISA are shown schematically, and the APOE protein levels are normalized to the total cellular protein levels. For each group of neurons, n=3 wells, unpaired t-test and two-tailed t-test were used. Figure 4NThe APOE protein level in the cell lysate of the primary neurons cultured for 14 days from the PS19-fE4 / GFAP-Cre mouse hippocampus measured by ELISA is shown schematically. After 14 days of culture, neurons were then treated with different recombinant proteins (Nrg3, HMGB1 and DPP10 were all 10 μg / ml) for 24 hours, and the APOE4 protein level in the cell lysate was measured by ELISA and normalized relative to the total cellular protein level. For each group of neurons, n=3 wells. Fig.4O The APOE protein levels in the cell lysates of primary neurons cultured for 14 days from the hippocampus of PS19-fE4 mice measured by ELISA are shown schematically. The primary neurons were cultured for 14 days and then treated with different recombinant proteins (Nrg3, HMGB1 and DPP10 were all 10 μg / ml) for 24 hours, and the APOE4 protein levels in the cell lysates were measured by ELISA and normalized to the total cellular protein levels. For each group of neurons, n=3 wells, single-factor ANOVA and Tukey post hoc multiple comparison tests were used. All data are expressed as mean ± SEM. Ex neurons, excitatory neurons; In neurons, inhibitory neurons; OPC, oligodendrocyte precursor cells.

[0015] Figures 5A to 5J It was shown that HMGB1 nuclear-cytoplasmic translocation and release from hippocampal neurons was significantly reduced by depletion of neuronal APOE4 and, to a lesser extent, by depletion of astrocytic APOE4. Figure 5A Representative images of immunostaining with anti-HMGB1 and DAPI in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre are shown (scale bar, 40 μm). Figure 5B Representative high magnification images (60×+3× magnification) of immunostaining with anti-HMGB1 and DAPI in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre are shown (scale bar, 10 μm). Figure 5C Diagrammatically shown are the nuclear integrated densities of HMGB1 immunostaining in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 5D Diagrammatically shown are the cytoplasmic integrated density of HMGB1 immunostaining in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Figure 5E Schematic representation of nuclear HMGB1 in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre + The average number of spots. Fig. 5FDiagram showing cytoplasmic HMGB1 in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre + Average number of spots. For quantification in c to f, n = 6 mice / group, and all data are presented as mean ± SEM using one-way ANOVA and Tukey's post hoc multiple comparison test. Figure 5G Graphically shown are total HMGB1 protein levels in the hippocampal interstitial fluid (ISF) of 8.5 month old PS19-fE4 mice with and without Cre as measured by ELISA. Figure 5H Graphically shown are HMGB1 protein levels in each collected ISF fraction of 8.5 month old PS19-fE4 mice with and without Cre as measured by ELISA. Figures 5G to 5H Fractions 1 to 2 were not included in the analysis because the artificial CSF was circulated at a higher flow rate during the first two hours to prevent clogging of the tube. Figures 5G to 5H Data in included n = 2 mice / genotype and are presented as mean ± SEM using one-way ANOVA and Tukey post hoc multiple comparison test. Fig.5I The quantified Iba1 coverage percentage ratio between injected:uninjected hippocampal sides 6 days after injection is shown graphically.Interstitial fluid (ISF) without HMGB1 (n=3) or enriched with HMGB1 (n=3) was injected into the hippocampus of 8.5 month old wild-type mice and the level of Iba1 was measured 6 days after injection. Figure 5J The diagram shows the quantified percentage ratio of GFAP coverage area between injected and uninjected hippocampus sides 6 days after injection. Interstitial fluid (ISF) without HMGB1 (n=3) or enriched with HMGB1 (n=3) was injected into the hippocampus of 8.5-month-old wild-type mice and the level of Iba1 was measured 6 days after injection. Figures 5I to 5J For quantification in , n=3 mice were used for the control (HMGB1-free) and experimental (HMGB1-enriched) groups.

[0016] Figures 6A to 6R It was shown that treatment with an HMGB1 inhibitor blocked the nuclear-cytoplasmic translocation of HMGB1 in PS19-fE4 mice and improved gliosis, Tau pathology, myelin defects, and neurodegeneration in PS19-fE4 mice. Fig. 6ASchematic diagram showing the experimental design of the HMGB1 inhibitor study, which took place over a 12-week period in which PS19-fE4 and PS19-fE3 mice were treated with either 0.9% saline vehicle or an HMGB1 inhibitor (a mixture of 80 mg / kg of ethyl pyruvate and 20 mg / kg of glycyrrhizic acid dissolved in 0.9% saline), starting at 6.5 months of age and ending at 9.5 months of age. Figure 6B Shown are representative high magnification images (60×+3× magnification) of immunostaining with anti-HMGB1 and DAPI in the dentate gyrus of 9.5-month-old PS19-fE4 and PS19-fE3 mice after treatment with saline or HMGB1 inhibitor (scale bar, 10 μm). Figure 6C Graphically shown are quantifications of nuclear integrated density of HMGB1 immunostaining in the dentate gyrus of treated PS19-fE4 and PS19-fE3 mice at 9.5 months of age. Fig.6D Graphically shown are quantifications of the cytoplasmic integrated density of HMGB1 immunostaining in the dentate gyrus of treated PS19-fE4 and PS19-fE3 mice at 9.5 months of age. Fig. 6E Representative images of microglia immunostained with anti-Iba1 in the hippocampus of 9.5-month-old treated PS19-fE4 and PS19-fE3 mice are shown (scale bar, 500 μm). Fig. 6F Quantification of percent Iba1 coverage in the hippocampus of 9.5 month old treated PS19-fE4 and PS19-fE3 mice is graphically shown. Figure 6G Representative images of astrocytes immunostained with anti-GFAP in the hippocampus of 9.5-month-old treated PS19-fE4 and PS19-fE3 mice are shown (scale bar, 500 μm). Figure 6H Graphically shown are quantifications of percent GFAP coverage in the hippocampus of 9.5 month old treated PS19-fE4 and PS19-fE3 mice. Fig.6I Representative images of pTau immunostained with anti-AT8 in the hippocampus of 9.5-month-old treated PS19-fE4 and PS19-fE3 mice are shown (scale bar, 500 μm). Figure 6J Graphically shown are quantifications of percent pTau (AT8) coverage in the hippocampus of 9.5 month old treated PS19-fE4 and PS19-fE3 mice. Figure 6K Representative images of myelin stained with anti-MBP and DAPI in the stratum radiatum of the hippocampus beneath the pyramidal cell layer of CA1 in 9.5-month-old treated PS19-fE4 and PS19-fE3 mice are shown (scale bar, 50 μm). Figure 6LGraphically shown are quantifications of percent MBP coverage in the hippocampal CA1 subfield of 9.5 month old treated PS19-fE4 and PS19-fE3 mice. Figure 6M Representative images of the ventral hippocampus of 9.5-month-old treated PS19-fE4 and PS19-fE3 mice after staining with Sudan Black are shown (scale bar, 1 mm). Figure 6N Quantification of hippocampal volume in 9.5 month old treated PS19-fE4 and PS19-fE3 mice is graphically shown. Fig.6O Quantification of posterior lateral ventricle volume in 9.5 month old treated PS19-fE4 and PS19-fE3 mice is shown schematically. Figure 6C , 6D Quantification in 6F, 6H, 6J, 6L, 6N, 6O, PS19-fE4 saline: n = 16; PS19-fE4 HMGB1 inhibitor: n = 18; PS19-fE3 saline: n = 16; PS19-fE3 HMGB1 inhibitor: n = 14. All data are expressed as mean ± SEM, using one-way ANOVA and Tukey's post hoc multiple comparison test. Figure 6P Graphically shown are quantification of the number of HMGB1 immunostained nuclear spots in the dentate gyrus of 9.5 month old PS19-fE4 and PS19-fE3 mice after treatment with saline or HMGB1 inhibitors (ethyl pyruvate (EP) and glycyrrhizic acid (GA) (HMGB1-In)). Figure 6Q Graphically shown are quantification of the number of HMGB1 immunostained cytoplasmic puncta in the dentate gyrus of 9.5 month old PS19-fE4 and PS19-fE3 mice after treatment with saline or HMGB1 inhibitors (HMGB1-In) (ethyl pyruvate (EP) and glycyrrhizic acid (GA)). Figure 6R Quantification of percent CD68 coverage in the hippocampus of 9.5 month old treated PS19-fE4 and PS19-fE3 is shown graphically. Fig. 6A , 6B Quantification in 6D, PS19-fE4 saline: n = 16; PS19-fE4 HMGB1 inhibitor: n = 18; PS19-fE3 saline: n = 16; PS19-fE3 HMGB1 inhibitor: n = 14. All data are expressed as mean ± SEM, using one-way ANOVA and Tukey's post hoc multiple comparison test.

[0017] Figures 7A to 7DFeatures of the PS19-fE mouse model are shown, in which the APOE gene is specifically removed from neurons or astrocytes by cell type-specific Cre expression. Fig. 7A Representative images of cell type specificity of Cre recombinase expression in the hippocampus of 10-month-old PS19-fE4 mice with Syn1-Cre or GFAP-Cre as determined by co-immunostaining with anti-Cre, anti-GFAP, and anti-NeuN (scale bar, 50 μm). Figure 7B Representative images of cell type specificity of APOE expression determined by co-immunostaining with anti-ApoE, anti-GFAP, and anti-NeuN in the hippocampus of 10-month-old PS19-fE4 mice with and without Cre (scale bar, 50 μm). Figure 7C APOE in the hippocampus of 10-month-old PS19-fE4 / GFAP-Cre mice as determined by co-immunostaining with anti-ApoE and anti-Iba1 + Representative images of microglia (scale bar, 50 μm). Fig.7D .ApoE levels in hippocampal lysates of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre measured by ELISA (PS19-fE4: no Cre, n=21; Syn1-Cre, n=18; GFAP-Cre, n=17; PS19-fE3: no Cre, n=17; Syn1-Cre, n=11; GFAP-Cre, n=11). For each individual mouse, ApoE levels were normalized to the weight of dissected hippocampal tissue. Data are expressed as mean ± SEM, using one-way ANOVA and Tukey post hoc multiple comparison test.

[0018] Fig. 8A 8F show that the AAV2 virus does not directly penetrate the non-injected hippocampus and that microgliosis is reduced following ablation of neuronal APOE4 or astrocytic APOE4 in the non-injected hippocampus of fE4 mice that received a unilateral injection of AAV2-Tau-P301S. Fig. 8A Representative images of the injected and non-injected hippocampal sides of 10-month-old fE4 mice 2 weeks after unilateral injection with AAV2-GFP virus (1.0E+13 vg / mL) and immunostained with anti-GFP (scale bar, 500 μm). Figure 8B Representative high magnification images of the dentate gyrus of the injected and non-injected hippocampal sides of 10-month-old fE4 mice 2 weeks after unilateral injection with AAV2-GFP virus and immunostained with anti-GFP and DAPI (scale bar, 100 μm). Figure 8CRepresentative images of microglia immunostained with anti-Iba1 in the non-injected hippocampus of 13-month-old fE4 mice with and without Cre or fE3 mice without Cre 12 weeks after unilateral injection of AAV2-Tau-P301S (2.10E+13 vg / mL) (scale bar, 500 μm). Fig.8D Quantification of percent Iba1 coverage area in the non-injected hippocampal side of 13 month old fE mice with and without Cre 12 weeks after unilateral injection of AAV2-Tau-P301S. Fig. 8E . Representative images of astrocytes immunostained with anti-GFAP in the non-injected hippocampus of 13-month-old fE mice with and without Cre 12 weeks after unilateral injection of AAV2-Tau-P301S (scale bar, 500 μm). Figure 8F. Quantification of percent GFAP coverage in the non-injected hippocampus of 13-month-old fE mice with and without Cre 12 weeks after unilateral injection of AAV2-Tau-P301S. Fig.8D Quantification in A, F, n = 8 mice / genotype group, and all data are expressed as mean ± SEM using one-way ANOVA and Tukey post hoc multiple comparison test.

[0019] Figures 9A to 9B It was shown that removal of neuronal APOE4 but not astroglial APOE4 ameliorated neuronal hyperexcitability in the hippocampus of PS19-fE4 mice. Fig.9A Mean normalized fPSP slope in CA1 stratum radiatum in response to Schaffer collateral incremental stimulation Neuronal APOE4 expression renders the CA3-CA1 network hyperexcitable as demonstrated by the potentiation of responses to synaptic stimulation. Fig. 9B . Calculated individual fPSP slope gain values ​​for all experiments in (a). PS19-fE3: n = 13, N = 2; PS19-fE4: n = 20, N = 4; PS19-fE4 / Syn1-Cre: n = 25, N = 4; PS19-fE4 / GFAP-Cre: n = 13, N = 2. Data are presented as mean ± SEM, and one-way ANOVA with Tukey's post hoc multiple comparison test was performed.

[0020] Figures 10A to 10F It was shown that myelin defects and OPC pool depletion were significantly reduced after APOE4 removal from neurons but not astrocytes. Fig. 10A Representative images of myelin stained with anti-MBP and DAPI in the stratum radiatum of the hippocampus beneath the pyramidal cell layer of CA1 in 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre (scale bar, 50 μm). Fig. 10BQuantification of percent MBP coverage in the hippocampal CA1 subfield of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Fig. 10C Representative images of OPCs in the hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre by immunostaining with anti-NG2 (scale bar, 500 μm). Fig. 10D Quantification of percent NG2 coverage in the hippocampus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre. Fig.10E Representative images of myelin stained with anti-MBP and DAPI in the stratum radiatum of the hippocampus in 10-month-old fE4 and fE3 mice. Fig.10F Representative images of OPCs stained with anti-NG2 in the hippocampus of 10-month-old fE4 and fE3 mice. Fig. 10B Quantification in A and D, n = 6 mice / genotype group, and all data are expressed as mean ± SEM using one-way ANOVA and Tukey post hoc multiple comparison test.

[0021] Figures 11A to 11J Shown is snRNA-seq analysis characterizing hippocampal cell clusters and the relevance of some clusters to pathological conditions. Fig.11A . Dot plots showing the normalized mean expression of selected marker genes for all 33 different hippocampal cell clusters. Fig. 11B . Characteristic graphs showing the relative levels of normalized human APOE gene expression in all 33 hippocampal cell clusters in PS19-fE4 mice without Cre (n=4), with Syn1-Cre (n=4), or with GFAP-Cre (n=3) and PS19-fE3 mice without Cre (n=3). Fig. 11C . UMAP plots highlighting cells in hippocampal clusters 17 and 25 for each mouse genotype group. Fig.11D Boxplot of the proportion of oligodendrocyte cluster 17 per sample by mouse genotype group. Each dot represents one mouse. Fig.11E .Dot plot of the top 20 KEGG pathways significantly enriched for differentially expressed genes in oligodendrocyte cluster 17 compared to other oligodendrocyte clusters 1 and 2. P values ​​are based on hypergeometric tests and adjusted for multiple testing using the Benjamini-Hochberg method. The size of the dots is proportional to the number of genes in a given gene set. Gene ratios represent the proportion of genes considered differentially expressed in the respective gene set using the FindMarkers function in Seurat. Fig.11F Boxplot of the proportion of excitatory neuron clusters 25 per sample by mouse genotype group. Each dot represents one mouse. Fig.11GTo J. Targeting oligodendrocyte cluster 17 ( Fig.11G , H) and excitatory neuron cluster 25 ( Fig.11I The proportion of cells in each sample (J) compared with the hippocampal volume (mm 3 Figure 3 is a scatter plot of the measurements of pTau (AT8) and pTau (AT8) coverage area (%). Samples are colored according to the mouse genotype group to which they belong, and the strain with the best genotype-specific fit is included.

[0022] Fig.12 A to 12S show snRNA-seq analysis demonstrating astrocyte and microglial subclusters and the relevance of some subclusters to pathological conditions. Fig.12 A. UMAP plots highlighting cells in astrocyte subclusters 4 and 7 in PS19-fE4 mice without Cre (n=4), with Syn1-Cre (n=4), or with GFAP-Cre (n=3) and PS19-fE3 mice without Cre (n=3). Fig.12 B. Characteristic graph showing the normalized relative levels of human APOE gene expression in all astrocyte subclusters for each mouse genotype group. Fig. 12C Boxplot of the proportion of cells per sample within each mouse genotype group for astrocyte subcluster 4. Each dot represents one sample. Fig.12D , E. Scatter plots of hippocampal volume (mm3) (d) and pTau (AT8) coverage area (%) (e) compared to the proportion of cells in each sample in astrocyte subcluster 4. Samples are colored according to the mouse genotype group to which they belong, and the strain with the best genotype-specific fit is included. Fig.12F . For astrocyte subcluster 7, boxplots of the proportion of cells per sample within each mouse genotype group. Figure 12G , H. Hippocampal volume (mm 3 )(g) and pTau(AT8) coverage area (%) (h) compared to the proportion of cells in each sample in astrocyte subcluster 7. Fig.12I . UMAP plots highlighting cells in microglial subclusters 4 and 8 for each mouse genotype group. Fig.12J Characteristic graph showing the normalized relative levels of human APOE gene expression in all microglial subclusters for each mouse genotype group. Figure 12K . Boxplot of the proportion of cells per sample within each mouse genotype group for microglial subcluster 4. Figure 12L , M. Hippocampal volume (mm 3 )(l) and dot plots of pTau(AT8) coverage area (%) (m) compared to the proportion of cells in each sample in microglial subcluster 4. Fig.12N. Boxplot of the proportion of cells per sample within each mouse genotype group for microglial subcluster 8. Fig.12O , P. Hippocampal volume (mm 3 Scatter plots of the proportion of cells in each sample in microglial subcluster 8 ( )(o) and pTau(AT8) coverage area (%) (p) compared to the proportion of cells in each sample in microglial subcluster 8. Figure 12Q , R. Dot plots of the top 20 KEGG pathways for differentially expressed genes significantly enriched for astrocyte subcluster 4 compared to all other astrocyte subclusters (q) and microglia subcluster 4 compared to all other microglia subclusters (r). P values ​​are based on hypergeometric tests and adjusted for multiple testing using the Benjamini-Hochberg method. The size of the point is proportional to the number of genes in a given gene set. Gene ratios represent the ratio of genes considered to be differentially expressed in each gene set using the FindMarkers function in Seurat. Figure 12S Principal component analysis plots of clusters 17 and 25 and the astrocyte and microglia subclusters. PC1 and PC2 show the overall relationship between clusters based on the similarity of the estimated log odds ratios per unit change in the eight pathological states for each cluster / subcluster. AS, astrocytes; MG, microglia.

[0023] Figures 13A to 13 I shows astrocyte APOE regulation of neuronal APOE expression. Fig.13A .UMAP plots of 22 different hippocampal cell clusters from the GEO:GSE164507 dataset. Figure B. Characteristic plot showing the normalized relative levels of human APOE gene expression in all 22 hippocampal cell clusters in the following: floxed APOE3 (FE3) mice, floxed APOE4 (FE4) mice, oil-treated P301S-Tau / Aldh1l1-Cre / APOE3flox / flox mice (TAFE3_oil), oil-treated P301S-Tau / Aldh1l1-Cre / APOE4 flox / flox (TAFE4_Oil), P301S-Tau / Aldh1l1-Cre / APOE3 treated with tamoxifen flox / flox Mice (TAFE3_tam), P301S-Tau / Aldh1l1-Cre / APOE4 treated with tamoxifen flox / flox (TAFE4_tam). Fig. 13C A, D. Histograms of APOE expression in cluster 3 excitatory (Ex) neurons (c) and cluster 5 inhibitory (In) neurons (d) from FE3, FE4, TAFE3_oil, and TAFE4_oil mice. For LogFC and p-values. Fig.13E , F. Histograms of APOE expression in cluster 3 excitatory (Ex) neurons (e) and cluster 5 inhibitory (In) neurons (f) from TAFE3_oil, TAFE4_oil, TAFE3_tam, and TAFE4_tam mice. For LogFC and p-values. Figure 13G .APOE protein levels in cell lysates of 14-day cultured primary neurons from the hippocampus and cortex of PS19-fE4 and PS19-fE4 / GFAP-Cre mice were measured by ELISA and normalized to total cellular protein levels. For each group, n=3 wells of neurons, unpaired t-test and two-tailed t-test were used. Fig.13 H, I. Primary neurons from the hippocampus of PS19-fE4 / GFAP-Cre (h) or PS19-fE4 (i) mice were cultured for 14 days and then treated with different recombinant proteins (Nrg3, HMGB1 and DPP10 were all 10 μg / ml) for 24 hours. After treatment, APOE4 protein levels in cell lysates were measured by ELISA and normalized to total cellular protein levels. For each group, n = 3 wells of neurons, single-factor ANOVA and Tukey post hoc multiple comparison tests were used. All data are expressed as mean ± SEM. Ex neurons, excitatory neurons; In neurons, inhibitory neurons; OPC, oligodendrocyte precursor cells.

[0024] Figures 14A to 14F It is shown that HMGB1 -enriched ISF fractions induce microgliosis in wild-type mice following unilateral injection. Fig.14A Representative high magnification images (60×+3× magnification) of immunostaining with anti-HMGB1 and DAPI in the dentate gyrus of 10-month-old fE4 and fE3 mice (scale bar, 10 μm). Fig. 14B Representative images of immunostaining with anti-HMGB1 and DAPI in the dentate gyrus of 10-month-old PS19-fE4 and PS19-fE3 mice with and without Cre (scale bar, 40 μm). Fig. 14C Representative images of microglia stained with anti-Iba1 in the hippocampus of 8.5-month-old wild-type mice after unilateral injection of ISF without HMGB1 (n=3) or enriched with HMGB1 (n=3) (scale bar, 500 μm). Fig.14D Quantification of the ratio of percent Iba1 coverage area between injected:uninjected hippocampal side 6 days after injection. Fig.14E Representative images of astrocytes stained with anti-GFAP in the hippocampus of 8.5-month-old wild-type mice following unilateral injection of HMGB1-free or HMGB1-enriched ISF (scale bar, 500 μm). Fig.14F . Quantification of the ratio of percentage GFAP coverage between the injected hippocampus side: the uninjected hippocampus side 6 days after injection. For quantification in d and f, n=3 mice were used for the control (without HMGB1) group and the experimental (enriched with HMGB1) experimental group. All data are expressed as mean ± SEM, using one-way ANOVA and Tukey post hoc multiple comparison test.

[0025] Figures 15A to 15D It was shown that treatment with an HMGB1 inhibitor reduced HMGB1 plaque formation and improved microglial activation in PS19-fE4 mice. Fig.15A , B. Quantification of the number of nuclear (a) and cytoplasmic (b) HMGB1 immunostained puncta in the dentate gyrus of 9.5-month-old PS19-fE4 and PS19-fE3 mice after treatment with saline or HMGB1 inhibitors. Fig. 15C ) Representative images of activated microglia immunostained with anti-CD68 in the hippocampus of 9.5-month-old treated PS19-fE4 and PS19-fE3 mice (scale bar, 500 μm). Fig.15D ) Quantification of percent CD68 coverage in the hippocampus of 9.5-month-old treated PS19-fE4 and PS19-fE3. For quantification in a, b, d, PS19-fE4 saline: n = 16; PS19-fE4 HMGB1 inhibitor: n = 18; PS19-fE3 saline: n = 16; PS19-fE3 HMGB1 inhibitor: n = 14. All data are expressed as mean ± SEM, using one-way ANOVA and Tukey post hoc multiple comparison test.

[0026] Figures 16A to 16D The "APOE4-HMGB1-inflammation-degeneration" cascade hypothesis / model for APOE4-related AD and other tauopathies is presented. Fig.16A.Schematic overview of the pathogenic mechanism of cell type specific APOE4 pathogenesis. In the presence of both neuronal APOE4 and astrocyte APOE4, as shown in PS19-fE4 mice, the pathogenic cascade begins with the neuronal expression of APOE4, which can be induced by a variety of neuronal stressors. Neuronal APOE4 has a potent effect on the accumulation and propagation of Tau pathological conditions, which can further induce neuronal APOE4 expression. Elevated neuronal APOE4 synergizes Tau pathological conditions to trigger nuclear-cytoplasmic translocation and the release of HMGB1 from neurons. Astrocyte APOE4 does not have a direct effect on the accumulation and propagation of Tau pathological conditions, however, it indirectly enhances the APOE4 expression in neurons by promoting the release of astrocyte factors (such as DPP10) that can regulate the expression of neuronal APOE4, thereby promoting the HMGB1 translocation induced by APOE4 / Tau pathological conditions and the release of HMGB1 from neurons. After HMGB1 is released from neurons, it acts as an inflammatory cytokine that induces gliosis. Extensive gliosis, especially the accumulation of toxic astrocyte and microglial subtypes, leads to abnormal phagocytosis of neurons, synapses, and oligodendrocyte-derived myelin, and subsequent neurodegeneration, neurological dysfunction, and oligodendrocyte degeneration. Fig. 16B .Shows a schematic diagram of the protective mechanism of neuronal APOE4 removal against the proposed cell type-specific APOE4 pathogenic mechanism. Removal of neuronal APOE4, as shown in PS19-fE4 / Syn1-Cre mice, results in a dramatic reduction in the accumulation and spread of Tau pathology in neurons. The reduction of Tau pathology and the lack of neuronal APOE4 exist together as a hindrance to the release of HMGB1 from neurons. Since astrocyte APOE4 contributes to the release of HMGB1 by indirectly enhancing neuronal APOE4 expression, the lack of neuronal APOE4 blocks the indirect effect of astrocyte APOE4 on triggering neuronal HMGB1 release. In the absence of sufficient neuronal HMGB1 release, gliosis, particularly gliosis of toxic microglia and astrocyte subtypes, is not induced, thus resulting in reduced neurodegeneration, neurological dysfunction, and oligodendrocyte degeneration. Therefore, removal of neuronal APOE4 results in complete prevention or rescue of all observed pathologies. Fig. 16C.Shows a schematic diagram of the protective mechanism of astrocyte APOE4 removal against the proposed cell type-specific APOE4 pathogenic mechanism. Astrocyte APOE4 removal, as shown in PS19-fE4 / GFAP-Cre mice, results in a significant but incomplete reduction in neuronal APOE4 expression. Reduced levels of neuronal APOE4 are still sufficient to induce significant accumulation and propagation of Tau pathology, which together with low levels of neuronal APOE4 trigger low levels of HMGB1 release. In the absence of sufficiently high HMGB1 release, the overall level of gliosis is greatly reduced. Nevertheless, low levels of neuronal APOE4 and HMGB1 release are sufficient to promote the presence of toxic astrocyte subtypes and microglial subtypes. The overall reduction in gliosis leads to reduced neurodegeneration, although subpopulations of disease-associated neurons still exist due to low levels of neuronal APOE4. The presence of toxic glial subtypes still leads to a lack of oligodendrocytes, probably due to the higher sensitivity of oligodendrocytes to lower levels of toxic glial subtypes. High levels of Tau pathology and the presence of toxic glial subtypes can sustain neurological dysfunction. Thus, ablation of astrocytic APOE4 resulted in partial prevention or rescue of the observed pathology. Fig.16D .Shows a schematic diagram of the protective mechanism of HMGB1 inhibitor treatment against the proposed cell type specific APOE4 pathogenic mechanism. In the presence of HMGB1 inhibitors, the release of HMGB1 from neurons is blocked. In the absence of released HMGB1 acting as an inflammatory cytokine, gliosis is significantly reduced, resulting in a significant reduction in neurodegeneration and oligodendrocyte degeneration. The lack of gliosis and the lack of possible toxic glial cell subtypes also lead to a significant reduction in Tau pathology, which together with the reduced neurodegeneration may improve neurological dysfunction. Therefore, HMGB1 inhibitor treatment also leads to complete prevention or rescue of all observed pathological states.

[0027] Figures 17A to 17M APOE4 was shown to promote cellular HMGB1 release to induce robust and sustained gliosis in the mouse hippocampus. Fig.17A .HMGB1 protein levels in the hippocampal interstitial fluid (ISF) of 10-month-old PS19-E4 and PS19-E3 mice measured by ELISA. Fig. 17B . HMGB1 protein levels in each collected ISF fraction of 10 month old PS19-E4 mice measured by ELISA. Fractions 1 and 2 were excluded from the analysis in A, B because the artificial CSF was circulated at a higher flow rate during the first two hours to prevent clogging of the tube. Fig. 17C. HMGB1 protein levels in each of the collected ISF fractions of one PS19-E4 mouse used for experiments in D and F to I as measured by ELISA. Fractions 3 to 7 were designated as HMGB1-free (HMGB1 - Fractions 19 to 22 were designated as HMGB1-enriched (HMGB1 + ). Figure 17D. Experimental design of the study involving injection of HMGB1-enriched or HMGB1-free ISF into the hippocampus of 10-month-old WT mice and assessment of acute gliosis 6 days after the initial injection. Figure 17E. Experimental design of the study involving two injections of recombinant HMGB1 (rHMGB1) or saline into the hippocampus of 3-month-old PS19-E4 mice and assessment of gliosis 8 weeks after the initial injection. Fig.17F . After unilateral injection of HMGB1-free (HMGB1) collected from 10-month-old PS19-E4 mice - ) or enriched in HMGB1 (HMGB1 + Representative images of microglia stained with anti-Iba1 in the hippocampus of 10-month-old WT mice after ISF (scale bar, 500 μm). Figure 17G Quantification of the ratio of percent Iba1 covered area between injected and non-injected hippocampal sides 6 days after injection. Fig.17H Representative images of astrocytes stained with anti-GFAP in the hippocampus of 10-month-old wild-type mice after unilateral injection of HMGB1-free or HMGB1-enriched ISF collected from 10-month-old PS19-E4 mice (scale bar, 500 μm). Fig.17I Quantification of the ratio of percent GFAP coverage between injected and non-injected hippocampal sides 6 days after injection. Fig.17J Representative images of microglia stained with anti-Iba1 in the hippocampus of 5-month-old PS19-E4 mice after two unilateral injections of rHMGB1 or saline (scale bar, 500 μm). Figure 17K Quantification of the ratio of percent Iba1 covered area between injected and non-injected hippocampal sides 8 weeks after the initial injection. Figure 17L Representative images of astrocytes stained with anti-GFAP in the hippocampus of 3-month-old PS19-E4 mice after two unilateral injections of rHMGB1 or saline (scale bar, 500 μm). Fig.17M Quantification of the ratio of percent GFAP coverage between the injected and uninjected hippocampal sides 8 weeks after the initial injection. Data in A, B include n=7 mice / genotype. For quantification in G, I, the ratio of percent GFAP coverage between the injected and uninjected hippocampal sides was 0.77 ± 0.13 % (P < 0.05). - ) groups and experiments (HMGB1 +) group used n=9 mice. For quantification in K, M, n=8 mice were used for rHMGB1 and saline control groups. All data are expressed as mean±SEM with unpaired two-sided t-test. ISF, interstitial fluid; WT, wild type; rHMGB1, recombinant HMGB1.

[0028] Figures 18A to 18B It is shown that the treatment of PS19-E4 mice with HMGB1 inhibitors significantly reduced the release of HMGB1 to ISF. The experimental design of the HMGB1 inhibitor study included treating PS19-E4 and PS19-E3 mice with 0.9% saline vehicle or HMGB1 inhibitors (a mixture of 80mg / kg of ethyl pyruvate and 20mg / kg of glycyrrhizic acid dissolved in 0.9% saline), three doses per week (Monday, Wednesday and Friday) for three weeks. After treatment, microdialysis was performed to collect interstitial fluid (ISF) from each mouse hippocampus. Fig.18A . HMGB1 protein levels in ISF of HMGB1 inhibitor-treated or saline-treated PS19-E4 mice measured by ELISA. Fig.18B .HMGB1 protein levels in each collected ISF fraction of PS19-E4 mice treated with HMGB1 inhibitors measured by ELISA. Fractions 1 and 2 were excluded from the analysis in E, F. Quantified data (PS19-E4, n=5; PS19-E4 HMGB1-In, n=5) are expressed as mean ± SEM with unpaired two-sided t-test. HMGB1-In, HMGB1-inhibitor.

[0029] Figures 19A to 19C Enhanced nuclear-cytoplasmic translocation of HMGB1 in neurons of human apoE4 / 4AD brain compared to human apoE3 / 3AD brain is shown. Fig.19A Immunofluorescence staining of HMGB1 protein on sections from human apoE4 / 4AD brain and human apoE3 / 3AD brain. Note the enhanced nuclear-cytoplasmic translocation of HMGB1 in neurons from human apoE4 / 4AD brain compared to neurons from human apoE3 / 3AD brain. Fig.19B and C. Quantification of nuclear integrated density (B) and extranuclear integrated density (C) of HMGB1 immunostaining in neurons from human apoE4 / 4AD brain and human apoE3 / 3AD brain. Quantified data (n=5 / genotype group) are expressed as mean±SEM using unpaired two-sided t-test. DETAILED DESCRIPTION

[0030] As described herein, inhibitors of high mobility group box protein 1 (HMGB1) can significantly reduce HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, Tau pathological conditions and myelin defects in objects with APOE4 alleles. For example, the method can be used for the treatment of tau diseases. Tau diseases are neurodegenerative disorders characterized by the deposition of abnormal tau proteins in the brain. Such tauopathies include Alzheimer's disease, Pick's disease, progressive supranuclear palsy, cortical basal degeneration and argyrophilic grain disease, primary age-related tau diseases, chronic traumatic encephalopathy or frontotemporal dementia.

[0031] As shown in this article, neuronal APOE4 promotes the release of HMGB1 from neurons, which triggers and / or exacerbates inflammation in the brain, induces gliosis, and causes neurodegeneration and myelin defects. In subjects with APOE4 alleles, HMGB1 protein is abnormally translocated from the nucleus of hippocampal neurons to the cytosol and released from neurons into the interstitial fluid of the hippocampus. The experiment described herein shows that the administration of two HMGB1 inhibitors (ethyl pyruvate and glycyrrhizic acid) blocks the HMGB1 nuclear-cytoplasmic translocation in mice and significantly reduces the degree of gliosis, neurodegeneration, Tau pathological conditions and myelin defects in APOE4 mice, while APOE3 mice do not have a discernible effect.

[0032] The method described herein may include administering an HMGB1 inhibitor to a subject. The subject may be a subject whose genome comprises an APOE4 allele. For example, the subject to be treated is a subject expressing an APOE4 protein. In some cases, the method includes administering a therapeutically effective amount of an HMGB1 inhibitor to a subject. Such a therapeutically effective amount of an HMGB1 inhibitor may reduce gliosis, neurodegeneration, Tau deposition, and myelin defects. For example, such a therapeutically effective amount of an HMGB1 inhibitor may reduce one or more of gliosis, neurodegeneration, Tau deposition, and myelin defects by at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99%.

[0033] High mobility group box protein 1 (HMGB1)

[0034] High mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein that is released by glial cells and neurons after inflammasome activation and activates the receptor for advanced glycation end products (RAGE) and toll-like receptor (TLR) 4 on target cells. The HMGB1 / TLR4 axis is a key initiator of neuroinflammation. As shown in this article, neuronal APOE4 promotes the release of HMGB1 from neurons, triggering and / or exacerbating inflammation in the brain, inducing gliosis, and leading to neurodegeneration and myelin defects.

[0035] The sequence of human HMGB1 protein is shown below as SEQ ID NO: 1 (UNIPROT accession number: P09429).

[0036]

[0037] The cDNA sequence encoding such HMGB1 protein is shown below as SEQ ID NO: 2 (NCBI accession number: X12597.1).

[0038]

[0039] The human HMGB1 gene is present on chromosome 13 (position 13q12.3; NC_000013.11 (30456704..30617597, complement)).

[0040] Variants and homologues of any of the sequences described herein may also be relevant to the methods and compositions described herein. For example, such variants and homologues may have less than 100% sequence identity with any of the sequences described herein. The variants and homologs may have about at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or 60% to 99% sequence identity, or 70% to 99% sequence identity, or 80% to 99% sequence identity, or 90% to 95% sequence identity, or 90% to 99% sequence identity, or 95% to 97% sequence identity, or 97% to 99% sequence identity, or 100% sequence identity with any of the sequences described herein.

[0041] HMGB1 inhibitors

[0042] HMGB1 inhibitors refer to any compound or molecule that can inhibit the expression, function, or extracellular release of HMGB1. Such HMGB1 inhibitors may include chemical compounds, antibodies, proteins, small RNA molecules, small DNA molecules, etc. that target HMGB1 protein or HMGB1 nucleic acid.

[0043] The chemical compound may be a synthetic compound or a naturally occurring compound. One example of an HMGB1 inhibitor that may be used is glycyrrhizin, also known as glycyrrhizic acid (structure shown below).

[0044]

[0045] Glycyrrhizin is an HMGB1 inhibitor that can bind one or two HMG boxes in HMGB1 and can inhibit the chemoattractant activity and mitogenic activity of HMGB1. After oral intake, glycyrrhizin is hydrolyzed by intestinal bacteria to 18β-glycyrrhetinic acid (enoxolone) and after absorption from the intestine, 18β-glycyrrhetinic acid is metabolized to 3β-monoglucuronyl-18β-glycyrrhetinic acid in the liver. 3β-monoglucuronyl-18β-glycyrrhetinic acid metabolites circulate in the bloodstream. Therefore, its oral bioavailability can be poor. Therefore, glycyrrhizin, 18β-glycyrrhetinic acid (enoxolone) and / or 3β-monoglucuronyl-18β-glycyrrhetinic acid can be administered intravenously, or topically to selected target tissues, such as to the brain.

[0046] In other examples, HMGB1 inhibitors include but are not limited to ethyl pyruvate, nicotine, (-) epigallocatechin gallate (EGCG), tanshinone, chlorogenic acid, rhamnoside-6-O-β-D-glucoside, rosmarinic acid, isorhamnetin-3-O-galactoside, polygonum multiflorum, forsythiaside B, chloroquine, actiside, shikonin, carbenoxolone, quercetin, lycopene, nafamostat mesylate, gabexate mesylate, sivelestat sodium, HMGB1 monoclonal antibody (m2G7 or #10-22), recombinant HMGB1 box A protein, acetylcholine, nicotinic acetylcholine receptor subtype α7 agonist GTS-21, peptide P5779, resveratrol, metformin, and derivatives thereof, which can also be used in the present invention. These compounds were found to have an inhibitory effect on HMGB1 secretion and can be administered by any convenient method (e.g., orally, topically or intravenously). One or more of these compounds can be administered in combination. Glycyrrhizin can also be administered together with one or more of these compounds.

[0047] In some cases, the HMGB1 inhibitor can be an RNA molecule such as siRNA, short hairpin RNA, microRNA, antisense RNA or guide RNA. The RNA molecule can be combined with the HMGB1 nucleic acid sequence (e.g., SEQ ID NO: 2). The RNA molecule can be modified according to actual needs. Other suitable RNA molecules that can target the HMGB1 gene can also be used in the present invention. Some examples of HMGB1 siRNA shown below can be used to target human HMGB1.

[0048]

[0049] Apolipoprotein E4

[0050] The APOE gene encodes apolipoprotein E, which is a protein that combines with lipids (including cholesterol) to form lipoproteins. There are a large number of different APOE but the main alleles are e2, e3, and e4 alleles. The most common allele is e3, which is present in more than half of the general population. All apoE isoforms have about 299 amino acids-the only difference is a single amino acid change. However, there are obvious functional differences because apoE3 (a common isoform) is not related to Alzheimer's disease.

[0051] Apolipoprotein E4 (APOE4) is the strongest risk factor for sporadic late-onset Alzheimer's disease (AD), which accounts for the vast majority of AD cases. APOE4 differs from APOE2 and APOE3 at amino acid positions 112 and 158 and has a unique conformation that affects its lipid-binding and receptor-binding properties. ApoE4 contains an arginine residue at position 112, while apoE3 has a cysteine ​​at this position.

[0052] The sequence of the Homo sapiens apolipoprotein E isoform b precursor is shown below as SEQ ID NO: 8 (NCBI accession number: NP_000032.1).

[0053]

[0054] SEQ ID NO:8 The apolipoprotein E sequence includes an eighteen amino acid signal peptide (highlighted above in bold and underlined). The amino acid sequence of the Homo sapiens apolipoprotein E isoform b precursor without the signal sequence is shown below in SEQ ID NO:9.

[0055]

[0056] As noted above, ApoE4 contains an arginine residue at position 112, while apoE3 has a cysteine ​​at this position (highlighted above in bold and underlined).

[0057] Therefore, a sequence of Homo sapiens apolipoprotein E4 is shown below as SEQ ID NO:10.

[0058]

[0059] The human APOE gene is present on chromosome 19 (position 19q13.32; NC_000019.10 (44905796..44909393) or NC_060943.1 (47730492..47734089)). The APOE4 allele is present in approximately 10% to 15% of the population. Subjects with other APOE (non-APOE4) alleles generally have a lower likelihood of developing Alzheimer's disease or other tauopathies. Everyone has two copies of the APOE gene: people with the APOE E2 / E2 alleles have the lowest overall risk of developing Alzheimer's disease, and those with APOE E4 / E4 have the highest risk. Other APOE combinations—E2 / E3, E2 / E4, E3 / E3, and E3 / E4—fall in between.

[0060] A variety of tests can be used to determine whether a subject has an APOE4 allele. Such tests can include genome sequencing, polymerase chain reaction amplification, restriction enzyme mapping, single nucleotide polymorphism detection (see, for example, any of the following websites: 23andme.com; empowerdxlab.com; nebula.org; alzheimersorganization.org; testing.com). Any such test can be used to determine whether a subject has at least one APOE4 allele.

[0061] Dipeptidyl Peptidase 10 (DPP10)

[0062] Dipeptidyl peptidase 10 is a protein encoded by the DPP10 gene in humans. The DPP10 protein is a single-channel type II membrane protein and a member of the S9B family in the SC family of serine proteases. The protein does not have detectable protease activity, most likely due to the lack of conserved serine residues that are usually present in the catalytic domain of serine proteases. However, the protein does bind to specific voltage-gated potassium channels and alter their expression and biophysical properties.

[0063] The sequence of human DPP10 protein can be obtained from the UNIPROT database under accession number Q8N608 (shown below as SEQ ID NO: 11).

[0064]

[0065]

[0066] The cDNA encoding the aforementioned human DPP10 protein has the following nucleotide sequence (NCBI accession number NM_020868.6; SEQ ID NO: 12).

[0067]

[0068]

[0069]

[0070]

[0071] Variants, isoforms, and homologs of any of the DPP10 sequences described herein may also be relevant to the methods and compositions described herein. For example, such variants and homologs may have less than 100% sequence identity with any of the sequences described herein. The variants and homologs may have about at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or 60% to 99% sequence identity, or 70% to 99% sequence identity, or 80% to 99% sequence identity, or 90% to 95% sequence identity, or 90% to 99% sequence identity, or 95% to 97% sequence identity, or 97% to 99% sequence identity, or 100% sequence identity with any of the sequences described herein.

[0072] As shown herein, increased levels of DPP10 are indicative of and can cause increased levels of APOE4. Thus, increased levels of DPP10 in a sample from a subject indicates that the subject will benefit from treatment with at least one HMGB1 inhibitor, at least one DPP10 inhibitor, or a combination of one or more HMGB1 inhibitors and one or more DPP10 inhibitors. For example, a subject may benefit from treatment with such an HMGB1 inhibitor and / or DPP10 inhibitor when the level of DPP10 in a sample from the subject is increased by at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.1-fold, at least 1.2-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold.

[0073] DPP10 can be detected using antibodies that specifically bind to DPP10 protein, and / or probes / primers that specifically bind to DPP10 mRNA. Antibodies that bind to DPP10 protein can be obtained from, for example, Alomone Labs, Invitrogen, SigmaAldrich, and ThermoFisher Scientific. Probes and primers that specifically bind to DPP10 mRNA can include a segment of about 15 to 100 nucleotides that has at least 90% sequence identity or is complementary to a DPP10 coding region (e.g., a DPP10 cDNA having a sequence of SEQ ID NO: 12).

[0074] DPP10 inhibitors include any compound or molecule capable of inhibiting the expression, function or extracellular release of DPP10. Such DPP10 inhibitors may include chemical compounds, antibodies, proteins, small RNA molecules, small DNA molecules, etc. that target DPP10 protein or DPP10 nucleic acid.

[0075] Composition

[0076] The present invention also relates to such a composition, which comprises one or more active agents, such as any of the HMGB1 inhibitor compounds described herein. Such an active agent may include a polypeptide, a nucleic acid encoding a polypeptide (e.g., in an expression cassette or expression vector), a modified cell, an inhibitory nucleic acid, a small molecule, a compound identified by the methods described herein, or a combination thereof. The composition may be a pharmaceutical composition. In some embodiments, the composition may include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt are compatible with the other ingredients of the preparation and are harmless to the recipient.

[0077] The compositions may be formulated in any convenient form.

[0078] In some embodiments, the active agent is administered in a "therapeutically effective amount". Such a therapeutically effective amount is an amount sufficient to obtain a desired physiological effect (e.g., to reduce at least one symptom of a tauopathy disease). Such symptoms may include HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, Tau pathology, and myelin defects.

[0079] For example, the active agent may reduce the symptoms of HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, Tau pathology, or myelin deficiency by 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 80%, or 90%, 95%, or 97%, or 99%, or any numerical percentage between 5% and 100%.

[0080] In order to realize the desired effect, activating agent can be used in single dose or separated dosage.For example, activating agent can be used in following dosage: at least about 0.01mg / kg body weight to about 500 to 750mg / kg body weight, at least about 0.01mg / kg body weight to about 300 to 500mg / kg body weight, at least about 0.1mg / kg body weight to about 100 to 300mg / kg body weight or at least about 1mg / kg body weight to about 50 to 100mg / kg body weight, but other dosages also can provide useful results.The amount applied will change according to many factors, including but not limited to for the type of using selected small molecule or compound, mammalian disease, body weight, physical condition, health status and age.Such factor can be easily determined by clinician using animal model or other test systems available in this area.

[0081] The administration of the activating agent according to the present invention can be in a single dose, multiple doses, continuous or intermittent mode, depending on, for example, the physiological condition of the recipient, whether the administration purpose is therapeutic or preventive and other factors known to skilled practitioners. The administration of the activating agent and composition of the present invention can be substantially continuous in a preselected time period, or can be a series of spaced doses. Both local and systemic administration are considered.

[0082] In order to prepare the composition, synthesize or otherwise obtain small molecules or compounds and purify as needed or desired. These small molecules or compounds and other medicaments can be suspended in a pharmaceutically acceptable carrier and / or lyophilized or otherwise stabilized. These active agents can be adjusted to appropriate concentrations and optionally combined with other medicaments. The absolute weight of a given small molecule or compound and / or other medicament contained in a unit dose can vary widely. For example, at least one molecule, compound, and / or other medicament of about 0.01 to about 2g, or about 0.1 to about 500mg, or a variety of molecules, compounds, and / or other medicaments can be administered. Alternatively, the unit dose can be about 0.01g to about 50g, about 0.01g to about 35g, about 0.1g to about 25g, about 0.5g to about 12g, about 0.5g to about 8g, about 0.5g to about 4g or about 0.5g to about 2g.

[0083] The daily dose of the activating agent of the present invention can also vary. Such daily dose can be, for example, from about 0.1 g / day to about 50 g / day, from about 0.1 g / day to about 25 g / day, from about 0.1 g / day to about 12 g / day, from about 0.5 g / day to about 8 g / day, from about 0.5 g / day to about 4 g / day, and from about 0.5 g / day to about 2 g / day.

[0084] It should be understood that the amount of active agent used for treatment varies not only with the specific carrier selected, but also with the route of administration, the nature of the disease or condition being treated, and the age and condition of the subject. Ultimately, the attending health care provider can determine the appropriate dosage. In addition, the pharmaceutical composition can be formulated into a single unit dosage form.

[0085] Therefore, one or more suitable unit dosage forms comprising an active agent can be administered by a variety of routes, including parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), oral, rectal, cutaneous, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. The active agent can also be formulated for sustained release (e.g., using microencapsulation, see WO 94 / 07529 and U.S. Patent No. 4,962,091). Where appropriate, the preparation can be conveniently presented in discrete unit dosage forms and can be prepared by any method known in the pharmaceutical field. Such a method may include mixing the active agent with a liquid carrier, a solid matrix, a semi-solid carrier, a finely divided solid carrier or a combination thereof, and then, if necessary, introducing or shaping the product into a desired delivery system. For example, the active agent can be connected to a convenient carrier (e.g., nanoparticles, albumin, polyalkylene glycol), or provided in the form of a prodrug. The active agent and its combination can be combined with a carrier and / or encapsulated in a vesicle such as a liposome.

[0086] The compositions of the invention can be prepared in a variety of forms, including aqueous solutions, suspensions, tablets, hard or soft gelatin capsules, and liposomes and other sustained-release formulations, such as shaped polymer gels. Administration of the inhibitor may also involve parenteral or topical administration of an aqueous solution or sustained-release carrier.

[0087] Thus, although active agents and / or other agents can sometimes be administered in an oral dosage form, the oral dosage form can be formulated to protect the small molecule, compound, other agent, and combination thereof from degradation or decomposition before the small molecule, compound, other agent, and combination thereof provide therapeutic utility. For example, in some cases, the small molecule, compound, and / or other agent can be formulated to be released into the intestine after passing through the stomach. Such formulations are described, for example, in U.S. Patent No. 6,306,434 and references contained therein.

[0088] Liquid pharmaceutical compositions can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, dry powders constructed with water or other suitable carriers before use. Such liquid pharmaceutical compositions may contain conventional additives, such as suspending agents, emulsifiers, non-aqueous carriers (which may contain edible oils) or preservatives. Pharmaceutical compositions may be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, and may contain formulation agents, such as suspending agents, stabilizers and / or dispersants. Suitable carriers include saline solutions, encapsulating agents (e.g., liposomes) and other substances. In the presence or absence of a carrier, active agents and / or other medicaments can be prepared in dry form (e.g., in lyophilized form). If a carrier is desired, the carrier may be included in the pharmaceutical preparation, or may be packaged separately in a separate container for addition to an inhibitor packaged in a convenient liquid in dry form, suspended form or soluble concentrated form.

[0089] The active agent and / or other agents may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers, or multi-dose containers, with an added preservative.

[0090] The composition may also contain other ingredients, such as active agents, antiviral agents, antibacterial agents, antimicrobial agents and / or preservatives.

[0091] The present description is further illustrated by the following examples, which should not be construed as limiting in any way.

[0092] Example 1: Materials and Methods

[0093] This example illustrates the materials and methods used to develop the present invention.

[0094] Mouse

[0095] Human LoxP-floxed APOE knock-in (fE) mice with conditional deletion of the human APOE gene were generated as described by Knoferle et al. (J. Neurosci. 34, 14069-14078 (2014)). Briefly, homozygous fE3 and fE4 mice (Wang et al. Neuron 109, 1657-1674.e7 (2021)) were crossed with synapsin 1-Cre transgenic mice [B6.Cg-Tg (Syn1-Cre) 671Jxm / J] (The Jackson Laboratory) (Zhu et al., Genes Dev. 15, 859-876 (2001)) or GFAP-Cre transgenic mice [B6.Cg-Tg (GFAP-Cre) 8Gtm] (National Cancer Institute Mouse Repository) (Bajenaru et al. Mol. Cell Biol. 22, 5100-5113 (2002); Uhlmann et al., Ann. Neurol. 52, 285-296 (2002)). The fE / Cre mice were further hybridized with Tau-P301S (PS19) transgenic mice [B6; C3-Tg (Prnp-MAPT * P301S) PS19Vle / J] (The Jackson Laboratory) expressing human P301S1N4RTau driven by the PrP promoter to generate PS19-fE4 and PS19-fE3 mice without Cre, with Syn1-Cre or with GFAP-Cre. The littermates that were negative for Syn1-Cre or GFAP-Cre were used as PS19-fE controls. In order to generate the PS19-fE / Syn1-Cre strain, only female Syn1-Cre mice were used for breeding purposes because germline recombination has been reported to occur in the offspring of male Syn1-Cre mice (Rempe et al., Genesis 44, 44-49 (2006)). Wild-type (WT) mice [C57BL / 6J] were obtained from Jackson Laboratory. All mice were of pure C57BL / 6 genetic background and were housed in a pathogen-free barrier facility at 19 to 23°C and 30% to 70% humidity with a 12-hour light cycle. Animals were identified by ear punch under brief isoflurane anesthesia and genotyped by polymerase chain reaction (PCR) of tail clipping. All animals except those reported in this study did not undergo surgery.All animal experiments were performed according to the guidelines and regulations of the National Institutes of Health, the University of California, and the Gladstone Institute under protocol AN176773.

[0096] For brain tissue collection, mice were deeply anesthetized by intraperitoneal injection of Avertin (Henry Schein) and cardiac perfusion of 0.9% saline for 1 minute. The brain was fixed as a whole brain or half brain according to the study. The right hemibrain was drop-fixed in 4% paraformaldehyde (16% PFA diluted in MilliQ H2O) (Electron Microscopy Sciences) for 48 hours, rinsed in 1×PBS (Corning) for 24 hours, and cryopreserved in 30% sucrose (Sigma) at 4°C for 48 hours. The fixed hemibrain was cut into 30 μm thick coronal sections on a freezing sliding microtome (Leica) and stored in a cryoprotectant solution (30% ethylene glycol, 30% glycerol, 40% 1×PBS) at -20°C. The left hemibrain was quickly frozen on dry ice and stored at -80°C.

[0097] Immunohistochemistry

[0098] For immunofluorescence staining, several sections (separated by approximately 300 μm) from each mouse are transferred to 12 well plates in 1 × PBS-T (PBS+0.1% Tween-20) (Millipore Sigma) and washed 3 × 5 minutes in PBS-T to remove cryoprotectant solution. Sections are incubated 5 minutes in boiling antigen retrieval buffer (Tris buffer, pH 7.6) (TEKNOVA) and washed 2 × 5 minutes in PBS-T. Then sections are incubated at room temperature in blocking solution (5% normal donkey serum (Jackson Labs), 0.2% Triton-X (Millipore Sigma) in 1 × PBS) for 1 hour to prevent non-specific antibody from binding. After blocking, sections are washed 1 × 5 minutes in PBS-T and in mice on mice (Mouse-on-Mouse, MOM) blocking buffer (1 drop of MOM IgG / 4mL PBS-T) (Vector Labs) at room temperature, incubated 1 hour. After MOM blocking, sections were incubated overnight at 4°C in primary antibodies diluted to optimal concentrations (anti-APOE 1:200 (Cell Signaling); anti-CD68 1:100 (Bio-Rad); anti-Cre 1:800 (Cell Signaling); anti-GFAP 1:800 (Millipore Sigma); anti-GFP 1:5000 (Thermofischer); anti-HMGB1 1:100 (Abcam); anti-Iba1(rbt) 1:200 (Wako); anti-Iba1(gt) 1:200 (Abcam); anti-MBP 1:500 (Abcam); anti-NeuN 1:500 (Millipore Sigma); anti-NG2 1:500 (Abcam); anti-S100β 1:200 (Abcam)). After primary antibody incubation, the sections were washed 3×5 minutes in PBS-T and then incubated in a fluorescently labeled secondary antibody (Abcam, Jackson Immuno, 1:1000 in PBS-T) diluted in PBS-T for 1 hour at room temperature in the dark. The sections were then washed 2×5 minutes in PBS-T and incubated in DAPI (1:50,000 in PBS-T) (Thermofisher) for 8 minutes at room temperature in the dark. The sections were then washed 2×5 minutes in PBS-T, mounted on microscope slides (Fisher Scientific), coverslipped with ProLong Gold mounting medium (Vector Laboratories), and sealed with clear nail polish.Images were acquired at 10×, 20×, 40×, or 60× magnifications, depending on staining, using a FV3000 confocal laser scanning microscope (Olympus) or an Aperio VERSA slide scanning microscope (Leica). Image analysis of percentage coverage was performed using open source Fiji (ImageJ) software after setting a standard threshold for all images. The investigators were also blinded to the samples to exclude the possibility of bias.

[0099] For DAB (3,3'-diaminobenzidine) staining, several sections from each mouse (about 300 μm apart) were transferred to a 12-well plate in 1× PBS-T and then washed 3×5 minutes in PBS-T to remove the cryoprotectant solution. The sections were then incubated in boiling antigen retrieval buffer (1× PBS, 0.1 M sodium citrate, 0.1 M citric acid) (FisherScientific, Fluka) for 5 minutes and washed 2×5 minutes in PBS-T. Next, the sections were incubated in endogenous peroxidase buffer (1×PBS, 10% methanol (Fisher Scientific), 3% H2O2 (Sigma) for 15 min and washed 3×5 min in PBS-T. The sections were then incubated in blocking solution (1×PBS-T, 5% normal donkey serum, 1% skim milk powder) for 1 h at room temperature. After blocking, the sections were washed 2×5 min in PBS-T and then incubated in avidin / biotin blocking reagent (4 drops per block) (Vector Laboratories) for 15 min and then washed 2×5 min in PBS-T. The sections were incubated in MOM blocking buffer (1 drop of MOM IgG / 4 mL PBS-T) (Vector Labs) for 1 h at room temperature. After MOM blocking, the sections were washed 2×5 min and diluted to the optimal concentration in PBS-T at 4°C (anti-pTau (AT8) 1:100 (Invitrogen); anti-HT7 1:200 (Peter Labs) Davies)) overnight. After the primary antibody incubation, the sections were washed 3×5 minutes in PBS-T and then incubated in a biotinylated secondary antibody (1:200; Jackson Immuno) for 1 hour at room temperature. Next, the sections were washed 3×5 minutes in PBS-T and incubated in ABC buffer (Vector Laboratories), which was prepared 10 minutes before the incubation step. The sections were washed 2×5 minutes in PBS-T and 1×5 minutes in Tris buffer (pH 7.6). The sections were incubated in DAB buffer (5 mL 1×PBS, 2 drops of buffer stock solution, 2 drops of DAB, 2 drops of H2O2) (Vector Laboratories) for exactly 2 minutes. The staining was terminated by washing the sections in Tris buffer (pH7.6) for 3×5 minutes and 2×5 minutes in PBS-T. The sections were mounted on microscope slides and dried overnight at room temperature. Next, the mounted sections were immersed in Xylene (Fisher Scientific) for 2 x 5 min and coverslipped with DPX mounting medium (Sigma-Aldrich).Images were acquired using an Aperio VERSA slide scanning microscope (Leica) at 10× magnification.

[0100] Volume analysis

[0101] Continuous coronal hippocampal brain slices (7 slices per mouse, 30 μm thick, 300 μm apart) were mounted on microscope slides (Fisher Scientific) and dried at room temperature for 1 hour. 0.1% Sudan Black solution was prepared by adding an appropriate amount of Sudan Black powder (Sigma) to 70% ethanol (KOPTEC) and mixing the solution with a magnetic stirrer while protecting from light. The solution was then centrifuged at 3,000 RPM for 10 minutes and the collected supernatant was filtered using a 0.2 μm filter syringe (ThermoScientific) to remove undissolved dye. The slices were then stained with 0.1% Sudan Black solution for 10 minutes at room temperature and washed 3×2 minutes in 70% ethanol and 3×5 minutes in Milli-Q water. The slices were then coverslipped with ProLong Gold mounting medium (Invitrogen) and imaged at 10× magnification on an Aperio VERSA slide scanning microscope (Leica). For hippocampal and posterior lateral ventricle volume analysis, the area of ​​interest was outlined using the segmentation tool in ImageJ and the volume was calculated using the following formula: Volume = (sum of areas) * 0.3 mm (Uhlmann et al. Ann. Neurol. 52, 285-296 (2002)). The sum of area values ​​was obtained by taking the sum of the quantitative area measurements of all 7 brain sections for each mouse, roughly between coordinates AP = -1.2 and AP = -3.4.

[0102] Neuronal layer thickness measurement

[0103] Two brain sections (30 μm thick, 300 μm apart) were immunofluorescently stained as described above using the primary antibody NeuN (1:500) to visualize the neuronal cell layer of the hippocampus. The sections were imaged at 20× magnification using a FV3000 confocal laser scanning microscope (Olympus). The thickness of the CA1 pyramidal cell layer and the dentate gyrus granule cell layer of the hippocampus were measured on the Fiji (ImageJ) software by drawing a straight line perpendicular to the NeuN+ cell layer at two points in each hippocampal sub-region and taking the average value of each mouse.

[0104] Measurement of nuclear-cytoplasmic localization of HMGB1

[0105] As described above, two brain sections (30 μm thick, 300 μm apart) were immunostained with anti-HMGB1 (1:100) and DAPI (1:50,000). The sections were imaged at 40× and 60× magnification using a FV3000 confocal laser scanning microscope (Olympus). All image processing and quantification were performed on Fiji (ImageJ) software. Briefly, a 1-pixel median filter was applied to the DAPI channel and an appropriate threshold was set to create a mask for DAPI. The image calculator function was then used to overlay the DAPI mask and the HMGB1 channel, which provided HMGB1 staining that was localized only to the nucleus. After obtaining the values ​​for the integrated density and particles, the DAPI mask was subtracted from the HMGB1 using the image calculator, which provided HMGB1 staining that was excluded from the nucleus.

[0106] Biochemical extraction of brain tissue

[0107] The mouse hemibrain of quick freezing is thawed on ice and then the dissection of hippocampus is performed.Hippocampal tissue is weighed and homogenized with 10 μL / mg tissues using Polytron immersion disperser Polytron homogenizer (Kinematica AG) in ice-cold RAB buffer (GBiosciences), supplemented with phosphatase inhibitors (Roche) and protease inhibitors (Roche).Then Optima TLX ultracentrifuge (Beckman Coulter) is used to centrifuge the sample for 20 minutes at 4 ℃ with 50,000g, and collect supernatant as RAB soluble fraction.Precipitation is resuspended in ice-cold RIPA buffer (Thermo Scientific) with 10 μL / mg tissues and centrifuged for 20 minutes at 4 ℃ with 50,000g.Supernatant is collected as RIPA soluble fraction and precipitation is stored at -80 ℃ for further use.All fractions are stored at -80 ℃ until further analysis.

[0108] Western blot analysis

[0109] Biochemically extracted mouse hippocampal tissue lysates were loaded onto 12% Bis-Tris SDS-PAGE gels (Invitrogen) and separated by gel electrophoresis at 160 V using MOPS buffer. The separated proteins were transferred to a nitrocellulose membrane at 18 V for 60 min (Trans-Blot Turbo Transfer System (Bio-rad). The membrane was washed 3×5 min in PBS-T and then incubated in Intercept blocking buffer (LI-COR) at room temperature for 1 h to block nonspecific binding sites. After blocking, the membrane was washed 3×5 min in PBS-T and incubated with primary antibodies overnight at 4°C (AT8 1:3,000 (Invitrogen), TUJ1 1:15,000 (Biolegend)). The membrane was washed 3×5 min in PBS-T and incubated in fluorescently labeled secondary antibodies (1:20,000; LI-COR) for 1 h in the dark at room temperature. The resulting bands were detected with the Odyssey CLx infrared imaging system (LI-COR), and the fluorescence intensity of the bands was quantified as the ratio of AT8:TUJ1 signals using Image Studio software.

[0110] Sandwich ELISA

[0111] Biochemically extracted mouse hippocampal tissue lysates were diluted in Milli-Q H2O to the appropriate concentration and run according to the provided manufacturer's protocol (human APOE (Abcam); mouse HMGB1 (Novus Biologicals)). The sample reactions were read on a SpectraMaX M5 spectrophotometer (Molecular Devices) and the protein concentration was determined after interpolation of the standard curve and adjustment of the dilutions.

[0112] Primary neuronal cultures and recombinant protein treatment

[0113] Primary cultures of neurons were prepared from prenatal E20 pups of various genotypes. After collecting pup brains, the cortex and hippocampus were isolated and placed in ice-cold dissociation medium + kyneurenic acid media (DM / KY) (DM: Na2SO4 (81.8mM); K2SO4 (30mM); MgCl2 (5.8mM), CaCl2 (0.25mM); HEPES (1mM); glucose (20mM); phenol red (0.001%); NaOH (0.16mM) in distilled H2O) (KY: kynurenic acid (10mM); phenol red (0.0025%); HEPES (5mM); MgCl2 (100mM); NaOH (dropwise added until pH 7.4)). The resulting DM / KY medium was made by combining 90% DM medium with 10% KY medium. The isolated tissue was finely minced and then immersed in preheated papain solution (1 mL / brain) for 13 minutes while gently inverting, and then immersed in trypsin inhibitor solution for 5 minutes (5 mL for up to 10 brains) while gently inverting. The tissue pellet was washed with Optimem / glucose solution (20 mM glucose, 1 mL / brain) while gently inverting. Then, fresh Optimem / glucose solution was added and the tissue was gently ground until it was separated into single cells. After filtering the cells through a 40 μm cell strainer, the dissociated cells were separated at 1×10 6 cells / well of a 12-well plate or 3×10 5 Cells / well of a 24-well plate were plated in a 100U / mL medium supplemented with B27. -1 Penicillin G, 100 μg / mL -1 Streptomycin and 1% GlutaMAX in Neurobasal medium (B27 / Neurobasal). Every 3 to 4 days, half of the culture medium was removed and replaced with fresh B27 / Neurobasal medium. In some experiments, primary neurons were treated in vitro for 24 hours with Dulbecco PBS (dPBS) vehicle or target recombinant protein (10 μg / mL in one well of a 12-well plate) on the 14th day. After treatment, the culture medium was collected and the culture was harvested for analysis. The total protein level present in the cell lysate was obtained by BCA analysis (Pierce).

[0114] Stereotaxic surgery in mice

[0115] Mice were anesthetized with intraperitoneal injection of ketamine (60 mg / kg) and xylazine (30 mg / kg) and maintained at 0.8% to 1.0% isoflurane (Henry Schein). Mice were fixed in a stereotaxic alignment system model 940 using ear bars and tooth bars (Kopf Instruments). The scalp was prepared by removing hair using Nair and sterilizing with 70% ethanol. The scalp was then incised using a scalpel and sterilized with 70% ethanol. 3% hydrogen peroxide was used to better visualize the cranial sutures. After identifying the bregma, a 0.5 mm microburr (Fine Science Tools) was used to drill the unilateral stereotaxic site using coordinates X=+1.5, Y=-2.1, Z=-2.1 (where Z is measured from the surface of the brain). Mice were injected with 2 μL of the corresponding virus (AAV2 (Y444F) -smCBA-human_P301S_Tau-WPRE, 2.10E+13 vg / mL, Virovek; AAV2-synaptophysin-GFP, 1.0E+13 vg / mL, SignaGen) or interstitial fluid (ISF) fraction at a flow rate of 500 nL / min and allowed to diffuse for 3 minutes. After surgery, mice were sutured with nylon monofilament non-absorbable 6-0 sutures (Henry Schein) and analgesics buprenorphine (intraperitoneal 0.0375 mg / kg), ketophen (subcutaneous 5 mg / kg) and saline (intraperitoneal 500 μL). Mice were monitored on a heating pad until they were ambulatory and provided with hydrogel for hydration.

[0116] Brain slice electrophysiological recording and data analysis

[0117] For electrophysiological recording studies, 8-month-old PS19-fE3 mice and PS19-fE4 mice without Cre, with Syn1-Cre or with GFAP-Cre were anesthetized with isoflurane and decapitated. The brain was quickly removed from the skull and placed in an ice-cold (2 to 5 ° C) slice solution. The slice solution contained (in mM): 110 choline chloride, 2.5 KCl, 26 NaHCO3, 10 MgCl2, 1.25 NaH2PO4, 0.5 CaCl2, 10 glucose, 3 pyruvic acid Na, 1 L-ascorbic acid, pH 7.4. 350 μm thick sagittal sections were cut from both hemispheres using a vibratome (VT1200, Leica) and transferred to a 95% O2-CO2 vapor interface holding chamber (BSK5, Scientific Systems Design) containing artificial cerebrospinal fluid (ACSF), where they were allowed to recover for one hour at 34°C and then kept at room temperature (20 to 22°C). ACSF contained (in mM): 126 NaCl, 2.5 KCl, 1.5 CaCl2, 1.5 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, and 1.5 L-ascorbic acid, pH 7.4.

[0118] For input / output recording studies, a concentric bipolar stimulating electrode (FHC) connected to a constant voltage separation and placed in the CA2 stratum radiatum was used to induce local field postsynaptic potentials (fPSPs) by stimulating Schaffer collaterals in the forward direction. fPSPs were recorded with a glass borosilicate microelectrode filled with ACSF and placed in the CA1 stratum radiatum. The signal was collected and digitized by a MultiClamp 700B amplifier and a Digidata 1550B1 acquisition system (Molecular Devices) with pClamp10 software, and analyzed by running a custom macro using IgorPro6 software (Wavemetrics). The fPSP slope was analyzed as a linear fit slope value of 10% to 90% of the fPSP peak. The input-output relationship was recorded as the fPSP slope value in response to the increased stimulation intensity (20 to 60 μA), and the fPSP slope gain was calculated as the linear slope of the resulting input-output curve.

[0119] Microdialysis of the mouse hippocampus

[0120] In vivo microdialysis of hippocampus is used to collect brain tissue interstitial fluid.Surgical operation (including preoperative and postoperative care) is performed for stereotaxic surgery as described above.During the operation, unilateral stereotaxic site is drilled with 1.2mm bone drill (BASi), and AtmosLM guide cannula PEG-4 (Amuza) is stereotaxically implanted above the right hippocampus at coordinates X=+1.5, Y=-2.1, Z=-1.1. Dental cement (GC America) is used to fix the cannula in place, and a temporary PEG-4AtmosLM dummy probe (dummy probe) (Amuza) is inserted and fixed with an AC-5 cap nut screw (Eicom). Two days after the operation, mice are placed in a microdialysis independent system (BASi) overnight to assimilate, and the 1000kDaAtmosLM collection probe (Eicom) is inserted into the hippocampus by the guide cannula in the afternoon of the second day, which further extends 1mm downward to Z=-2.1 to target the dentate gyrus. Artificial CSF (Harvard Apparatus) prepared with 0.15% BSA (Thermo Scientific) was circulated through the system at a flow rate of 0.5 μL / min using a push-pull method, and interstitial fluid (ISF) was collected in a refrigerated fraction collector (BASI) every hour for approximately 24 hours. In order to prevent the clogging of the tube, the pump was operated at a 10× collection speed for the first two hours before the pump was adjusted to a flow rate of 0.5 μL / min. After completing the ISF collection, mice were euthanized as described above and perfused with 0.9% saline. The brain was dissected into hemispheres, the right hemisphere was post-fixed in 4% PFA for 48 hours and the left hemisphere was freshly frozen. The interstitial fluid (ISF) fraction was frozen at -80°C for further analysis.

[0121] Treatment with HMGB1 inhibitors

[0122] At 6.5 months of age, male and female PS19-fE4 and PS19-fE3 mice were randomly assigned to a control group or a treatment group. Mice were injected intraperitoneally with a mixture of sterile 0.9% saline (Fisher Scientific) or the following HMGB1 inhibitors: ethyl pyruvate (80mg / kg) (Sigma-Aldrich) and glycyrrhizic acid (20mg / kg) (Sigma-Aldrich) dissolved in 0.9% saline. Mice were injected three times a week for 12 weeks, starting at 6.5 months of age until they arrived at 9.5 months of age. The weight changes, licking changes and postures of all mice were monitored during the experiment and no changes were observed. After treatment, animals were perfused and their brain tissues were processed for histopathological analysis as described above.

[0123] 10× loaded single nucleus preparation

[0124] Mouse hippocampi were dissected on ice and placed in a pre-cooled 2 mL Dounce containing 1 mL of cold 1× homogenization buffer (1×HB) (250 mM sucrose, 25 mM KCL, 5 mM MgCl2, 20 mM Tricine-KOH pH7.8, 1 mM DTT, 0.5 mM Sermidine, 0.15 mM Sermine, 0.3% NP40, 0.2 units / μL RNase inhibitor, 0.2 units / μL protease inhibitor). Pestle loosely with Dounce "A" (about 10 pestles) and then tightly with "B" (about 15 pestles). The homogenate was filtered using a 70 μM Flowmi filter (Eppendorf) and transferred to a pre-cooled 2 mL LoBind tube (Fischer Scientific). The nuclei were precipitated by centrifugation at 350 RCF for 5 minutes at 4°C. The supernatant was removed and the nuclei were resuspended in 400 μL of 1×HB. Next, 400 μL of 50% iodixanol solution was added to the nuclei and then slowly layered with 600 μL of 30% iodixanol solution at 25% mixture, and then layered with 600 μL of 40% iodixanol solution at 30% mixture. The nuclei were then centrifuged at 3,000 g for 20 minutes at 4°C in a pre-cooled swing bucket centrifuge. The 200 μL nuclear band at the 30% to 40% interface was collected and transferred to a new tube. Then, 800 μL of 2.5% BSA in PBS plus 0.2 units / μL RNase inhibitor was added to the nuclei and then centrifuged at 500 RCF for 10 minutes at 4°C. The nuclei were resuspended with 2% BSA in PBS plus 0.2 units / μL RNase inhibitor to reach approximately 500 nuclei / μL. The nuclei were then filtered with a 40 μM Flowmi filter. Nuclei were counted and approximately 13,000 nuclei / sample were subsequently loaded onto a 10× Genomics Next GEM Chip G. snRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 (10× Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer at the UCSF CAT Core.

[0125] Custom reference genomes

[0126] The PS19 tau mutant floxed APOE knock-in mouse model (Bien-Ly et al., J. Neurosci. 32, 4803-4811 (2012)) was used for single-nucleus RNA sequencing (snRNA-seq). Homo sapiens microtubule associated protein tau (MAPT) (NCBI reference sequence: NM_001123066.4) (Agarwala et al. Nucleic Acids Res. 44, D7-D19 (2016)) and Homo sapiens APOE were the target genes of this study. These genes are not expected to be part of the mouse reference genome, so to quantify reads aligned to these target genes, a custom mouse reference genome was made using the following: a reference mouse genome sequence (GRCm38) from Ensembl (Release 98) (Howe et al. Ensembl 2021. Nucleic Acids Res. 49, D884-D891 (2021)) and a mouse gene annotation file from GENCODE (Release M23) (Frankish et al. GENCODE 2021. Nucleic Acids Res. 49, D916-D923 (2021)), similar to those used in the 10× Genomics Cell Ranger mouse reference package mm10 2020-A. The header of the Ensembl reference mouse genome sequence fasta file with chromosome names was modified to match the chromosome names in the fasta file from GENCODE. The annotation GTF file contains entries from non-polyA transcripts overlapping with protein-coding genes. These reads were marked as multiply mapped and not counted by the 10×Genomics Cell Ranger v6.1.1 counting process (Zheng et al.Nat.Commun.8,14049(2017)). To avoid this, the GTF file was modified to (1) remove the version suffix from the transcript, gene, and exon ids to match the Cell Ranger reference package, and (2) remove non-polyA transcripts. The Homo sapiens MAPT sequence and Homo sapiens APOE sequence were appended to the end of the mouse reference genome sequence as separate chromosomes and the corresponding gene annotations were appended to the filtered mouse reference gene annotation GTF file. The modified fasta file and GTF file were used to create a custom reference genome using the 10×Genomics Cell Ranger v6.1.1mkref process.

[0127] Preprocessing and clustering of mouse snRNA-seq samples

[0128] The snRNA-seq sample includes a total of 16 samples, wherein four genotype groups (PS19-fE4, PS19-fE4Syn1-Cre, PS19-fE4GFAP-Cre and PS19-fE3) each have four mice. There are two male mice and two female mice in each group of four mice. As described in the Cell Ranger document, the demultiplexing fastq files of these samples are compared with the custom mouse reference genome (see the other description of the custom reference genome method) using 10 × Genomics Cell Rangerv6.1.1 counting process (Zheng et al.Nat.Commun.8,14049(2017)).The flag comprising introns of the counting process is set to true (true) to count the reads mapped to the intron region. The Cell Ranger counting website summary shows the "Low Fraction Reads in Cells" error of two samples-one from the PS19-fE4 GFAP-Cre group and one from the PS19-fE3 group. These two samples have only about 40% of the reads assigned to the cell-related barcode and <80% of the reads mapped to the genome. For the other 14 samples, these metrics are much higher. Checking the experimental records shows that these two samples have problems in the nuclear separation step, and due to the use of expired old batch sample preparation reagents, lower cDNA is recovered. All other 14 samples are prepared with new batches of sample preparation reagents. Therefore, these two samples are excluded and only the remaining 14 samples are used for the downstream analysis performed with Seurat.

[0129] The filtered count matrices generated by the Cell Ranger counting process for 14 samples were processed using the R package for single-cell nuclear analysis Seurat v4.0.5 (Hao et al. Cell 184, 3573-3587.e29 (2021)). Each sample was preprocessed into a Seurat object, and the top 1% cells of each sample with a large number of unique genes, cells with <= 200 unique genes, and cells with >= 0.25% mitochondrial genes were filtered out for each sample. The 14 samples were merged into a single Seurat object and normalized and variance stabilized using sctransform (Hafemeister & Satija, Genome Biol. 20, 296 (2019)) with the "glmGamPoi" (Bioconductor package version 1.6.0) method (Ahlmann-Eltze & Huber, Bioinformatics 36, 5701-5702 (2021)) for initial parameter estimation.

[0130] Graph-based clustering was performed using the Seurat v4.0.5 functions FindNeighbors and FindClusters. First, cells were built into a k-nearest neighbor (KNN) graph based on the Euclidean distance in PCA space. The edge weights between two cells were further modified using Jaccard similarity. Next, clustering was performed using the Louvain algorithm implementation in the FindClusters Seurat function. All combinations of 10, 15, and 20 PCs with 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 resolutions were clustered. Clustering with 15 PCs and 0.7 resolution produced 33 different biologically relevant clusters, which were used for further analysis.

[0131] Cell type assignment

[0132] Data visualization of 14 samples in UMAP space using Seurat v4.0.5 shows that there is no batch effect for age, sex, genotype, date of birth or nuclear separation date. The FindAllMarkers Seurat function is used to identify marker genes for each cluster for SCT assay data. The algorithm uses the Wilcoxon rank sum test to iteratively identify differentially expressed genes of a cluster for all other clusters. The marker genes are filtered to retain only positively expressed genes, which are detected in at least 25% of cells in any population and have at least 0.5log2 fold changes. Cell clusters are assigned identities by matching cell clusters with known cell types, and the known cell types have expression of typical cell type-specific genes, expression of genes identified in publicly available mouse hippocampal single-cell RNA-seq data sets, and expression of marker genes for each cluster in publicly available whole-brain in situ hybridization image resources, as reported previously by us (Zalocusky et al., Nat. Neurosci. 24, 786-798 (2021)).

[0133] Subclustering of astrocyte and microglia sn-RNA-seq data

[0134] Annotate hippocampal cell cluster 10 as astrocytes and hippocampal cell clusters 11, 21, and 29 as microglia. Both cell types are sub-clustered. Use sctransform 82 Normalization and variance stabilization were performed using the “glmGamPoi” (Bioconductor package version 1.6.0) method (Ahlmann-Eltze & Huber, Bioinformatics 36, 5701-5702 (2021)) for initial parameter estimation. Graph-based clustering was performed using the Seurat v4.0.5 functions FindNeighbors and FindClusters. First, cells were built into a k-nearest neighbor (KNN) graph based on the Euclidean distance in PCA space. The edge weights between two cells were further modified using Jaccard similarity. Next, clustering was performed using the Louvain algorithm implemented in the FindClusters Seurat function. All combinations of 10, 15, 20, 25, and 30 PCs with resolutions of 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 were clustered. Subclustering with 15 PCs and 0.9 resolution produced 18 different biologically relevant astrocyte subclusters. Subclustering with 15 PCs and 0.9 resolution yielded 18 distinct biologically relevant microglial subclusters.

[0135] Gene set enrichment analysis

[0136] The FindMarkers Seurat function was used to identify differentially expressed genes between the target clusters for SCT assay data. The algorithm used the Wilcoxon rank sum test to identify differentially expressed genes between two populations. Differentially expressed genes were limited to genes detected in at least 10% of cells in either population and had at least a 0.1 log2 fold change. ClusterProfiler v4.2.1 (Wu et al. Innov. (NY) 2, 100141 (2021)) was used for overexpression (or enrichment) analysis to find gene sets of mice associated with differentially expressed genes in the KEGG database (Kanehisa et al. Nucleic Acids Res. 44, D457-D462 (2016)). The p value was adjusted for multiple testing based on the hypergeometric test and using the Benjamini-Hochberg method (Benjamini et al., JR Stat. Soc. Ser. B Stat. Methodol. 57, 289-300 (1995)). Significantly enriched gene sets were filtered to have an adjusted p-value less than 0.8 and at least 10 differentially expressed genes in the gene set.Gene set enrichment analysis of astrocyte subclusters and microglia subclusters was performed using the same method.

[0137] Correlation between clusters and genotypes

[0138] A generalized linear mixed effects model (GLMM_AM) was implemented in the lme4 (v1.1-27.1) R package (Bates et al., J.Stat.Soft.67, 1-48 (2015)) to assess the correlation with the animal model, and the generalized linear mixed effects model was used to estimate the correlation between cluster members and mouse models. These models were run for each cell cluster separately. The GLM model was run when the family parameter was set to a binomial probability distribution and the bobyqa control optimizer was used for maximum likelihood estimation. Depending on whether the cell belongs to the cluster under consideration, the cluster member of each cell was simulated as a 0-1 response variable. The corresponding mouse id from which the cell was derived was a random effect variable, and the animal model of the mouse id was included as a fixed variable. The reference animal model was set to PS19fE4. The p-values ​​of the log odds ratios estimated in the three animal models (relative to PS19 fE4) and clusters were adjusted for multiple testing using the Benjamini-Hochberg method (Benjamini et al., JR Stat. Soc. Ser. B Stat. Methodol. 57, 289-300 (1995)). The same method was used to estimate the inter-cluster correlation of the genotypes with astrocyte subcluster and microglia subcluster.

[0139] Correlation between the proportions of cell types and histopathological parameters

[0140] Generalized linear mixed effects models (GLMM_histopathology) were implemented in the lme4 (v1.1-27.1) R package (Bates et al., J. Stat. Soft. 67, 1-48 (2015)) to assess the association with histopathology and were used to identify cell types whose proportions were significantly associated with histopathological changes in the samples. These models were run separately for each cell cluster combination and the following eight histological parameters: hippocampal volume (mm 3), AT8 coverage area percentage, GFAP coverage area percentage, S100β coverage area percentage, IBA1 coverage area percentage, CD68 coverage area percentage, MBP coverage area percentage, and oligodendrocyte progenitor cell (OPC) coverage area percentage. GLM model is performed when the family parameter is set to the binomial probability distribution family and the 'bobyqa' control optimizer is used for maximum likelihood estimation. Depending on whether the cell belongs to the cluster under consideration, the cluster member of each cell is simulated as a 0-1 response variable. The corresponding mouse model from which the cell is derived is included as a random effect, and the mouse id in a given mouse model is also further simulated as a random effect. Note that this represents the hierarchical nature of this data for GLMM, and first assumes that the mouse model is sampled from a "universal" mouse model, and then samples mice in each mouse model. In contrast to fixed effects, the modeling selection of including the mouse model as a random effect means increasing the degrees of freedom (or maximizing statistical power) to detect the correlation of purpose, especially considering the relatively small amount of repetitions (3 to 4 times) of each animal model. The considered histological parameters were simulated as fixed effects in the model.

[0141] After adjusting the p-value distribution in the histopathological parameters across cell types with Benjamini-Hochberg multiple testing correction (Benjamini et al., J RStat.Soc.Ser.B Stat.Methodol.57,289-300 (1995)), a subset of the target cell types was selected and the log odds ratio estimates (derived from the GLMM fit) were visualized in a heat map (Fig. 4d) using the pheatmap package 1.0.12. The process was applied to astrocyte subtypes and microglia subtypes and the correlation between the target astrocyte subtype and microglia subtype and the eight histopathological parameters in Fig. 4h and Fig. 4i were visualized respectively. The first five principal component coordinates were estimated using eight log odds ratios of unit changes in the histopathological parameters of each cell type and the target astrocyte and microglia subtypes. This was implemented using prcomp (scale = T, center = T) in the stats R package. We visualized the first two PCs using fviz_pca_ind() implemented in the factoextra 1.0.7R package.

[0142] Mouse snRNA-seq dataset from GEO:GSE164507

[0143] Mouse snRNAseq data (accession number GSE164507) available on the Gene Expression Omnibus database at ncbi.nlm.nih.gov / geo were reanalyzed. Download publicly available data sets, which for each sample contain a filtered gene matrix based on cell expression, a file with a barcode, and a file with expressed genes. Full details of the study are available in the original publication (Wang et al., Neuron 109, 1657-1674.e7 (2021)). In short, the study detected P301S mutant Tau transgenic mice carrying floxed APOE-ε4 or APOE-ε3 alleles. These mice were hybridized with mice expressing Cre recombinase under the regulation of tamoxifen-inducible ER elements and Aldh1l1 astrocyte-specific promoters. At 5.5 months of age after the onset of tau pathology, these Aldh1l1-CreERT2 mice were administered tamoxifen to induce Cre recombinase expression, or vehicle was administered. Single nuclei isolated from the hippocampus of these mice were sequenced using 10×Genomics Chromium single-cell sequencing, and the data were processed using the Cell Ranger single-cell software suite (v3.0.2). The filtered count matrices generated by the Cell Ranger counting process for all 8 samples were processed using Seurat v4.0.4 (Hao et al. Cell 184:3573-3587.e29 (2021)). The samples were filtered to include only cells with 500 to 2,000 genes detected and <5% mitochondrial reads. The filtered samples were merged into a single Seurat object, which contained a matrix of 33,457 genes by 63,248 nuclei. Normalization and variance stabilization were performed using sctransform (Hafemeister & Satija, Genome Biol. 20, 296 (2019)). Clustering was determined as implemented in Seurat v4.0.4 using the RunPCA(), FindNeighbors(), and FindClusters() functions. The nearest neighbor distances were calculated using up to the first 15 PCs. The algorithm builds cells into a k-nearest neighbor graph based on the Euclidean distance in PCA space. The edge weights between any two cells were further refined using Jaccard similarity. Clustering was performed using the Louvain algorithm with default settings and a resolution of 0.7, resulting in a set of 22 different clusters.Differential gene expression was detected using the Wilcoxon rank sum test in Seurat, implemented by the FindMarkers() function with min.pct=0.1, test.use="wilcox", and logfc.threshold=0.05.

[0144] General statistical analysis

[0145] The differences between the genotype groups were evaluated by common one-way ANOVA and Tukey's multiple comparison test afterwards, wherein the mean value of each column was compared with the mean value of every other column. All drawn data are shown as mean ± SEM. Simple linear regression was used to analyze the correlation between two data in the same genotype group and plotted as mean ± SEM. GraphPad Prism version 9.2.0 was used to analyze and create a graph.

[0146] Example 2: Neuron-specific or astrocyte-specific ablation of the APOE gene in human APOE knock-in mice expressing human mutant Tau

[0147] The present inventors and colleagues have previously generated mouse strains expressing the following: floxed human APOE3 or APOE4 genes (Bien-Ly et al., J. Neurosci. 32, 4803-4811 (2012)) and the Cre recombinase gene under the control of the neuron-specific synapsin-1 promoter (Syn1-Cre) (Zhu et al. Genes Dev. 15, 859-876 (2001)) or the Cre recombinase gene under the control of the astrocyte-specific glial fibrillary acidic protein promoter (GFAP-Cre) (Bajenaru et al., Mol. Cell Biol. 22, 5100-5113 (2002); Uhlmann et al., Ann. Neurol. 52, 285-296 (2002)).

[0148] These floxed APOE-KI (fE) mice express homozygous people APOE3 or APOE4 instead of endogenous mouse Apoe.People APOE gene is flanked by a pair of LoxP sites, to allow accurate excision in the presence of cell type-specific Cre recombinase expression (Knoferle et al., J.Neurosci.34, 14069-14078 (2014)).The fE mice without Cre, with Syn1-Cre or with GFAP-Cre and the mice (PS19 strain) (Yoshiyama et al., Neuron 53, 337-351 (2007)) of mutant 1N4R people microtubule-associated protein Tau (MAPT) that has been widely used as tau disease mouse model are hybridized. The resulting composite mice were referred to as PS19-fE, PS19-fE / Syn1-Cre, or PS19-fE / GFAP-Cre mice.

[0149] fE / Syn1-Cre and fE / GFAP-Cre mice are strictly characterized to verify the specificity of Cre recombinase expression under neuron-specific Syn1 or astrocyte-specific GFAP promoter (Knoferle et al. J. Neurosci. 34, 14069-14078 (2014)). PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice are further verified by immunohistochemical analysis of brain slices from 10-month-old PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice. In order to confirm the specificity of Cre recombinase expression in hippocampal neurons and astrocytes when driven under Syn1 or GFAP promoters, NeuN and GFAP antibodies are used together with Cre recombinase antibodies for co-immunostaining. In PS19-fE4 / Syn1-Cre mice, Cre recombinase is expressed only in NeuN positive neurons and not in GFAP positive astrocytes. In PS19-fE4 / GFAP-Cre mice, Cre recombinase is expressed only in GFAP-positive astrocytes and not in NeuN-positive neurons. Co-immunostaining using antibodies against APOE, GFAP, and NeuN illustrates that PS19-fE4 mice without Cre have APOE expression in GFAP-positive astrocytes and some NeuN-positive neurons, while PS19-fE4 / Syn1-Cre mice have APOE expression in astrocytes and lack APOE expression in neurons, and PS19-fE4 / GFAP-Cre mice have APOE expression in some neurons and lack APOE expression in astrocytes. PS19-fE4 / GFAP-Cre mice exhibit some APOE-positive cells that are negative for NeuN and GFAP but positive for the microglial marker Iba1, indicating that they are microglia expressing APOE.

[0150] To quantitatively determine the levels of APOE protein within these various mouse models, hippocampal lysates from 10-month-old mice were analyzed by sandwich ELISA against human APOE. PS19-fE4 / Syn1-Cre mice showed approximately 20% reduction in APOE levels relative to PS19-fE4 mice ( Figure 1A), which is consistent with previous data showing that neuronal APOE contributes approximately 20% to 30% of total APOE protein levels in the hippocampus and cortex. PS19-fE4 / GFAP-Cre mice exhibited approximately 70% reduction in APOE levels relative to PS19-fE4 mice, consistent with the recognized role of astrocytes as the major producers of APOE within the CNS. Similarly, PS19-fE3 / Syn1-Cre mice exhibited approximately 25% reduction in APOE levels relative to PS19-fE3 mice and PS19-fE3 / GFAP-Cre exhibited approximately 67% reduction in APOE levels relative to PS19-fE3 mice ( Figure 1A Together, these results provide strong evidence that APOE gene expression is abolished in neurons or astrocytes when Cre recombinase expression is driven under the Syn1 or GFAP promoter, respectively, in these composite mouse models.

[0151] Example 3: Removal of APOE4 from neurons but not astrocytes significantly reduces Tau pathology

[0152] To determine whether the removal of neuronal APOE or astrocyte APOE affects Tau pathology, the inventors evaluated 10-month-old mice (Yoshiyama et al., Neuron 53, 337-351 (2007)) when PS19 mice exhibit extensive Tau pathology throughout the hippocampus. The accumulation of pTau in the hippocampus was analyzed by immunohistochemical staining with a pTau-specific AT8 antibody and Tau pathology was quantified as the percentage of AT8 coverage in the hippocampus.

[0153] PS19-fE4 mice exhibited extensive Tau pathology throughout the hippocampus and the extent of Tau pathology was significantly lower in PS19-fE3 mice ( Figures 1B to 1C ), as previously reported (Shi et al., Nature 549, 523-527 (2017)). Relative to PS19-fE4 mice, PS19-fE4 / Syn1-Cre mice showed a significant reduction of about 81% in Tau pathology, while PS19-fE4 / GFAP-Cre mice had a slight reduction of about 30%, which did not reach statistical significance. Interestingly, the reduction in pTau coverage in PS19-fE4 / Syn1-Cre mice was similar to the extent of Tau pathology observed in PS19-fE3 mice ( Figures 1B to 1C There was no significant difference in Tau pathology between PS19-fE3 with Cre and without Cre, probably because Tau pathology in PS19-fE3 mice was already low.

[0154] Western blot analysis was also used to assess pTau levels in mouse hippocampal tissue after sequential biochemical extraction with RAB and RIPA buffers containing highly soluble and less soluble Tau proteins, respectively (Shi et al., Nature 549, 523-527 (2017); Ishihara et al., Neuron 24, 751-762 (1999)) ( Figures 1D to 1G In the RAB fraction, there was no significant difference in pTau levels between the various genotype groups ( Figure 1D , 1F However, PS19-fE4 / Syn1-Cre and PS19-fE3 mice showed significantly reduced pTau levels in RIPA fractions relative to PS19-fE4 mice ( Figure 1E , 1G ). PS19-fE4 / GFAP-Cre mice showed a trend of about 40% reduction in pTau levels in RIPA fractions relative to PS19-fE4 mice, which did not reach statistical significance. In summary, these data indicate that neuronal APOE4 expression is a strong driver of Tau pathology, while astrocyte APOE4 plays a minimal role in Tau pathology.

[0155] Example 4: Propagation of Tau pathology is reduced following APOE4 removal in neurons but not in astrocytes

[0156] To further investigate the mechanism by which neuronal APOE4 drives Tau pathology, the effect of cell type-specific APOE4 expression on the spread of Tau pathology was determined. Previous studies have shown that after various forms of Tau protein were directly injected into the mouse brain, pathological Tau can spread to anatomically connected brain regions (Boluda et al., Acta Neuropathol. 129, 221-237 (2015); Rauch et al., Nature. 580, 381-385 (2020); Guo et al., J. Exp. Med. 213, 2635-2654 (2016); Kaufman et al., Neuron 92, 796-812 (2016)).

[0157] The extent of Tau spread was analyzed after unilateral injection of adeno-associated virus-2 encoding human P301S mutant Tau (AAV2-Tau-P301S) into the right dorsal hippocampus of fE mice without Cre, with Syn1-Cre, or with GFAP-Cre ( Figures 1H to 1IfE mice lack human P301S mutant Tau and instead express endogenous mouse Mapt genes, allowing for more accurate detection of human Tau transmission because fE mice exhibit minimal Tau pathology. Mice were injected with AAV2-Tau-P301S virus at 10 months of age and evaluated at 13 months of age, 12 weeks after injection ( Figure 1H ).

[0158] To provide evidence that the observed Tau propagation phenotype could truly be attributed to the spread of pathological human Tau between neurons, as opposed to the unexpected result that the virus encoding Tau itself moved to the non-injected hippocampus, AAV2 encoding GFP of the same serotype (AAV2-GFP) injected unilaterally into the right dorsal hippocampus of 10-month-old fE4 mice was evaluated. Immunostaining with anti-GFP two weeks after injection showed that the GFP signal remained localized to neurons within the injected hippocampus ( Figure 1J ). The uninjected hippocampus had no evidence of any GFP signal in neuronal somata, although there were some GFP-positive neuronal projections that may have originated from neurons present on the injected hippocampus side. These results show that AAV2 itself does not spread between the right and left hippocampus after unilateral injection. In addition, for all immunohistochemical analyses, the number of neurons containing soma-positive Tau was quantified to more accurately reflect the spread of human mutant Tau between neurons and exclude confounding factors (e.g., Tau-positive commissural fibers from neurons originating from the injected side).

[0159] Immunohistochemical staining with human Tau antibody (HT7) showed robust spread of human Tau to the uninjected hippocampus in PS19-fE4 mice and minimal Tau spread in PS19-fE3 mice ( Figures 1K to 1L ), indicating that APOE4 promotes Tau propagation. Interestingly, PS19-fE4 / Syn1-Cre mice had limited propagation of human Tau to the uninjected hippocampus, whereas PS19-fE4 / GFAP-Cre mice had extensive human Tau propagation, similar to the propagation of Tau pathology observed in PS19-fE4 mice ( Figures 1K to 1L ). Notably, after removal of neuronal APOE4, HT7 cells with Tau propagation + The number of cells was reduced by 55%, and after astrocyte APOE4 removal, HT7 cells with Tau propagation + There were no discernible differences in the number of cells ( Figures 1K to 1N ).

[0160] The extent of Tau propagation was further analyzed by immunostaining of pTau with AT8 antibody. Both PS19-fE4 and PS19-fE4 / GFAP-Cre mice showed robust propagation of pTau to the uninjected side of the hippocampus, whereas pTau propagation was significantly reduced in PS19-fE4 / Syn1-Cre and PS19-fE3 mice ( Figures 1M to 1N ).

[0161] Together, these data suggest that one mechanism by which neuronal APOE4 drives Tau pathology is by stimulating the propagation of pathological Tau across synaptically connected neurons. The data also further support the view that astrocytic APOE4 is not a key mediator of Tau pathology, as demonstrated by its lack of effect on Tau propagation.

[0162] Example 5: Network hyperexcitability is eliminated after removal of neuronal APOE4 but not astrocyte APOE4

[0163] To determine the cell type-specific effects of APOE4 on neuronal function in the context of tauopathy, neuronal network excitability was measured in the angular angular lateral ... Figure 2A ) 54 .

[0164] Compared with PS19-fE3 mice, PS19-fE4 mice had significant CA1 neuronal hyperexcitability ( Figure 2B ). Removal of neuronal APOE4 eliminated neuronal network hyperexcitability, whereas removal of astroglial APOE4 resulted in a slight reduction that did not reach significance ( Figure 2B ), suggesting that neuronal APOE4 drives neuronal dysfunction in the context of tauopathy.

[0165] Example 6: Neurodegeneration is reduced following removal of neuronal APOE4 or astrocyte APOE4

[0166] The extent of neurodegeneration in 10-month-old PS19-fE mice was evaluated after APOE was removed from neurons or astrocytes. Analysis of hippocampal volume and posterior lateral ventricle volume showed that PS19-fE4 mice exhibited extensive neurodegeneration relative to PS19-fE3 mice ( Figures 3A to 3CNeurodegeneration was significantly reduced in both PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice, as indicated by increased hippocampal volume and decreased posterior ventricular volume ( Figures 3A to 3C ), similar to those in PS19-fE3 mice. Removal of neuronal APOE3 or astroglial APOE3 did not significantly affect neurodegeneration ( Figures 3A to 3C ).

[0167] Quantification of the extent of neuronal cell loss in multiple subregions of the hippocampus showed that PS19-fE4 mice had extensive neuronal loss in the CA1 and dentate gyrus, whereas both PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice had significantly reduced neuronal cell loss in both the CA1 and dentate gyrus subregions of the hippocampus ( Figures 3D to 3G When APOE3 was removed from neurons or glial cells, there was no significant difference in neuronal cell layer thickness in PS19-fE3 mice ( Figures 3D to 3G ).

[0168] In PS19-fE4 mice, there was a weak but significant negative correlation between Tau pathology and hippocampal volume ( Figure 3H ), suggesting that Tau pathology contributes, at least in part, to neurodegeneration in these mice. There was also a strong negative correlation between hippocampal volume and posterior lateral ventricle volume, and a strong positive correlation between the thickness of the CA1 cell layer and hippocampal volume ( Figures 3I to 3J ). In summary, these data show that removal of neuronal APOE4 or astroglial APOE4 is protective against Tau-mediated neurodegeneration and results in reduced neuronal cell and hippocampal volume loss. This result is interesting considering that the Tau pathology data suggest that astroglial APOE4 has minimal effect on the accumulation and spread of Tau pathology.

[0169] Example 7: Glial proliferation is significantly reduced after removal of neuronal APOE4 or astrocyte APOE4

[0170] The extent of microgliosis and astrogliosis was investigated within genotype groups at 10 months of age to explore the role of neuronal APOE4 or astrocytic APOE4 in causing gliosis in the context of tauopathy. Immunohistochemical staining of microglia using the Iba1 antibody showed that PS19-fE4 mice had extensive microgliosis in the hippocampus, as indicated by a high percentage of microglial coverage ( Figure 4ABoth PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice showed a dramatic reduction in microglial proliferation ( Figure 4A In addition, PS19-fE3 mice without Cre, with Syn1-Cre, or with GFAP-Cre all had greatly reduced microgliosis relative to PS19-fE4 mice ( Figure 4A There was a strong negative correlation between Iba1 coverage and hippocampal volume in PS19-fE4 mice, suggesting that microgliosis is a good indicator and potential contributor to neurodegeneration ( Figure 4B ).

[0171] The microgliosis phenotype was further defined by immunohistochemical staining for CD68, a marker of activated microglia. There were significant differences in the area covered by activated microglia between genotype groups, with PS19-fE4 mice showing extensive microglial activation and all other genotype groups having minimal microglial activation ( Figure 4C Interestingly, of all the pathological correlations made in PS19-fE4 mice, the area covered by CD68+ activated microglia had the strongest negative correlation with hippocampal volume ( Figure 4D ), indicating that the extent of microglial activation is the strongest indicator and potential contributor to APOE4-enhanced neurodegeneration in tauopathies. Taken together, these data indicate that APOE4 strongly enhances microgliosis relative to APOE3 in tauopathies, and that removal of APOE4 from neurons or astrocytes attenuates the extent of microgliosis.

[0172] The extent of astrogliosis was also assessed after APOE was removed from neurons or astrocytes. As detected by immunohistochemical staining with an astrocyte GFAP antibody, PS19-fE4 mice showed considerable astrogliosis in the hippocampus relative to PS19-fE3 mice ( Figure 4E After removing either neuronal APOE4 or astrocyte APOE4, the extent of astrogliosis was greatly reduced ( Figure 4E ). The activated astrocyte marker S100β 56 These observations were also supported by immunohistochemical staining performed ( Figure 4F There was no significant difference in the extent of astrogliosis after APOE3 was removed from either cell type relative to PS19-fE3 mice ( Figures 4E to 4FStrikingly, and in stark contrast to microgliosis, neither astrocyte coverage nor activated astrocyte coverage was significantly correlated with hippocampal volume ( Figures 4G to 4H ). These data suggest that APOE4 strongly enhances astrogliosis relative to APOE3 in tauopathy, and that removal of APOE4 from neurons or astrocytes reduces astrocyte activation and / or recruitment. In addition, it is shown that the extent of astrogliosis may not be a valuable indicator or contributor to the neurodegeneration enhanced by APOE4 in tauopathy.

[0173] To further reveal the relationship between Tau pathology and gliosis, we investigated whether Tau propagation also induces gliosis in the non-injected hippocampus (refer to Figures 1H to 1N Indeed, fE4 mice also exhibited higher microgliosis in the uninjected hippocampus than fE3 mice, along with higher Tau propagation ( Fig. 4I ). Interestingly, after the removal of neuronal APOE4 or astrocytic APOE4, there was a significant reduction in microglial proliferation in the non-injected hippocampus. This suggests that regardless of the extent of Tau propagation, the removal of neuronal APOE4 or astrocytic APOE4 reduces microglial proliferation, because fE4 / GFAP-Cre mice still have relatively high levels of Tau propagation to the non-injected hippocampus.

[0174] These findings suggest that astrocytic APOE4 acts downstream of Tau pathology to promote microgliosis. The extent of astrocyte coverage in the non-injected hippocampus was also assessed and no discernible differences in astrogliosis were observed after removal of either neuronal APOE4 or astrocytic APOE4 ( Figures 4I to 4J ), possibly because of the relatively low astrogliosis in the non-injected hippocampus even in fE4 mice in this Tau propagation model.

[0175] Example 8: Myelin defects and depletion of oligodendrocyte progenitor cells are reduced following APOE4 removal from neurons but not astrocytes

[0176] Myelin degeneration and oligodendrocyte deficiency have been observed in human AD brain (Benitez et al., Neuroimage Clin. 4:64-71 (2013); Dean et al., JAMA Neurol. 74, 41-49 (2017); Nasrabady et al., Acta Neuro. Comms. 6, 22 (2018)) and in mouse models of Alzheimer's disease and tauopathy (Desai et al., Glia 57, 54-65 (2009); Zhang et al., Sci. Adv. 6, eabb8680 (2020); Shi et al., Neuron 109, 2413-2426.e7 (2021)).

[0177] The inventors studied the effect of neuronal APOE4 and astrocyte APOE4 removal on maintaining myelin integrity and the density of hippocampal oligodendrocyte progenitor cells (OPC). In order to determine the integrity of myelin in the hippocampus, myelin basic protein (MBP) was immunostained with MBP-specific antibodies. Quantification of the percentage coverage area of ​​MBP in the stratum radiatum below the pyramidal cell layer of CA1 showed that PS19-fE4 mice had extensive myelin loss relative to PS19-fE3 mice ( Figure 4K Strikingly, a significant reduction in myelin loss was observed in PS19-fE4 / Syn1-Cre mice, which was similar to the phenotype of PS19-fE3 mice, while PS19-fE4 / GFAP-Cre mice showed considerable myelin loss, which was similar to the phenotype of PS19-fE4 mice ( Figure 4K These data suggest that in the context of tauopathy, neuronal APOE4 plays a major detrimental role in promoting myelin defects, whereas astrocytic APOE4 does not affect this phenotype. There were no significant differences in myelin integrity in PS19-fE3 mice following ablation of either neuronal APOE3 or astrocytic APOE3.

[0178] To further characterize the effects of neuronal APOE4 and astrocyte APOE4 on biological components involved in the myelination process, cells were immunostained with NG2 antibodies to search for oligodendrocyte progenitor cells (OPCs), which are thought to help repair damaged myelin in the context of CNS injury and neurodegeneration (Shi et al. Glia 57, 54-65 (2009); Tripathi et al., J. Neurosci 30, 16383-16390 (2010)). A significant decrease in percentage OPC coverage was observed in the hippocampus of PS19-fE4 mice compared to PS19-fE3 mice ( Figure 4L Strikingly, removal of neuronal APOE4 significantly reduced the percent OPC coverage area to levels similar to those in PS19-fE3 mice ( Figure 4L In contrast, PS19-fE4 / GFAP-Cre mice had significantly lower OPC coverage relative to PS19-fE4 / Syn1-Cre mice and even showed a trend toward decreased OPCs relative to PS19-fE4 mice, although this did not reach statistical significance ( Figure 4L After ablation of either neuronal APOE3 or astrocytic APOE3 in PS19-fE3 mice, there was no significant difference in OPC coverage ( Figure 4L ).

[0179] Immunohistochemical staining of myelin and OPCs in fE4 and fE3 mice showed that APOE4 mice lacking the human mutant Tau-P301S did not exhibit myelin defects and had similar levels of OPCs in the hippocampus as fE3 mice, indicating that the effects of APOE4 on these phenotypes are specific to the context of tauopathy.

[0180] Collectively, these findings suggest that neuronal APOE4 plays a critical role in depleting the hippocampal OPC pool and causing myelin defects in this composite tauopathy mouse model, whereas astrocytic APOE4 does not contribute significantly to either pathological process.

[0181] Example 9: snRNA-seq identifies neurodegenerative disease-associated subpopulations of neurons and oligodendrocytes that are largely eliminated by removal of neuronal APOE4 but not astrocyte APOE4

[0182] In order to obtain a deep understanding of the cell type-specific effects of APOE4 in different types of hippocampal cells at the transcriptome level, single-cell nuclear RNA sequencing (snRNA-seq) was performed in the hippocampus isolated from PS19-fE4 mice and PS19-fE3 mice without Cre, with Syn1-Cre or with GFAP-Cre at 10 months of age. After normalization and filtering for quality control, the snRNA-seq data set includes 119,317 cores covering 26,285 genes. Clustering performed by shared nearest neighbor (shared nearest neighbor, SNN) and visualization performed by uniform manifold approximation and projection (uniformmanifold approximation and projection, UMAP) show 33 different cell clusters. Based on the expression of their marker genes, these clusters are assigned to:

[0183] 16 excitatory (Ex) neuron clusters (3 to 5, 7, 12, 15, 16, 18 to 20, 23, 25 to 28, 33);

[0184] 7 inhibitory (In) neuron clusters (6, 8, 9, 13, 22, 24, 30);

[0185] · 3 oligodendrocyte clusters (1, 2, 17);

[0186] An astrocyte cluster (10);

[0187] · 3 microglial clusters (11, 21, 29);

[0188] · 2 oligodendrocyte progenitor cell (OPC) clusters (14, 32); and

[0189] · 1 unknown cluster (31).

[0190] APOE is highly expressed in astrocytes in PS19-fE4 and PS19-fE3 mice, and its expression is significantly reduced in astrocytes in PS19-fE4 / GFAP-Cre mice. The inventors and colleagues have reported (Zalocusky et al., Nat. Neurosci. 24, 786-798 (2021)), and some neurons also express APOE in PS19-fE4 and PS19-fE3 mice. As described herein, neuronal APOE expression is eliminated in PS19-fE4 / Syn1-Cre mice.

[0191] The logarithmic odds ratio estimates from the generalized linear mixed effects model (GLMM_AM) for assessing the correlation with the animal model were used to identify the cell clusters that were changed in PS19-fE3, PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice compared to those in PS19-fE4 mice. The analysis showed that the probability of oligodendrocyte cluster 17 having cells from PS19-fE4 / Syn1-Cre mice was lower than the probability of having cells from PS19-fE4 mice, wherein it was almost completely eliminated in PS19-fE4 / Syn1-Cre mice. Based on differentially expressed (DE) gene analysis, cells in cluster 17 showed a dramatic upregulation of the expression of three major heat shock protein genes and a significant downregulation of the expression of myelin basic protein (Mbp) and myelin associated oligodendrocyte basic protein (Mobp) genes relative to other oligodendrocyte clusters. Differentially expressed (DE) pathway analysis showed enrichment of Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways associated with general neurodegeneration, AD, and other neurodegenerative diseases, indicating that cluster 17 represents oligodendrocytes associated with neurodegenerative diseases.

[0192] In addition, the probability of excitatory neuron cluster 25 having cells from PS19-fE4 / Syn1-Cre and PS19-fE3 mice is significantly lower than the probability of having cells from PS19-fE4 mice, wherein it is almost completely eliminated in PS19-fE4 / Syn1-Cre mice. Differential expression (DE) gene analysis shows that the expression of three major heat shock protein genes, human MAPT gene and amyloid precursor protein (App) gene of cells in cluster 25 is significantly upregulated. Differential expression (DE) pathway analysis shows the enrichment of KEGG pathways related to general neurodegeneration, Alzheimer's disease and other neurodegenerative diseases, indicating that cluster 25 represents neurons related to neurodegenerative diseases.

[0193] Similarly, the log odds ratio estimates from another generalized linear mixed effects model (GLMM_histopathology) for assessing the correlation with histopathology show that the ratio of cells in clusters 17 and 25 shows a significant negative correlation with hippocampal volume and a significant positive correlation with the coverage area of ​​pTau and with astrocyte proliferation and microgliosis. Correlation is largely driven by the PS19-fE4 group, and to a lesser extent by the PS19-fE4 / GFAP-Cre group. The ratio of cells in cluster 17 also has a significant negative correlation with the MBP coverage area.

[0194] Together, these findings show that ablation of neuronal APOE4, but not astrocytic APOE4, leads to a dramatic reduction in the amounts of disease-associated oligodendrocyte and neuronal subpopulations within the hippocampus that are neurodegenerative, and that both populations are strongly associated with Tau pathology, gliosis, neurodegeneration, and oligodendrocyte deficiency.

[0195] Example 10: Neurodegenerative disease-associated astrocyte and microglial subsets are largely eliminated by removing neuronal APOE4 but not astrocyte APOE4

[0196] Further sub-clustering of astrocytes identified 18 subgroups. The logarithmic odds ratio estimates from GLMM_AM show that the probability of astrocyte subclusters 4 and 7 having cells from PS19-fE4 / Syn1-Cre mice is lower than the probability of having cells from PS19-fE4 mice, and subcluster 7 is completely eliminated in PS19-fE4 / Syn1-Cre mice. Interestingly, astrocyte subclusters 4 and 7 highly express APOE. Differentially expressed (DE) genes and pathway analysis show the enrichment of KEGG pathways related to general neurodegeneration, Alzheimer's disease and other neurodegenerative diseases in subcluster 7, indicating that subcluster 7 represents neurodegenerative disease-related astrocytes. Subcluster 4 shows the enrichment of KEGG pathways related to cAMP signaling, synaptic function and long-term potentiation, indicating that subcluster 4 represents synaptic dysfunction-related astrocytes.

[0197] Further sub-clustering of microglia also identified 18 subgroups. The log odds ratio estimates from GLMM_AM show that the probability of microglia subclusters 4 and 8 having cells from PS19-fE4 / Syn1-Cre mice is lower than the probability of having cells from PS19-fE4 mice, and subcluster 8 is completely eliminated in PS19-fE4 / Syn1-Cre mice. Interestingly, microglia subclusters 4 and 8 highly express APOE. Differentially expressed (DE) genes and pathway analysis show the enrichment of KEGG pathways related to general neurodegeneration, AD and other neurodegenerative diseases in subcluster 8, indicating that subcluster 8 represents neurodegenerative disease-related microglia. Subcluster 4 shows the enrichment of KEGG pathways related to synaptic function, cAMP signaling and long-term potentiation, indicating that subcluster 4 represents synaptic dysfunction-related microglia.

[0198] Similarly, the log odds ratio estimates from GLMM_histopathology show that the ratio of cells in astrocyte subcluster 7 and microglia subcluster 8 shows a significant negative correlation with hippocampal volume and a significant positive correlation with the coverage area of ​​pTau and with astrocyte proliferation and microgliosis. Correlation is driven by the PS19-fE4 group to a large extent, and is driven by the PS19-fE4 / GFAP-Cre group to a lower extent. Interestingly, the PCA clustering of oligodendrocyte cluster 17 and excitatory neuron cluster 25 together with all astrocytes and microglia subclusters shows that disease-related astrocyte subcluster 7 and microglia subcluster 8 have similar contributions to two disease-related clusters of oligodendrocytes (cluster 17) and neurons (cluster 25) for eight pathological parameters.

[0199] Together, these findings show that ablation of neuronal APOE4, but not astroglial APOE4, leads to a dramatic reduction in the amounts of neurodegenerative disease-associated astrocyte and microglial subsets within the hippocampus, and that both glial subsets are strongly associated with Tau pathology, gliosis, and neurodegeneration.

[0200] Example 11: Removal of APOE4 from astrocytes secondary reduces neuronal APOE4 expression

[0201] During the snRNA-seq data analysis, the inventors noted that the removal of astrocyte APOE significantly reduced APOE expression in neurons in addition to its predicted elimination of APOE expression in astrocytes. To confirm this observation, a similar composite mouse model carrying a floxed knock-in human APOE gene and astrocyte-specific Cre (Aldh1l1-Cre / ERT2 BAC transgenic) was used to further analyze the publicly available snRNA-seq data set (GEO: GSE164507), which was further analyzed in the presence or absence of P301S mutant human Tau during adulthood in a tamoxifen-inducible manner to make the human APOE gene in astrocytes (Wang et al., Neuron 109, 1657-1674.e7 (2021)).

[0202] In the snRNA-seq data set, neuron APOE expression is observed in both FE3 and FE4 mice, which is further improved in PS19-FE4 (TAFE4_oil) and PS19-FE3 (TAFE3_oil) mice, indicating that tau protein disease improves neuron APOE expression. After induced removal of astrocyte APOE by tamoxifen treatment during adulthood, relative to oil-treated mice, in PS19-fE4 / Aldh111-Cre / ERT2 (TAFE4_tam) and PS19-fE3 / Aldh111-Cre / ERT2 (TAFE3_tam) mice, it is also clearly observed that neuron APOE in both excitatory neurons and inhibitory neurons is sharply reduced. The effect of removing astrocyte APOE on neuron APOE reduction is observed in all neuron subtypes, indicating that astrocyte APOE is generally regulated to neuron APOE expression.

[0203] Thus, the initial observation that ablation of astrocytic APOE secondary reduces neuronal APOE expression was confirmed in different tauopathy models with human APOE, even when ablation of astrocytic APOE was driven by different astrocyte-specific promoters and its ablation was induced at different time points in the lifespan of the mouse models.

[0204] To determine whether this observation could be validated at the protein level, the levels of APOE protein in cultured primary neurons isolated from the cortex and hippocampus of prenatal pups were quantitatively evaluated by sandwich ELISA. This analysis showed that primary neurons from PS19-fE4 / GFAP-Cre mice exhibited a significant 50% reduction in APOE protein levels relative to those from PS19-fE4 mice ( Figure 4M ).

[0205] These data provide strong evidence that ablation of astrocytic APOE4 leads to a dramatic reduction in neuronal APOE4 expression and protein production and consolidates this regulatory relationship between astrocytic APOE4 and neuronal APOE4. Therefore, the limited protective effect of ablation of astrocytic APOE4 may be due to a secondary reduction in neuronal APOE4 rather than complete elimination.

[0206] Early studies by the inventors and colleagues reported that neuronal APOE expression is regulated by astrocyte release factors through regulating the ERK pathway in neurons (Zhao et al., Nat. Commun. 11, 5540 (2020)). In-depth comparison of snRNA-seq data between astrocytes from PS19-fE4 / GFAP-Cre mice and those from PS19-fE4 mice showed that after astrocyte APOE4 was removed, some of the top downregulated genes were clear activators of the ERK / MAPK pathway, including dipeptidyl peptidase 10 (DDP10) and neuregulin-3 (Neuregulin-3, Nrg3).

[0207] The inventors hypothesized that astrocyte APOE4 can regulate neuronal APOE4 expression by promoting the release of these factors that activate neuronal ERK pathways, resulting in increased neuronal APOE4 expression. In order to test this possibility, primary neurons isolated from PS19-fE4 and PS19-fE4 / GFAP-Cre mice were treated with 10 μg / mL of recombinant DPP10 or Nrg3 protein or dPBS control. The APOE protein level in primary neuronal cell lysates was then quantitatively evaluated by sandwich ELISA.

[0208] The APOE protein level in PS19-fE4 / GFAP-Cre primary neurons treated with DPP10 was significantly increased by 98% compared with that in dPBS control ( Figure 4N On the other hand, there was no significant difference in APOE levels in PS19-fE4 primary neurons after DPP10 treatment ( Fig.4O ). These data suggest that primary neurons from PS19-fE4 / GFAP-Cre mice (in which astrocyte APOE4 is deleted) are sensitive to DPP10 upregulation of APOE expression, whereas primary neurons from PS19-fE4 mice are not. We did not observe differences in APOE levels in PS19-fE4 / GFAP-Cre or PS19-fE4 primary neurons after Nrg3 treatment ( Figures 4N to 4O ), indicating that Nrg3 is not involved in the regulation of neuronal APOE4 expression.

[0209] Example 12: Neuronal release of HMGB1 is completely abolished by removal of neuronal APOE4 but not astrocyte APOE4

[0210] In the process of searching for proinflammatory cytokines that can trigger Tau-induced and neurodegeneration-induced gliosis, the inventors observed that the expression of the high mobility group box protein 1 (Hmgb1) gene in cells in neurodegenerative disease-related neuronal clusters 25 was significantly increased. HMGB1 protein is required for glial cell activation (Paudel et al. Front. Neurosci. 12, 628 (2018)). In pathological conditions, HMGB1 translocates from the nucleus to the cytoplasm of stressed or dying cells, and is subsequently released to act as a proinflammatory cytokine.

[0211] Immunohistochemical staining of HMGB1 protein and nuclear marker DAPI in 10-month-old mice showed that PS19-fE4 mice had significantly high amounts of HMGB1 protein that was translocated from the nuclei of neurons in the dentate gyrus of the hippocampus to the cytoplasm, as a significant portion of the protein was found outside the nuclei of hippocampal neurons ( Figures 5A to 5D On the other hand, HMGB1 protein in PS19-fE3 mice was largely retained in the nucleus, with minimal translocation to the cytoplasm ( Figures 5A to 5D Interestingly, removal of APOE4 from neurons or astrocytes attenuated this phenotype observed in PS19-fE4 mice, in which the majority of HMGB1 protein remained localized to the nucleus ( Figures 5A to 5D ).

[0212] In addition, PS19-fE4 mice contained a larger number of HMGB1-positive puncta in both the nucleus and cytoplasm compared with the other genotype groups ( Figures 5E to 5F Notably, fE4 and fE3 mice lacking the human mutant Tau-P301S have HMGB1 protein localized in the nucleus, indicating that nuclear-to-cytoplasmic translocation of HMGB1 in APOE4 mice requires the coexistence of both APOE4 and tauopathy.

[0213] In order to evaluate the extent of disease-related HMGB1 release from hippocampal neurons in these multiple genotype groups, in vivo microdialysis was used to collect hippocampal interstitial fluid (ISF) from 8.5-month-old mice over a 24-hour period (Yamada et al., J. Neurosci. 31: 13110-13117 (2011)). Sandwich ELISA was used to quantify the level of HMGB1 protein in interstitial fluid (ISF). Although PS19-fE4 mice showed high levels of HMGB1 protein in their hippocampal ISF, the removal of neuronal APOE4 reduced the HMGB1 protein level in ISF to undetectable levels ( Figures 5G to 5H ), indicating that neuronal APOE4 plays a role in controlling the release of HMGB1 from neurons. Ablation of astrocytic APOE4 also resulted in an 86% reduction in HMGB1 protein in ISF, but not complete elimination ( Figures 5G to 5H ), which may be due to a secondary reduction in neuronal APOE4 expression rather than complete elimination.

[0214] Experiments were also performed to determine whether HMGB1 is a potential regulator of neuronal APOE4 expression. To this end, primary neurons isolated from PS19-fE4 / GFAP-Cre and PS19-fE4 mice were treated with 10 μg / mL of recombinant HMGB1 protein or dPBS control and APOE protein levels in cell lysates were quantitatively evaluated by sandwich ELISA. After treatment with HMGB1, no significant differences in APOE protein levels were observed in PS19-fE4 / GFAP-Cre or PS19-fE4 primary neurons, indicating that HMGB1 is not involved in the regulation of neuronal APOE4 expression.

[0215] To evaluate the importance of neuron-released HMGB1 in triggering gliosis in APOE4-related tauopathy, interstitial fluid (ISF) collected from the hippocampus of 8.5-month-old PS19-fE4 mice was injected into the hippocampus of 8.5-month-old wild-type mice. The injected ISF fraction was enriched in relatively high concentrations of HMGB1 protein ( Figure 5H fractions 19 to 22 in the sample) or had undetectable levels of HMGB1 protein as a control ( Figure 5H Wild-type mice received a unilateral injection of HMGB1 into the right dorsal hippocampus. + or control ISF and were analyzed 6 days after injection to assess dramatic changes in gliosis.

[0216] The control (HMGB1 -) ISF-injected wild-type mice exhibited relatively low levels of microgliosis and astrogliosis on both the injected and uninjected hippocampal sides, showing that most of the gliosis induced by the injection procedure itself resolved 6 days after injection ( Figures 5I to 5J ). In contrast, HMGB1 + Wild-type mice injected with ISF showed significantly increased levels of microgliosis on the injected hippocampus relative to the uninjected side ( Fig.5I Interestingly, compared with the uninjected side, the HMGB1-injected + ISF did not lead to a significant increase in astrogliosis in the injected hippocampus ( Figure 5J Together, these data suggest that neuronal release of HMGB1 represents a novel mechanism by which neuronal APOE4 promotes glial activation.

[0217] Example 13: Treatment with HMGB1 inhibitors significantly reduces APOE4-driven gliosis, Tau pathology, and degeneration

[0218] Based on the finding that neuronal APOE4 is a potent driver of neuronal HMGB1 release, experiments were performed to test the therapeutic efficacy of HMGB1 inhibitors to combat APOE4-driven pathogenesis in the context of tauopathies.

[0219] Two HMGB1 inhibitors, ethyl pyruvate (EP) and glycyrrhizic acid (GA), were tested as selective inhibitors of HMGB1 translocation and release (Ulloa et al., Proc. Natl. Acad. Sci. USA 99: 12351-12356 (2002); Sun et al., Front. Immunol. 9, 1518 (2018); Mollica et al., Chem Biol. 14, 431-41 (2007); Dave et al., J. Leukoc. Biol. 86, 633-643 (2009)). A mixed solution of ethyl pyruvate (80 mg / kg) and glycyrrhizic acid (20 mg / kg) or saline vehicle was administered to PS19-fE4 and PS19-fE3 mice (3 doses per week for 12 weeks) by intraperitoneal injection. Fig. 6A Treatment was started at 6.5 months of age, around the onset of adverse pathology, and completed at 9.5 months of age, when severe neurodegeneration and pathology are usually present, as shown in this study and others (Shi et al., Nature 549, 523-527 (2017); Yoshiyama et al., Neuron 53: 337-351 (2007)).

[0220] The effectiveness of ethyl pyruvate (EP) and glycyrrhizic acid (GA) in blocking the nuclear-cytoplasmic translocation of HMGB1 protein was first confirmed by immunostaining with anti-HMGB1 and DAPI. PS19-fE4 mice treated with saline showed extensive HMGB1 translocation out of the nucleus, while PS19-fE4 mice treated with HMGB1 inhibitor had no discernible HMGB1 translocation ( Figures 6B to 6D ; 6P to 6Q). In PS19-fE3 mice, there was no significant difference in HMGB1 translocation between saline-treated and inhibitor-treated groups.

[0221] The extent of microgliosis and astrogliosis present in the mice after treatment was then assessed. + Microglia ( Figures 6E to 6F ) and CD68 + As exemplified by the high coverage of activated microglia (Figure 9R), saline-treated PS19-fE4 mice showed considerable microgliosis throughout the hippocampus. PS19-fE4 mice treated with HMGB1 inhibitors showed significantly reduced microgliosis. In addition, immunostaining with anti-GFAP showed that saline-treated PS19-fE4 mice had extensive astrocyte proliferation throughout the hippocampus, while PS19-fE4 mice treated with HMGB1 inhibitors had significantly reduced astrocyte proliferation (Figure 9R). Figures 6G to 6H PS19-fE3 mice showed no significant differences in microgliosis or astrogliosis whether treated with saline or HMGB1 inhibitor.

[0222] The extent of Tau pathology in these mice was then assessed by immunostaining for pTau. PS19-fE4 mice treated with saline showed significant Tau pathology throughout the hippocampus, whereas PS19-fE4 mice treated with the HMGB1 inhibitor showed a dramatic reduction in Tau pathology ( Figures 6I to 6J ). In addition, the effectiveness of HMGB1 inhibitor administration in combating myelin defects was determined by immunostaining for MBP. PS19-fE4 mice treated with saline showed severe myelin loss in the stratum radiatum of CA1, while PS19-fE4 mice treated with HMGB1 inhibitor showed rescue of the defect with high MBP coverage ( Figures 6K to 6L There were no discernible differences in myelin coverage or Tau pathology between saline-treated and inhibitor-treated PS19-fE3 mice.

[0223] The effects of HMGB1 inhibition on neurodegeneration were also determined by quantifying hippocampal and posterior lateral ventricle volumes. Saline-treated PS19-fE4 mice exhibited considerable neurodegeneration, whereas HMGB1 inhibitor-treated PS19-fE4 mice exhibited rescue of neurodegeneration relative to saline-treated PS19-fE4 mice as evidenced by a significant increase in hippocampal volume and a significant decrease in posterior lateral ventricle volume ( Figures 6M to 6O There were no significant differences in neurodegeneration between saline-treated and HMGB1 inhibitor-treated PS19-fE3 mice.

[0224] In summary, these findings show that treating PS19-fE4 mice with HMGB1 inhibitors can effectively prevent the occurrence of obvious pathological conditions driven by APOE4 (including gliosis, Tau pathological conditions, neurodegeneration and myelin defects) by blocking nuclear-cytoplasmic translocation and subsequent release of HMGB1 protein. The data support the conclusion that HMGB1 translocation and release play a role in inducing and worsening these multiple APOE4-driven pathological conditions.

[0225] Embodiment 14

[0226] Apolipoprotein E4 (APOE4) is a major genetic risk factor for Alzheimer's disease (AD), however, its underlying cellular and molecular mechanisms remain elusive. In the brain, APOE is produced primarily by astrocytes and at a lower level by neurons. Described herein is a rigorous comparison of the effects of neuronal APOE4 and astrocyte APOE4 on AD-related pathologies by selectively removing APOE4 from any cell type of mice carrying a human P301S mutant Tau transgene and a floxed knock-in human APOE gene. APOE4 mice have significantly more Tau pathology, gliosis, neurodegeneration, neurological dysfunction, and myelin defects than APOE3 mice. Single-cell nuclear RNA sequencing identified the enrichment of neurodegenerative disease-associated subpopulations of neurons, oligodendrocytes, astrocytes, and microglia in APOE4 mice. Ablation of neuronal APOE4 significantly reduced all of these pathologies and largely eliminated disease-associated cell subsets, whereas ablation of astrocytic APOE4 reduced only gliosis and neurodegeneration. The limited protective effect of ablation of astrocytic APOE4 was attributed to secondary reduction rather than complete elimination of neuronal APOE4. Mechanistically, neuronal APOE4 promoted the release of HMGB1 from neurons to induce gliosis and subsequent neurodegeneration and myelin defects, which could be effectively blocked by treatment with HMGB1 inhibitors. Therefore, neuronal APOE4 drives Tau-mediated inflammation and degeneration by promoting neuronal HMGB1 release, and HMGB1 inhibitors represent a new approach for treating APOE4-associated AD and other tauopathies.

[0227] introduce

[0228] Tauopathy is defined by a class of neurodegenerative disorders of abnormal intracellular accumulation of hyperphosphorylated Tau (pTau) protein. Alzheimer's disease (AD) represents the main type of tauopathy, is a highly prevalent disease, its pathological characteristics are the accumulation of amyloid plaques and Tau tangles and clinical characteristics are the loss of memory and other cognitive functions. In these two main pathological signs of AD, only Tau tangles are strongly associated with neurodegeneration and cognitive decline. Other AD pathological signs under study include neuroinflammation and gliosis, which have recently been determined to be the main contributing factors of neurodegeneration. In addition, oligodendrocyte deficiency and myelin degeneration have been observed in the mouse model of AD brain and AD and tauopathy. Therefore, AD represents a complex pathological condition group, although the connection between these pathological conditions is still unclear. It is important to understand the relationship between pathological conditions and to clarify the potential mechanism responsible for its induction or deterioration for developing better treatment strategies (alone or in combination) targeting these pathological conditions.

[0229] Epidemiological and genome-wide association studies have identified apolipoprotein E4 (APOE4) as the main genetic risk factor for AD. The human APOE gene exists as three common alleles (including ε2, ε3 and ε4). APOEε4 is considered to be the most harmful allele because it increases AD risk and reduces the age of disease onset in a dose-dependent manner. Great efforts have been made to understand how APOE4 causes AD risk to increase, and a large amount of work has shown that APOE4 worsens many obvious AD-related pathological conditions relative to APOE. In particular, APOE4 has been shown to accelerate hippocampal volume loss in human patients and improve neurodegeneration in mice with or without tau disease. In addition to its well-studied role in promoting amyloid pathological conditions, recent studies have found that APOE4 also increases the Tau load in the human brain and promotes the accumulation of pTau in mice and human neuron models. In addition, APOE4 increases neuroinflammation and gliosis in human AD brain and tau disease mouse models. It has also been reported that APOE4 is associated with reduced myelination and white matter integrity in the human brain. In summary, these studies show clear evidence that APOE4 is associated with promoting Tau pathology, gliosis, neurodegeneration, and myelin degeneration in AD and other tauopathies. Nevertheless, the underlying mechanisms responsible for the widespread effects of APOE4 on these multiple pathologies remain unclear.

[0230] Recently, there has been increasing interest in determining the cell type-specific effects of APOE4 in various aspects of AD pathogenesis. In the central nervous system (CNS), APOE acts as a major lipid transporter and is primarily produced by astrocytes. Stress or injury conditions can induce APOE expression in neurons and microglia. Previous studies have shown that the deleterious effects of APOE4 may depend on its cell source. However, there is a gap in the understanding of the exact role of neuronal APOE4 and astrocyte APOE4 in triggering and / or exacerbating a variety of AD pathological conditions. In the current study, extensive analysis was performed: directly comparing the effects of removing APOE4 from neurons or astrocytes on the occurrence of major AD pathological conditions (including Tau pathological conditions, gliosis, neurodegeneration, neurological dysfunction and myelin defects) to better understand the cell type-specific effects of APOE4 in the pathogenesis of AD and other tau diseases. The aim is to uncover the key cellular and molecular mechanisms involved in the cascade that triggers AD-related pathologies and to determine whether these mechanisms are regulated by neuronal APOE4 or astrocytic APOE4.

[0231] result

[0232] Neuron-specific or astrocyte-specific ablation of the APOE gene in human APOE-KI mice expressing human mutant Tau

[0233] Under the control of neuron-specific synapsin-1 promoter (Syn1-Cre) or astrocyte-specific glial fibrillary acid protein promoter (GFAP-Cre), a mouse strain expressing floxed human APOE3 or APOE4 gene and Cre recombinase gene is produced. These floxed APOE-KI (fE) mice express homozygous human APOE3 or APOE4 instead of endogenous mouse Apoe, and the flank of the human APOE gene is a pair of LoxP sites, allowing its precise excision in the presence of cell type-specific Cre recombinase expression. The fE mice without Cre, with Syn1-Cre or with GFAP-Cre are hybridized with mice (PS19 strains) expressing mutant 1N4R human microtubule-associated protein Tau (MAPT) encoding disease-related P301S mutations that have been widely used as tau disease mouse models. The resulting composite mice were referred to as PS19-fE, PS19-fE / Syn1-Cre, or PS19-fE / GFAP-Cre mice.

[0234] fE / Syn1-Cre and fE / GFAP-Cre mice have been rigorously characterized to validate the specificity of Cre recombinase expression under the neuron-specific Syn1 or astrocyte-specific GFAP promoter. PS19-fE / Cre mice were further validated by immunohistochemical analysis of brain sections from 10-month-old PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice. To confirm the specificity of Cre recombinase expression in hippocampal neurons and astrocytes when driven under the Syn1 or GFAP promoter, respectively, NeuN and GFAP antibodies were used together with the Cre recombinase antibody. In PS19-fE4 / Syn1-Cre mice, Cre recombinase is expressed only in NeuN-positive neurons and not in GFAP-positive astrocytes ( Fig. 7A In PS19-fE4 / GFAP-Cre mice, Cre recombinase is expressed only in GFAP-positive astrocytes but not in NeuN-positive neurons ( Fig. 7ACo-immunostaining with antibodies against APOE, GFAP, and NeuN showed that PS19-fE4 mice without Cre had APOE expression in GFAP-positive astrocytes and some NeuN-positive neurons, while PS19-fE4 / Syn1-Cre mice had APOE expression in astrocytes and lacked APOE expression in neurons, and PS19-fE4 / GFAP-Cre mice had APOE expression in some neurons and lacked APOE expression in astrocytes ( Figure 7B PS19-fE4 / GFAP-Cre mice are negative for NeuN and GFAP ( Figure 7B ) but positive for the microglial marker Iba1 ( Figure 7C ), indicating that they are APOE-expressing microglia.

[0235] To quantitatively determine the levels of APOE protein within these various mouse models, hippocampal lysates from 10-month-old mice were analyzed by sandwich ELISA against human APOE. PS19-fE4 / Syn1-Cre mice showed approximately 20% reduction in APOE levels relative to PS19-fE4 mice ( Fig.7D ), which is consistent with previous reports indicating that neuronal APOE contributes approximately 20% to 30% of total APOE protein levels in the hippocampus and cortex. PS19-fE4 / GFAP-Cre mice exhibited a dramatic reduction in APOE levels of approximately 70% relative to PS19-fE4 mice, consistent with the recognized role of astrocytes as the major producers of APOE within the CNS. Similarly, PS19-fE3 / Syn1-Cre mice exhibited approximately 25% reduction in APOE levels and PS19-fE3 / GFAP-Cre exhibited approximately 67% reduction in APOE levels relative to PS19-fE3 mice ( Fig.7D Together, these results provide strong evidence that APOE gene expression is abolished in neurons or astrocytes when Cre recombinase expression is driven under the Syn1 or GFAP promoter, respectively, in these composite mouse models.

[0236] Removal of APOE4 from neurons but not astrocytes significantly reduces Tau pathology

[0237] In order to determine whether the removal of neuronal APOE or astrocyte APOE affects Tau pathology, 10-month-old mice were evaluated when PS19 mice showed extensive Tau pathology in the entire hippocampus. The accumulation of pTau in the hippocampus was analyzed by immunohistochemical staining with pTau-specific AT8 antibodies and the Tau pathology was quantified as the AT8 coverage area percentage in the hippocampus. PS19-fE4 mice showed extensive Tau pathology in the entire hippocampus and the extent of Tau pathology was significantly lower in PS19-fE3 mice. Relative to PS19-fE4 mice, PS19-fE4 / Syn1-Cre mice showed a significant reduction (about 81%) in Tau pathology, while PS19-fE4 / GFAP-Cre mice had a slight reduction (about 30%), which did not reach statistical significance. Interestingly, the reduction of pTau coverage in PS19-fE4 / Syn1-Cre mice was similar to the extent of Tau pathology observed in PS19-fE3 mice. There was no significant difference in Tau pathology between PS19-fE3 with Cre and without Cre, probably because Tau pathology in PS19-fE3 mice was already low.

[0238] After continuous biochemical extraction with RAB and RIPA buffers containing highly soluble and less soluble Tau proteins, Western blot analysis was used to assess the pTau levels in mouse hippocampal tissue. In RAB fractions, there was no significant difference in the pTau levels between various genotype groups. However, PS19-fE4 / Syn1-Cre and PS19-fE3 mice showed a significant decrease in pTau levels relative to PS19-fE4 mice in RIPA fractions. PS19-fE4 / GFAP-Cre mice showed a trend of reduced pTau levels (about 40%) in RIPA fractions relative to PS19-fE4 mice, which did not reach statistical significance. In short, these data show that neuronal APOE4 expression is a strong driver of Tau pathological conditions, while astrocyte APOE4 plays a minimal role in Tau pathological conditions.

[0239] The spread of tau pathology is reduced after APOE4 removal in neurons but not in astrocytes

[0240] In order to further study the mechanism of neuronal APOE4 driving Tau pathological conditions, the effect of cell type specific APOE4 expression on the propagation of Tau pathological conditions was determined.Previous studies have shown that after various forms of Tau protein are directly injected into the mouse brain, pathological Tau can be propagated to the brain area of ​​anatomical connection.After the adeno-associated virus-2 (AAV2-Tau-P301S) encoding human P301S mutant Tau is unilaterally injected into the right dorsal hippocampus of fE mice without Cre, with Syn1-Cre or with GFAP-Cre, the degree of Tau propagation is analyzed.fE mice lack human P301S mutant Tau and express endogenous mouse Mapt gene instead, allowing more accurate detection of human Tau propagation, because fE mice show minimal Tau pathological conditions.Mice are injected with AAV2-Tau-P301S virus at 10 months of age and evaluated at 13 months of age 12 weeks after injection.

[0241] To provide evidence that the observed Tau propagation phenotype could truly be attributed to the spread of pathological human Tau between neurons, as opposed to the unexpected result that the virus encoding Tau itself moved to the non-injected hippocampus, AAV2 encoding GFP of the same serotype (AAV2-GFP) injected unilaterally into the right dorsal hippocampus of 10-month-old fE4 mice was evaluated. Immunostaining with anti-GFP two weeks after injection showed that the GFP signal remained localized to neurons within the injected hippocampus ( Fig. 8A To B). The uninjected hippocampus side does not have any obvious GFP signal in the neuronal somata, although there are some GFP-positive neuronal projections that may originate from neurons present on the injected hippocampus side. This shows that AAV2 itself does not spread between the right and left hippocampi after unilateral injection. In addition, for all immunohistochemical analyses, the number of neurons containing soma-positive Tau was quantified to more accurately reflect the spread of human mutant Tau between neurons and exclude confounding factors (such as Tau-positive commissural fibers from neurons originating from the injected side).

[0242] Immunohistochemical staining with human Tau antibody (HT7) showed robust spread of human Tau to the uninjected hippocampus in PS19-fE4 mice and minimal Tau spread in PS19-fE3 mice, indicating that APOE4 promotes Tau spread. Interestingly, PS19-fE4 / Syn1-Cre mice had limited spread of human Tau to the uninjected hippocampus, while PS19-fE4 / GFAP-Cre mice had extensive human Tau spread, similar to the spread of Tau pathology observed in PS19-fE4 mice. Notably, after removal of neuronal APOE4, HT7 mice with Tau spread +The number of cells was reduced by 55%, and after astrocyte APOE4 removal, HT7 cells with Tau propagation + There were no discernible differences in the number of cells.

[0243] The extent of Tau propagation was analyzed by immunostaining pTau with AT8 antibody. Both PS19-fE4 and PS19-fE4 / GFAP-Cre mice showed robust propagation of pTau to the uninjected hippocampal side, while the pTau propagation of PS19-fE4 / Syn1-Cre and PS19-fE3 mice was significantly reduced. In short, these data show that a mechanism for driving Tau pathological conditions by neuronal APOE4 is to pass through synaptically connected neurons by stimulating the propagation of pathological Tau. The data further support the view that astrocyte APOE4 is not a key mediator of Tau pathological conditions, as demonstrated by its lack of effect on Tau propagation.

[0244] Network hyperexcitability is abolished following removal of neuronal APOE4 but not astroglial APOE4

[0245] To determine the cell type-specific effects of APOE4 on neuronal function in the context of tauopathy, neuronal network excitability was measured in the angular angular lateral ... Fig.9A Compared with PS19-fE3 mice, PS19-fE4 mice had significant CA1 neuronal hyperexcitability ( Fig. 9B ). Removal of neuronal APOE4 eliminated neuronal network hyperexcitability, whereas removal of astroglial APOE4 resulted in a slight reduction that did not reach significance ( Fig. 9B ), suggesting that neuronal APOE4 drives neuronal dysfunction in the context of tauopathy.

[0246] Neurodegeneration is reduced after removal of either neuronal APOE4 or astroglial APOE4

[0247] Next, after removing APOE from neurons or astrocytes, the degree of neurodegeneration in 10-month-old PS19-fE mice was evaluated. Analysis of hippocampal volume and posterior lateral ventricle volume shows that PS19-fE4 mice show extensive neurodegeneration relative to PS19-fE3 mice. Increased hippocampal volume and reduced posterior lateral ventricle volume show that neurodegeneration in both PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice is significantly reduced, similar to those of PS19-fE3 mice. Removal of neuron APOE3 or astrocyte APOE3 does not significantly affect neurodegeneration.

[0248] Quantification of the extent of neuronal cell loss in multiple subregions of the hippocampus shows that PS19-fE4 mice have extensive neuronal loss in CA1 and dentate gyrus, while PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice both have significantly reduced neuronal cell loss in both CA1 and dentate gyrus subregions of the hippocampus. When APOE3 is removed from neurons or glial cells, there is no significant difference in the thickness of the neuronal cell layer in PS19-fE3 mice.

[0249] In PS19-fE4 mice, there is a weak but significant negative correlation between Tau pathology and hippocampal volume, indicating that Tau pathology promotes neurodegeneration in these mice at least to some extent. There is also a strong negative correlation between hippocampal volume and posterior lateral ventricle volume, and there is a strong positive correlation between the thickness of the CA1 cell layer and hippocampal volume. In short, these data show that removing neuronal APOE4 or astrocyte APOE4 is protective for Tau-mediated neurodegeneration and leads to reduced neuronal cells and hippocampal volume loss. Considering that the Tau pathology data show that astrocyte APOE4 has a minimal effect on the accumulation and propagation of Tau pathology, the result is interesting.

[0250] Glial proliferation was significantly reduced after ablation of either neuronal APOE4 or astrocytic APOE4

[0251] Next, the extent of microgliosis and astrogliosis in these multiple genotype groups at 10 months of age was studied to explore the effect of neuron APOE4 or astrocyte APOE4 causing gliosis in the context of tau protein disease. Immunohistochemical staining of microglia using Iba1 antibody showed that PS19-fE4 mice had extensive microgliosis in the hippocampus, as indicated by the high percentage coverage area of ​​microglia. Both PS19-fE4 / Syn1-Cre and PS19-fE4 / GFAP-Cre mice showed a sharp reduction in microgliosis. In addition, PS19-fE3 mice without Cre, with Syn1-Cre or with GFAP-Cre all had greatly reduced microgliosis relative to PS19-fE4 mice. There was a strong negative correlation between Iba1 coverage and hippocampal volume in PS19-fE4 mice, suggesting that microgliosis is a good indicator and potential contributor to neurodegeneration.

[0252] The microgliosis phenotype is further limited by immunohistochemical staining of CD68, which is a marker of activated microglia. Between genotype groups, there are significant differences in the coverage area of ​​activated microglia, where PS19-fE4 mice show extensive microglia activation and all other genotype groups have minimal microglia activation. Interestingly, among all pathological correlations made in PS19-fE4 mice, the coverage area of ​​CD68+ activated microglia has the strongest negative correlation with hippocampal volume, indicating that the degree of microglial activation is the strongest indicator and potential contributor of neurodegeneration enhanced by APOE4 in tau disease. In short, these data show that APOE4 strongly enhances microgliosis relative to APOE3 in tau disease, and removing APOE4 from neurons or astrocytes weakens the degree of microgliosis.

[0253] The extent of astrocyte proliferation after APOE is removed from neurons or astrocytes is assessed. As detected by immunohistochemical staining with astrocyte GFAP antibody, PS19-fE4 mice show considerable astrocyte proliferation in the hippocampus relative to PS19-fE3 mice. After removing neuronal APOE4 or astrocyte APOE4, the extent of astrocyte proliferation is greatly reduced. Immunohistochemical staining of activated astrocyte marker S100β also supports these observations. Relative to PS19-fE3 mice, after APOE3 is removed from either cell type, there is no significant difference in the extent of astrocyte proliferation. Remarkably, and in sharp contrast to microglial proliferation, neither the coverage area of ​​astrocytes nor the coverage area of ​​activated astrocytes is significantly correlated with hippocampal volume. These data suggest that APOE4 strongly enhances astrogliosis relative to APOE3 in tauopathies, and that removal of APOE4 from neurons or astrocytes reduces astroglial activation and / or recruitment. Furthermore, it is shown that the extent of astrogliosis may not be a valuable indicator or contributor to APOE4-enhanced neurodegeneration in tauopathies.

[0254] To further reveal the relationship between Tau pathology and gliosis, we investigated whether Tau propagation also causes gliosis in the non-injected hippocampus. Indeed, we observed that fE4 mice, together with higher Tau propagation, also exhibited higher microgliosis in the non-injected hippocampus than fE3 mice ( Figure 8C To D). Attractively, after removing neuronal APOE4 or astrocyte APOE4, there was a significant reduction in microglial proliferation in the uninjected hippocampal side. This suggests that regardless of the extent of Tau propagation, removing neuronal APOE4 or astrocyte APOE4 reduces microglial proliferation, because fE4 / GFAP-Cre mice still have relatively high levels of Tau propagation to the uninjected hippocampal side. These findings suggest that astrocyte APOE4 acts downstream of Tau pathology to promote microglial proliferation. In addition, we evaluated the extent of astrocyte coverage in the uninjected hippocampal side and did not observe discernible differences in astrocyte proliferation after removing neuronal APOE4 or astrocyte APOE4 ( Fig. 8E to F), possibly because of the relatively low astrogliosis in the non-injected hippocampus even in fE4 mice in this Tau propagation model.

[0255] Myelin defects and depletion of oligodendrocyte progenitors are reduced after removal of APOE4 from neurons but not astrocytes

[0256] Since myelin degeneration and oligodendrocyte deficiency have been observed in human AD brain and mouse models of AD and tauopathy, the effects of neuronal APOE4 and astrocytic APOE4 removal on maintaining myelin integrity and the density of hippocampal oligodendrocyte progenitor cells (OPCs) were studied. To determine myelin integrity in the hippocampus, myelin basic protein (MBP) was stained with an MBP-specific antibody. Quantification of the percentage coverage area of ​​MBP in the stratum radiatum beneath the pyramidal cell layer of CA1 showed that PS19-fE4 mice had extensive myelin loss relative to PS19-fE3 mice ( Fig. 10A To B). Strikingly, myelin loss was significantly reduced in PS19-fE4 / Syn1-Cre mice, similar to the phenotype of PS19-fE3 mice, whereas PS19-fE4 / GFAP-Cre mice exhibited considerable myelin loss, similar to the phenotype of PS19-fE4 mice ( Fig. 10A To B). This suggests that in the context of tauopathy, neuronal APOE4 plays a major detrimental role in promoting myelin defects, while astroglial APOE4 does not affect this phenotype. There was no significant difference in myelin integrity in PS19-fE3 mice after removal of either neuronal APOE3 or astroglial APOE3.

[0257] To further characterize the effects of neuronal APOE4 and astrocytic APOE4 on biological components involved in the myelination process, the NG2 antibody was used to interrogate OPCs, which are thought to help repair damaged myelin in the setting of CNS injury and neurodegeneration. The percentage OPC coverage area in the hippocampus of PS19-fE4 mice was significantly reduced compared to PS19-fE3 mice ( Fig. 10C To D). Strikingly, ablation of neuronal APOE4 significantly reduced the percent OPC coverage area to levels similar to those in PS19-fE3 mice ( Fig. 10C In contrast, PS19-fE4 / GFAP-Cre mice had significantly lower OPC coverage relative to PS19-fE4 / Syn1-Cre mice and even showed a trend toward decreased OPCs relative to PS19-fE4 mice, although this did not reach statistical significance ( Fig. 10C To D). After ablation of neuronal APOE3 or astrocytic APOE3 in PS19-fE3 mice, there was no significant difference in OPC coverage ( Fig. 10CTo D). Immunohistochemical staining of myelin and OPCs in fE4 and fE3 mice showed that APOE4 mice lacking the human mutant Tau-P301S did not exhibit myelin defects and had similar levels of OPCs in the hippocampus as fE3 mice, indicating that the effects of APOE4 on these phenotypes are specific to the context of tauopathy ( Fig.10E to F). Collectively, these findings suggest that neuronal APOE4 plays a critical role in depleting the hippocampal OPC pool and causing myelin defects in this composite tauopathy mouse model, whereas astrocytic APOE4 does not contribute significantly to either pathological process.

[0258] snRNA-seq identifies neurodegenerative disease-associated subpopulations of neurons and oligodendrocytes that are largely eliminated by removal of neuronal APOE4 but not astrocytic APOE4

[0259] In order to obtain a deep understanding of the cell type-specific effects of APOE4 in different types of hippocampal cells at the transcriptome level, single-cell nuclear RNA sequencing (snRNA-seq) was performed in the hippocampus isolated from PS19-fE4 mice and PS19-fE3 mice without Cre, with Syn1-Cre or with GFAP-Cre at 10 months of age. After normalization and filtering for quality control, the snRNA-seq data set includes 119,317 cores covering 26,285 genes. Clustering performed by shared nearest neighbors (SNN) and visualization performed by unified manifold approximation and projection (UMAP) show 33 different cell clusters. Based on the expression of their marker genes, these clusters were assigned to 16 excitatory (Ex) neuron clusters (3 to 5, 7, 12, 15, 16, 18 to 20, 23, 25 to 28, 33), 7 inhibitory (In) neuron clusters (6, 8, 9, 13, 22, 24, 30); 3 oligodendrocyte clusters (1, 2, 17), one astrocyte cluster (10), 3 microglia clusters (11, 21, 29), 2 oligodendrocyte progenitor cell (OPC) clusters (14, 32), and 1 unknown cluster (31) ( Fig. Fig.11A As predicted, APOE was highly expressed in astrocytes in PS19-fE4 and PS19-fE3 mice, and its expression was significantly reduced in astrocytes in PS19-fE4 / GFAP-Cre mice ( Fig. 11B As previously reported, some neurons also expressed APOE in PS19-fE4 and PS19-fE3 mice, and neuronal APOE expression was eliminated in PS19-fE4 / Syn1-Cre mice ( Fig. 11B ).

[0260] The log odds ratio estimates from the generalized linear mixed effects model (GLMM_AM) for assessing the relevance to the animal model were used to identify cell clusters that were altered in PS19-fE3, PS19-fE4 / Syn1-Cre, and PS19-fE4 / GFAP-Cre mice compared to those in PS19-fE4 mice. The analysis showed that the probability of oligodendrocyte cluster 17 having cells from PS19-fE4 / Syn1-Cre mice was lower than the probability of having cells from PS19-fE4 mice, wherein the probability of almost completely eliminating in PS19-fE4 / Syn1-Cre mice ( Fig. 11C Based on differentially expressed (DE) gene analysis, cells in cluster 17 showed a dramatic upregulation of the expression of three major heat shock protein genes relative to other oligodendrocyte clusters ( Fig.11A ) and myelin basic protein (Mbp) and myelin-associated oligodendrocyte basic protein (Mobp) genes were significantly downregulated. DE pathway analysis showed enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to general neurodegeneration, AD and other neurodegenerative diseases (Figure 711E), indicating that cluster 17 represents oligodendrocytes associated with neurodegenerative diseases.

[0261] In addition, the probability of excitatory neuron cluster 25 having cells from PS19-fE4 / Syn1-Cre and PS19-fE3 mice was significantly lower than that of having cells from PS19-fE4 mice, with cluster 25 being almost completely eliminated in PS19-fE4 / Syn1-Cre mice ( Fig. 11C , F). DE gene analysis showed that the three major heat shock protein genes ( Fig.11A ), human MAPT gene and amyloid precursor protein (App) gene expression were significantly upregulated. DE pathway analysis showed the enrichment of KEGG pathways related to general neurodegeneration, AD and other neurodegenerative diseases (), indicating that cluster 25 represents neurons related to neurodegenerative diseases.

[0262] Similarly, log odds ratio estimates from another generalized linear mixed effects model (GLMM_Histopathology) to assess associations with histopathology showed that the proportion of cells in both clusters 17 and 25 showed significant negative correlations with hippocampal volume and significant positive correlations with pTau coverage and with astrogliosis and microgliosis. The correlations were largely driven by the PS19-fE4 group and, to a lesser extent, by the PS19-fE4 / GFAP-Cre group ( Fig.11G To J). The proportion of cells in cluster 17 also had a significant negative correlation with the MBP coverage area ( Fig.11D). Taken together, these findings show that ablation of neuronal APOE4, but not astroglial APOE4, leads to a dramatic reduction in the amounts of neurodegenerative disease-associated oligodendrocyte and neuronal subpopulations within the hippocampus, and that both cell populations have a strong correlation with Tau pathology, gliosis, neurodegeneration, and oligodendrocyte deficiency.

[0263] Ablation of neuronal, but not astroglial, APOE4 largely eliminates neurodegenerative disease-associated astrocyte and microglial subsets

[0264] Further subclustering of astrocytes identified 18 subclusters. The log odds ratio estimates from GLMM_AM showed that the odds of astrocyte subclusters 4 and 7 having cells from PS19-fE4 / Syn1-Cre mice were lower than the odds of having cells from PS19-fE4 mice, with subcluster 7 being completely eliminated in PS19-fE4 / Syn1-Cre mice ( Fig.12 A, C, F). Interestingly, astrocyte subclusters 4 and 7 highly expressed APOE ( Fig.12D ). DE gene and pathway analysis showed enrichment of KEGG pathways related to general neurodegeneration, AD and other neurodegenerative diseases in subcluster 7, indicating that subcluster 7 represents neurodegenerative disease-related astrocytes. Subcluster 4 showed enrichment of KEGG pathways related to cAMP signaling, synaptic function and long-term potentiation ( Figure 12Q ), indicating that subcluster 4 represents synaptic dysfunction-associated astrocytes.

[0265] Further subclustering of microglia also identified 18 subclusters. The log odds ratio estimates from GLMM_AM showed that the odds of microglial subclusters 4 and 8 having cells from PS19-fE4 / Syn1-Cre mice were lower than the odds of having cells from PS19-fE4 mice, with subcluster 8 being completely eliminated in PS19-fE4 / Syn1-Cre mice ( Fig.12I Interestingly, microglial subclusters 4 and 8 highly expressed APOE ( Fig.12J ). DE gene and pathway analysis showed enrichment of KEGG pathways related to general neurodegeneration, AD and other neurodegenerative diseases in subcluster 8, indicating that subcluster 8 represents neurodegenerative disease-associated microglia. Subcluster 4 showed enrichment of KEGG pathways related to synaptic function, cAMP signaling and long-term potentiation ( Figure 12R ), indicating that subcluster 4 represents synaptic dysfunction-associated microglia.

[0266] Similarly, log odds ratio estimates from GLMM_Histopathology showed that the proportion of cells in both astrocyte subcluster 7 and microglia subcluster 8 showed significant negative correlations with hippocampal volume and significant positive correlations with pTau coverage and with astrogliosis and microgliosis. The correlations were largely driven by the PS19-fE4 group and, to a lesser extent, by the PS19-fE4 / GFAP-Cre group ( Fig.12D Interestingly, PCA clustering of oligodendrocyte cluster 17 and excitatory neuron cluster 25 together with all astrocyte and microglia subclusters showed that disease-associated astrocyte subcluster 7 and microglia subcluster 8 had similar contributions to the eight pathological parameters as the two disease-associated clusters of oligodendrocytes (cluster 17) and neurons (cluster 25) ( Figure 12S ). Taken together, these findings show that ablation of neuronal APOE4, but not astroglial APOE4, leads to a dramatic reduction in the amounts of neurodegenerative disease-associated astrocyte and microglia subpopulations within the hippocampus, and that both glial subpopulations are strongly associated with Tau pathology, gliosis, and neurodegeneration.

[0267] Ablation of astrocyte APOE4 secondary reduces neuronal APOE4 expression

[0268] During analysis of the snRNA-seq data, ablation of astroglial APOE significantly reduced APOE expression in neurons in addition to its predicted elimination of APOE expression in astrocytes ( Fig. 10B ). To confirm this observation, a publicly available snRNA-seq dataset (GEO: GSE164507) was analyzed using a similar composite mouse model carrying a floxed knock-in human APOE gene and astrocyte-specific Cre (Aldh1l1-Cre / ERT2 BAC transgene) that deleted the human APOE gene in astrocytes in a tamoxifen-inducible manner during adulthood in the presence or absence of P301S mutant human Tau. In this snRNA-seq dataset ( Fig.13A ), neuronal APOE expression was observed in both FE3 and FE4 mice ( Fig. 13B ), which was further increased in PS19-FE4 (TAFE4_Oil) and PS19-FE3 (TAFE3_Oil) mice ( Fig.13A, B), indicating that tau protein disease increases neuronal APOE expression. After removing astrocyte APOE induced by tamoxifen treatment during adulthood, a sharp decrease in neuronal APOE in both excitatory and inhibitory neurons was also clearly observed in PS19-fE4 / Aldh111-Cre / ERT2 (TAFE4_tam) and PS19-fE3 / Aldh111-Cre / ERT2 (TAFE3_tam) mice relative to oil-treated mice ( Fig. 13B , E, F). The effect of astrocyte APOE removal on neuronal APOE reduction was observed in all neuronal subtypes, indicating a general regulation of neuronal APOE expression by astrocyte APOE. Thus, the initial observation that astrocyte APOE removal secondary reduced neuronal APOE expression was confirmed in different tauopathy models with human APOE, even when astrocyte APOE removal was driven by different astrocyte-specific promoters and its removal was induced at different time points in the lifespan of the mouse model.

[0269] To determine whether this observation could be validated at the protein level, the levels of APOE protein in cultured primary neurons isolated from the cortex and hippocampus of prenatal pups were quantitatively evaluated by sandwich ELISA. This analysis showed that primary neurons from PS19-fE4 / GFAP-Cre mice exhibited a significant 50% reduction in APOE protein levels relative to those from PS19-fE4 mice ( Figure 13G These data provide strong evidence that ablation of astrocytic APOE4 leads to a dramatic reduction in neuronal APOE4 expression and protein production and consolidates this regulatory relationship between astrocytic APOE4 and neuronal APOE4. Therefore, the limited protective effect of ablation of astrocytic APOE4 may be due to a secondary reduction in neuronal APOE4 rather than complete elimination.

[0270] Earlier studies have reported that neuronal APOE expression is regulated by factors released by astrocytes through regulation of the ERK pathway within neurons. In-depth comparison of snRNA-seq data between astrocytes from PS19-fE4 / GFAP-Cre mice and astrocytes from PS19-fE4 mice revealed that after astrocyte APOE4 removal, some of the top downregulated genes were clear activators of the ERK / MAPK pathway, including dipeptidyl peptidase 10 (DDP10) and neuregulin-3 (Nrg3). Therefore, it was hypothesized that astrocyte APOE4 could regulate neuronal APOE4 expression by promoting the release of these factors that activate the neuronal ERK pathway, resulting in increased neuronal APOE4 expression. To test this possibility, primary neurons isolated from PS19-fE4 and PS19-fE4 / GFAP-Cre mice were treated with 10 μg / mL of recombinant DPP10 or Nrg3 protein or dPBS control, and then APOE protein levels in cell lysates were quantitatively evaluated by sandwich ELISA. It was observed that APOE protein levels in PS19-fE4 / GFAP-Cre primary neurons treated with DPP10 were significantly increased by 98% (95% CI, 2.0-3.0) compared with those treated with dPBS control. Fig.13 H). On the other hand, there was no significant difference in APOE levels in PS19-fE4 primary neurons after DPP10 treatment ( Fig.13 I). The data suggest that primary neurons from PS19-fE4 / GFAP-Cre mice (in which astrocyte APOE4 is removed) are sensitive to DPP10 upregulation of APOE expression, while primary neurons from PS19-fE4 mice are not. No differences in APOE levels were observed in PS19-fE4 / GFAP-Cre or PS19-fE4 primary neurons after Nrg3 treatment ( Fig.13 H, I), indicating that Nrg3 is not involved in the regulation of neuronal APOE4 expression.

[0271] Neuronal release of HMGB1 was completely abolished by removal of neuronal APOE4 but not astrocytic APOE4

[0272] In the search for proinflammatory cytokines that trigger Tau-induced and neurodegeneration-induced gliosis, we observed that cells in neurodegenerative disease-associated neuronal clusters 25 had significantly increased expression of the high-mobility group box protein 1 (Hmgb1) gene ( Fig. 10A). HMGB1 protein is widely considered to be a factor of glial cell activation. In pathological cases, HMGB1 is translocated from the nucleus to the cytoplasm of stressed cells or dying cells, and it is subsequently released to serve as a proinflammatory cytokine. Immunohistochemical staining of HMGB1 protein and nuclear marker DAPI in 10-month-old mice shows that PS19-fE4 mice have a significant amount of HMGB1 protein, which is translocated from the nucleus of neurons in the dentate gyrus of the hippocampus to the cytoplasm, because it can be found that a considerable portion of the protein is located outside the nucleus of hippocampal neurons. On the other hand, the HMGB1 protein in PS19-fE3 mice is largely retained in the nucleus, and there is very little translocation to the cytoplasm. Interestingly, removing APOE4 from neurons or astrocytes weakens this phenotype observed in PS19-fE4 mice, wherein most of the HMGB1 protein is still located in the nucleus.

[0273] In addition, PS19-fE4 mice contained a large amount of HMGB1 positive spots in both the nucleus and cytoplasm relative to other genotype groups. It is worth noting that fE4 and fE3 mice lacking human mutant Tau-P301S have HMGB1 proteins located in the nucleus, indicating that the nuclear-cytoplasmic translocation of HMGB1 in APOE4 mice requires the coexistence of APOE4 and tau protein disease (Figure 14).

[0274] To evaluate the extent to which disease-related HMGB1 is released from hippocampal neurons within these multiple genotype groups, hippocampal interstitial fluid (ISF) was collected from 8.5-month-old mice over a 24-hour period using in vivo microdialysis. The level of HMGB1 protein in ISF was quantitatively determined using a sandwich ELISA. Although PS19-fE4 mice exhibited high levels of HMGB1 protein in their hippocampal ISF, the removal of neuronal APOE4 reduced HMGB1 protein levels in ISF to undetectable levels, indicating that neuronal APOE4 plays a role in controlling the release of HMGB1 from neurons. The removal of astrocyte APOE4 also resulted in an 86% reduction in HMGB1 protein in ISF rather than complete elimination, which may be due to a secondary reduction in neuronal APOE4 expression rather than complete elimination ( Fig. 13B , F, G).

[0275] To determine whether HMGB1 is a potential regulator of neuronal APOE4 expression, primary neurons isolated from PS19-fE4 / GFAP-Cre and PS19-fE4 mice were treated with 10 μg / mL of recombinant HMGB1 protein or dPBS control and APOE protein levels in cell lysates were quantitatively evaluated by sandwich ELISA. No significant differences in APOE protein levels were observed in PS19-fE4 / GFAP-Cre or PS19-fE4 primary neurons after HMGB1 treatment ( Fig.13 H, I), indicating that HMGB1 is not involved in the regulation of neuronal APOE4 expression.

[0276] In order to evaluate the importance of HMGB1 released by neurons in triggering gliosis in APOE4 related tau diseases, ISF collected from the hippocampus of 8.5 month-old PS19-fE4 mice was injected into the hippocampus of 8.5 month-old wild-type mice. As determined by sandwich ELISA, the ISF fractions injected were rich in relatively high concentrations of HMGB1 protein (fractions 19 to 22) or had undetectable levels of HMGB1 protein as a control (fractions 4 to 7). Wild-type mice were made to receive unilateral injections into the HMGB1 in the right dorsal hippocampus. + or control ISF and were analyzed 6 days after injection to assess dramatic changes in gliosis. - ) ISF-injected wild-type mice showed relatively low levels of microgliosis and astrogliosis on both the injected and uninjected hippocampal sides, indicating that most of the gliosis induced by the injection procedure itself subsided 6 days after injection ( Fig. 14C to F). In contrast, HMGB1 + Wild-type mice injected with ISF showed significantly increased levels of microgliosis on the injected hippocampus relative to the uninjected side ( Fig. 14C Interestingly, compared with the uninjected side, the HMGB1-injected + ISF did not lead to a significant increase in astrogliosis in the injected hippocampus ( Fig.14E to F). Taken together, these data suggest that neuronal release of HMGB1 represents a novel mechanism by which neuronal APOE4 promotes glial activation. Treatment with an HMGB1 inhibitor significantly reduced APOE4-driven gliosis, Tau pathology, and degeneration.

[0277] Based on the discovery that neuron APOE4 is an effective driving factor for neuron HMGB1 release, the treatment effect of HMGB1 inhibitors against the pathogenesis driven by APOE4 in the context of tau disease was tested. For this reason, two well-characterized HMGB1 inhibitors ethyl pyruvate (EP) and glycyrrhizic acid (GA) were tested, which are the selective inhibitors of HMGB1 translocation and release. The mixed solution or saline vehicle of ethyl pyruvate (80mg / kg) and glycyrrhizic acid (20mg / kg) was injected intraperitoneally with three doses per week, and the scheme for 12 weeks was applied to PS19-fE4 and PS19-fE3 mice. When mice were 6.5 months old, processing was started approximately when bad pathological conditions broke out, and when mice were 9.5 months old, serious neurodegeneration and pathological changes were usually completed when they existed, as shown in this study and other studies.

[0278] The effectiveness of EP and GA in blocking the nuclear-cytoplasmic translocation of HMGB1 protein was first confirmed by immunostaining with anti-HMGB1 and DAPI. PS19-fE4 mice treated with saline showed extensive HMGB1 translocation out of the nucleus, while PS19-fE4 mice treated with HMGB1 inhibitor had no discernible HMGB1 translocation ( Fig.15A , B). In PS19-fE3 mice, there was no significant difference in HMGB1 translocation between saline-treated and inhibitor-treated groups.

[0279] Next, the extent of microgliosis and astrogliosis present in the mice following treatment was assessed. + Microglia and CD68 + Saline-treated PS19-fE4 mice exhibited considerable microgliosis throughout the hippocampus, as exemplified by the high coverage of activated microglia ( Fig. 15C , D). PS19-fE4 mice treated with HMGB1 inhibitors showed a significant reduction in microgliosis. In addition, immunostaining with anti-GFAP showed that PS19-fE4 mice treated with saline had extensive astrocyte proliferation throughout the hippocampus, while astrocyte proliferation in PS19-fE4 mice treated with HMGB1 inhibitors was significantly reduced. Whether treated with saline or HMGB1 inhibitors, PS19-fE3 mice did not show significant differences in microgliosis or astrocyte proliferation.

[0280] The degree of Tau pathology was evaluated by immunostaining pTau in these mice. PS19-fE4 mice treated with saline showed significant Tau pathology throughout the hippocampus, while the Tau pathology of PS19-fE4 mice treated with HMGB1 inhibitors was sharply reduced. In addition, the effectiveness of HMGB1 inhibitors in counteracting myelin defects was evaluated by immunostaining MBP. PS19-fE4 mice treated with saline showed severe myelin loss in the stratum radiatum of CA1, while PS19-fE4 mice treated with HMGB1 inhibitors showed rescue of defects with high MBP coverage. There was no discernible difference in myelin coverage or Tau pathology between saline-treated and inhibitor-treated PS19-fE3 mice.

[0281] The effects of HMGB1 inhibitors on neurodegeneration were determined by quantifying hippocampal and posterior lateral ventricle volumes. Saline-treated PS19-fE4 mice exhibited considerable neurodegeneration, while HMGB1 inhibitor-treated PS19-fE4 mice exhibited rescue of neurodegeneration by significant increases in hippocampal volume and significant decreases in posterior lateral ventricle volume relative to saline-treated PS19-fE4 mice. There were no significant differences in neurodegeneration between saline-treated and HMGB1 inhibitor-treated PS19-fE3 mice.

[0282] In summary, these findings show that treating PS19-fE4 mice with HMGB1 inhibitors can effectively prevent the occurrence of obvious pathological conditions driven by APOE4 (including gliosis, Tau pathological conditions, neurodegeneration and myelin defects) by blocking nuclear-cytoplasmic translocation and subsequent release of HMGB1 protein. This supports the view that HMGB1 translocation and release play a role in inducing and exacerbating these multiple pathological conditions driven by APOE4.

[0283] discuss

[0284] Within the central nervous system, APOE is primarily produced by astrocytes; however, conditions of stress or injury can induce neuronal APOE expression. In this study, we demonstrated that APOE4 leads to a significant increase in Tau pathology, gliosis, neurodegeneration, neuronal dysfunction, and myelin defects, as well as enrichment of neurodegenerative disease-associated subpopulations of neurons, oligodendrocytes, astrocytes, and microglia. Ablation of neuronal APOE4 significantly reduced all of these observed pathologies and largely eliminated disease-associated cell subpopulations, while astrocyte APOE4 ablation only reduced gliosis and neurodegeneration, indicating that neuronal APOE4 is a major driver of many of these pathologies.

[0285] Strikingly, snRNA-seq and primary neuronal culture studies revealed that ablation of astrocytic APOE4 also resulted in a substantial, but not complete, reduction in neuronal APOE4 expression and protein production. This suggests that the limited protective effect of ablation of astrocytic APOE4 against gliosis and neurodegeneration may be attributed to this partial reduction in neuronal APOE4. The presence of tau pathology, neurological dysfunction, and myelin defects after ablation of astrocytic APOE4, and the subsequent reduction in neuronal APOE4, may indicate that these pathologies are sensitive to low levels of neuronal APOE4 and can only be fully rescued after its complete elimination. In addition, snRNA-seq analysis showed that even after ablation of astrocytic APOE4, there was still enrichment of disease-associated subpopulations of neurons, oligodendrocytes, astrocytes, and microglia. This finding shows that despite an overall reduction in gliosis and neurodegeneration following astrocyte APOE4 ablation, these neurodegenerative disease-associated cell subpopulations are sensitive to low levels of neuronal APOE4 and will remain unless neuronal APOE4 is completely eliminated.

[0286] In addition, neurodegenerative disease-associated neuronal subpopulations showed a significant increase in the expression of HMGB1, a nuclear protein known to induce neuroinflammation following its translocation to the cytoplasm and subsequent extracellular release. Mechanistically, it was shown for the first time that neuronal APOE4 promotes the release of HMGB1 from neurons, as its removal resulted in a complete abolition of HMGB1 release. In contrast, astrocyte APOE4 removal resulted in a substantial reduction in HMGB1 release, but not complete abolition, probably because it partially but not completely reduced neuronal APOE4. This HMGB1 release driven by neuronal APOE4 induces gliosis and subsequent neurodegeneration and myelin defects, all of which can be effectively blocked by treatment with HMGB1 inhibitors.

[0287] Based on all these findings, the "APOE4-HMGB1-inflammation-degeneration" cascade model of APOE4-related AD and other tau diseases was hypothesized (Figure 16G). In the presence of both neuronal APOE4 and astrocyte APOE4, such as PS19-fE4 mice ( Fig.16A), the pathogenic cascade begins with the neuronal expression of APOE4, which can be induced by a variety of neuronal stressors. Neuronal APOE4 has a potent effect on the accumulation and spread of Tau pathological conditions, which can further induce neuronal APOE4 expression. Elevated neuronal APOE4 cooperates with Tau pathological conditions to trigger nuclear-cytoplasmic translocation and the release of HMGB1 from neurons. Astrocyte APOE4 does not have a direct effect on the accumulation and spread of Tau pathological conditions, however, it indirectly enhances APOE4 expression in neurons by promoting the release of astrocyte factors (such as DPP10) that can regulate the APOE4 expression of neurons, thereby secondary promotion of the release of HMGB1 induced by APOE4 / Tau pathological conditions from neurons. After HMGB1 is released from neurons, it acts as an inflammatory cytokine that induces gliosis. Extensive gliosis, especially the accumulation of toxic astrocyte and microglial subtypes, leads to abnormal phagocytosis of neurons, synapses, and oligodendrocyte-derived myelin, and subsequent neurodegeneration, neurological dysfunction, and oligodendrocyte degeneration.

[0288] Ablation of neuronal APOE4, as shown in PS19-fE4 / Syn1-Cre mice ( Fig. 16B ), resulting in a dramatic reduction in the accumulation and spread of Tau pathology in neurons. The reduction of Tau pathology and the lack of neuronal APOE4 exist together as an obstacle to the release of HMGB1 from neurons. Since astrocyte APOE4 helps to release HMGB1 by indirectly enhancing the expression of APOE4 in neurons, the lack of neuronal APOE4 blocks the indirect effect of astrocyte APOE4 on triggering the release of neuronal HMGB1. In the absence of sufficient neuronal HMGB1 release, gliosis, particularly gliosis of toxic microglia and astrocyte subtypes, will not be induced, thus leading to reduced neurodegeneration, neurological dysfunction and oligodendrocyte degeneration. Therefore, the removal of neuronal APOE4 leads to the complete prevention or rescue of all observed pathological states.

[0289] Ablation of APOE4 in astrocytes, as shown in PS19-fE4 / GFAP-Cre mice ( Fig. 16C), resulting in a significant but incomplete reduction in neuronal APOE4 expression. Reduced levels of neuronal APOE4 are still sufficient to induce significant Tau pathological state accumulation and propagation, which together with low levels of neuronal APOE4 triggers low levels of HMGB1 release. In the absence of sufficiently high levels of HMGB1 release, the total level of gliosis is greatly reduced. Nevertheless, low levels of neuronal APOE4 and HMGB1 release are sufficient to promote the accumulation of toxic astrocyte subtypes and microglial subtypes. The overall reduction in gliosis leads to a reduction in neurodegeneration, although subpopulations of disease-related neurons still exist due to low levels of neuronal APOE4. The presence of toxic glial subtypes still leads to a lack of oligodendrocytes, probably due to the higher sensitivity of oligodendrocytes to lower levels of toxic glial subtypes. High levels of Tau pathological state and the presence of toxic glial subtypes can sustainably lead to neurological dysfunction. Therefore, the removal of astrocyte APOE4 leads to partial prevention or rescue of the observed pathological state.

[0290] Importantly, pharmacological studies suggest that HMGB1 inhibitors represent a novel and effective approach for treating APOE4-associated AD and other tauopathies. Fig.16D ), the release of HMGB1 from neurons is blocked. In the case where the released HMGB1 does not act as an inflammatory cytokine, gliosis is significantly reduced, resulting in a significant reduction in neurodegeneration and oligodendrocyte degeneration. The lack of gliosis and the potential reduction of toxic glial cell subtypes also lead to a significant reduction in Tau pathological states, which together with the reduced neurodegeneration may improve neurological dysfunction. Therefore, HMGB1 inhibitor treatment also leads to the complete prevention or rescue of all observed pathological states.

[0291] Although inhibition of HMGB1 has been investigated as a viable treatment option for a variety of neurological disorders, this study provides the first evidence that treatment with an HMGB1 inhibitor is a valuable and effective treatment for counteracting APOE4-driven effects on many of the apparent AD pathologies. Furthermore, our findings also suggest that developing treatments that target neuronal APOE4 removal may offer greater therapeutic value than astrocyte APOE4 removal. As our studies and those of others have shown, while removal of either neuronal APOE4 or astrocyte APOE4 significantly reduced gliosis and neurodegeneration by the 10-month time point we analyzed, the long-term consequences of the presence of neurodegenerative disease-associated glial subpopulations and high levels of Tau pathology that occur in the absence of astrocyte APOE4 are unclear and may lead to neurodegeneration or disrupt other important processes at later time points. Furthermore, removal of neuronal APOE4 provides the additional benefit of reducing Tau pathology, neurological dysfunction, myelin degeneration, and OPC depletion. In conclusion, our study identifies neuronal APOE4 as a central player in triggering the pathogenic cascade in APOE4-related AD and other tauopathies and provides a potential target for the development of anti-AD and tauopathies drugs.

[0292] Example 15: Method

[0293] Mice. Human LoxP-floxed APOE knock-in (fE) mice with conditional deletion of the human APOE gene were generated. Briefly, homozygous fE3 and fE4 mice 44 were crossed with synapsin 1-Cre transgenic mice [B6.Cg-Tg (Synl-Cre) 671Jxm / J (The Jackson Laboratory) 45 or GFAP-Cre transgenic mice [B6.Cg-Tg (GFAP-Cre) 8Gtm] (National Cancer Institute Mouse Repository). The fE / Cre mice were further hybridized with Tau-P301S (PS19) transgenic mice [B6; C3-Tg (Prnp-MAPT * P301S) PS19Vle / J] (The Jackson Laboratory) expressing human P301S1N4R Tau driven by the PrP promoter to generate PS19-fE4 and PS19-fE3 mice without Cre, with Syn1-Cre or with GFAP-Cre. The littermates born to Syn1-Cre or GFAP-Cre negative were used as PS19-fE controls. In order to generate the PS19-fE / Syn1-Cre strain, only female Syn1-Cre mice were used for breeding purposes because germline recombination has been reported to occur in the offspring of male Syn1-Cre mice. Wild-type (WT) mice [C57BL / 6J] were obtained from the Jackson Laboratory. All mice were of pure C57BL / 6 genetic background and were housed in a pathogen-free barrier facility at 19 to 23°C and 30% to 70% humidity with a 12-hour light cycle. Animals were identified by ear punching under brief isoflurane anesthesia and genotyped by polymerase chain reaction (PCR) of tail shearing. Except for the animals reported in this study, all other animals did not undergo surgery. All animal experiments were performed according to the guidelines and regulations of the National Institutes of Health, the University of California, and the Gladstone Institutes, according to protocol AN176773.

[0294] For brain tissue collection, mice were deeply anesthetized by intraperitoneal injection of Avertin (Henry Schein) and cardiac perfusion of 0.9% saline for 1 minute. The brain was fixed as a whole brain or half brain according to the study. The right hemibrain was drop-fixed in 4% paraformaldehyde (16% PFA diluted in MilliQ H2O) (Electron Microscopy Sciences) for 48 hours, rinsed in 1×PBS (Corning) for 24 hours, and cryopreserved in 30% sucrose (Sigma) at 4°C for 48 hours. The fixed hemibrain was cut into 30 μm thick coronal sections on a freezing sliding microtome (Leica) and stored in a cryoprotectant solution (30% ethylene glycol, 30% glycerol, 40% 1×PBS) at -20°C. The left hemibrain was quickly frozen on dry ice and stored at -80°C.

[0295] Immunohistochemistry.For immunofluorescence staining, several sections (separated by approximately 300 μm) from each mouse are transferred to 12-well plates in 1 × PBS-T (PBS+0.1% Tween-20) (Millipore Sigma) and washed 3 × 5 minutes in PBS-T to remove cryoprotectant solution. Sections are incubated for 5 minutes in boiling antigen retrieval buffer (Tris buffer, pH 7.6) (TEKNOVA) and washed 2 × 5 minutes in PBS-T. Then sections are incubated at room temperature in blocking solution (5% normal donkey serum (Jackson Labs), 0.2% Triton-X (Millipore Sigma) in 1 × PBS) for 1 hour to prevent non-specific antibody binding. After blocking, sections are washed 1 × 5 minutes in PBS-T and incubated at room temperature in mice on mice (Mouse-on-Mouse, MOM) blocking buffer (1 drop of MOM IgG / 4mL PBS-T) (VectorLabs) for 1 hour. After MOM blocking, sections were incubated overnight at 4°C in primary antibodies diluted to optimal concentrations (anti-APOE 1:200 (Cell Signaling); anti-CD68 1:100 (Bio-Rad); anti-Cre 1:800 (Cell Signaling); anti-GFAP 1:800 (Millipore Sigma); anti-GFP 1:5000 (Thermofischer); anti-HMGB1 1:100 (Abcam); anti-Iba1(rbt) 1:200 (Wako); anti-Iba1(gt) 1:200 (Abcam); anti-MBP 1:500 (Abcam); anti-NeuN 1:500 (Millipore Sigma); anti-NG2 1:500 (Abcam); anti-S100β 1:200 (Abcam)). After the primary antibody incubation, the sections were washed 3×5 minutes in PBS-T and then incubated in a fluorescently labeled secondary antibody (Abcam, Jackson Immuno, 1:1000 in PBS-T) diluted in PBS-T for 1 hour at room temperature in the dark. The sections were then washed 2×5 minutes in PBS-T and incubated in DAPI (1:50,000 in PBS-T) (Thermofisher) for 8 minutes at room temperature in the dark. The sections were then washed 2×5 minutes in PBS-T, mounted on microscope slides (FisherScientific), coverslipped with ProLong Gold mounting medium (Vector Laboratories), and sealed with clear nail polish.Images were acquired at 10×, 20×, 40×, or 60× magnifications, depending on staining, using a FV3000 confocal laser scanning microscope (Olympus) or an Aperio VERSA slide scanning microscope (Leica). Image analysis of percentage coverage was performed using open source Fiji (ImageJ) software after setting a standard threshold for all images. The investigators were also blinded to the samples to exclude the possibility of bias.

[0296] For DAB (3,3'-diaminobenzidine) staining, several sections from each mouse (about 300 μm apart) were transferred to a 12-well plate in 1× PBS-T and then washed 3×5 minutes in PBS-T to remove the cryoprotectant solution. The sections were then incubated in boiling antigen retrieval buffer (1× PBS, 0.1 M sodium citrate, 0.1 M citric acid) (FisherScientific, Fluka) for 5 minutes and washed 2×5 minutes in PBS-T. Next, the sections were incubated in endogenous peroxidase buffer (1×PBS, 10% methanol (Fisher Scientific), 3% H2O2 (Sigma) for 15 min and washed 3×5 min in PBS-T. The sections were then incubated in blocking solution (1×PBS-T, 5% normal donkey serum, 1% skim milk powder) for 1 h at room temperature. After blocking, the sections were washed 2×5 min in PBS-T and then incubated in avidin / biotin blocking reagent (4 drops per block) (Vector Laboratories) for 15 min and then washed 2×5 min in PBS-T. The sections were incubated in MOM blocking buffer (1 drop of MOM IgG / 4 mL PBS-T) (Vector Labs) for 1 h at room temperature. After MOM blocking, the sections were washed 2×5 min and diluted to the optimal concentration in PBS-T at 4°C (anti-pTau (AT8) 1:100 (Invitrogen); anti-HT7 1:200 (Peter Labs) Davies)) overnight. After the primary antibody incubation, the sections were washed 3×5 minutes in PBS-T and then incubated in a biotinylated secondary antibody (1:200; Jackson Immuno) for 1 hour at room temperature. Next, the sections were washed 3×5 minutes in PBS-T and incubated in ABC buffer (Vector Laboratories), which was prepared 10 minutes before the incubation step. The sections were washed 2×5 minutes in PBS-T and 1×5 minutes in Tris buffer (pH 7.6). The sections were incubated in DAB buffer (5 mL 1×PBS, 2 drops of buffer stock solution, 2 drops of DAB, 2 drops of H2O2) (Vector Laboratories) for exactly 2 minutes. The staining was terminated by washing the sections in Tris buffer (pH7.6) for 3×5 minutes and 2×5 minutes in PBS-T. The sections were mounted on microscope slides and dried overnight at room temperature. Next, the mounted sections were immersed in Xylene (Fisher Scientific) for 2 x 5 min and coverslipped with DPX mounting medium (Sigma-Aldrich).Images were acquired using an Aperio VERSA slide scanning microscope (Leica) at 10× magnification.

[0297] Volume analysis. Continuous coronal hippocampal brain slices (7 slices per mouse, 30 μm thick, 300 μm apart) were mounted on microscope slides (Fisher Scientific) and dried at room temperature for 1 hour. 0.1% Sudan Black solution was prepared by adding an appropriate amount of SudanBlack powder (Sigma) to 70% ethanol (KOPTEC) and mixing the solution with a magnetic stirrer while protecting from light. The solution was then centrifuged at 3,000 RPM for 10 minutes and the collected supernatant was filtered using a 0.2 μm filter syringe (Thermo Scientific) to remove undissolved dye. The slices were then stained with 0.1% Sudan Black solution for 10 minutes at room temperature and washed 3×2 minutes in 70% ethanol and 3×5 minutes in Milli-Q water. The slices were then coverslipped with ProLong Gold mounting medium (Invitrogen) and imaged at 10× magnification on an Aperio VERSA slide scanning microscope (Leica). For hippocampal and posterior lateral ventricle volume analysis, the area of ​​interest was outlined using the segmentation line tool in ImageJ and the volume was calculated using the following formula: Volume = (sum of areas) * 0.3 mm23. The sum of area values ​​was obtained by taking the sum of the quantified area measurements of all 7 brain sections per mouse, approximately between coordinates AP = -1.2 and AP = -3.4.

[0298] Neuronal layer thickness measurement. Two brain slices (30 μm thick, 300 μm apart) were immunofluorescently stained as described above using the primary antibody NeuN (1:500) to visualize the neuronal cell layer of the hippocampus. The slices were imaged at 20× magnification using a FV3000 confocal laser scanning microscope (Olympus). The thickness of the CA1 pyramidal cell layer and the dentate gyrus granule cell layer of the hippocampus were measured on the Fiji (ImageJ) software by drawing a straight line perpendicular to the NeuN+ cell layer at two points in each hippocampal sub-region and taking the average value of each mouse.

[0299] Nuclear-cytoplasmic localization measurement of HMGB1. As described above, two brain slices (30 μm thick, 300 μm apart) were immunostained with anti-HMGB1 (1: 100) and DAPI (1: 50,000). The slices were imaged at 40× and 60× magnifications using a FV3000 confocal laser scanning microscope (Olympus). All image processing and quantification were performed on Fiji (ImageJ) software. In brief, a 1-pixel median filter was applied to the DAPI channel and an appropriate threshold was set to create a mask for DAPI. The image calculator function was then used to overlay the DAPI mask and the HMGB1 channel, which provided HMGB1 staining that was localized only to the nucleus. After obtaining the values ​​of the integrated density and particles, the DAPI mask was subtracted from the HMGB1 using an image calculator, which provided HMGB1 staining that was excluded from the nucleus.

[0300] Biochemical extraction of brain tissue.The quick-frozen mouse hemibrain was thawed on ice and then the dissection of the hippocampus was performed.The hippocampal tissue was weighed and homogenized with 10 μL / mg tissue using a Polytron immersion disperser Polytron homogenizer (Kinematica AG) in ice-cold RAB buffer (G Biosciences), supplemented with phosphatase inhibitors (Roche) and protease inhibitors (Roche).Then Optima TLX ultracentrifuge (Beckman Coulter) was used to centrifuge the sample for 20 minutes at 50,000g at 4°C, and the supernatant was collected as the RAB soluble fraction.The precipitate was resuspended in ice-cold RIPA buffer (Thermo Scientific) with 10 μL / mg tissue and centrifuged for 20 minutes at 50,000g at 4°C.The supernatant was collected as the RIPA soluble fraction and the precipitate was stored at -80°C for further use.All fractions were stored at -80°C until further analysis.

[0301] Western Blot Analysis. Biochemically extracted mouse hippocampal tissue lysates were loaded onto 12% Bis-Tris SDS-PAGE gels (Invitrogen) and separated by gel electrophoresis at 160 V using MOPS buffer. The separated proteins were transferred to a nitrocellulose membrane at 18 V for 60 min (Trans-Blot Turbo Transfer System (Bio-rad). The membrane was washed 3×5 min in PBS-T and then incubated in Intercept blocking buffer (LI-COR) for 1 h at room temperature to block nonspecific binding sites. After blocking, the membrane was washed 3×5 min in PBS-T and incubated with primary antibodies overnight at 4°C (AT8 1:3,000 (Invitrogen), TUJ1 1:15,000 (Biolegend)). The membrane was washed 3×5 min in PBS-T and incubated in fluorescently labeled secondary antibodies (1:20,000; LI-COR) for 1 h in the dark at room temperature. The resulting bands were detected with the Odyssey CLx infrared imaging system (LI-COR), and the fluorescence intensity of the bands was quantified as the ratio of AT8:TUJ1 signals using Image Studio software.

[0302] Sandwich ELISA. Biochemically extracted mouse hippocampal tissue lysates were diluted in Milli-Q H2O to the appropriate concentration and run according to the provided manufacturer's protocol (human APOE (Abcam); mouse HMGB1 (Novus Biologicals)). Sample reactions were read on a SpectraMaX M5 spectrophotometer (Molecular Devices) and protein concentrations were determined after interpolation of the standard curve and adjustment of dilutions.

[0303] Primary neuronal cultures and recombinant protein treatment. Primary cultures of neurons were prepared from prenatal E20 pups of various genotypes. After collecting pup brains, the cortex and hippocampus were isolated and placed in ice-cold dissociation medium + kynurenic acid medium (DM / KY) (DM: Na2SO4 (81.8mM); K2SO4 (30mM); MgCl2 (5.8mM), CaCl2 (0.25mM); HEPES (1mM); glucose (20mM); phenol red (0.001%); NaOH (0.16mM) in distilled H2O) (KY: kynurenic acid (10mM); phenol red (0.0025%); HEPES (5mM); MgCl2 (100mM); NaOH (added dropwise until pH 7.4)). The obtained DM / KY medium is made by combining 90% DM medium with 10% KY medium. The separated tissue is finely minced and then immersed in preheated papain solution (1 mL / brain) for 13 minutes, while gently inverting, and then immersed in trypsin inhibitor solution for 5 minutes (5 mL, for up to 10 brains), while gently inverting. Wash the tissue pellet with Optimem / glucose solution (20mM glucose, 1 mL / brain) while gently inverting. Then, add fresh Optimem / glucose solution and gently grind the tissue until it is separated into single cells. After filtering the cells through a 40 μm cell strainer, the dissociated cells are 1×10 6 cells / well of a 12-well plate or 3×10 5 Cells / well of a 24-well plate were plated in a 100U / mL medium supplemented with B27. -1 Penicillin G, 100 μg / mL -1 Streptomycin and 1% GlutaMAX in Neurobasal medium (B27 / Neurobasal). Every 3 to 4 days, half of the culture medium was removed and replaced with fresh B27 / Neurobasal medium. In some experiments, primary neurons were treated in vitro for 24 hours with Dulbecco PBS (dPBS) vehicle or target recombinant protein (10 μg / mL in one well of a 12-well plate) on the 14th day. After treatment, the culture medium was collected and the culture was harvested for analysis. The total protein level present in the cell lysate was obtained by BCA analysis (Pierce).

[0304] Stereotaxic surgery for mice. Mice were anesthetized with intraperitoneal injection of ketamine (60 mg / kg) and xylazine (30 mg / kg) and maintained at 0.8% to 1.0% isoflurane (Henry Schein). Mice were fixed in a stereotaxic alignment system model 940 using ear bars and tooth bars (Kopf Instruments). The scalp was prepared by removing hair using Nair and sterilizing with 70% ethanol. The scalp was then incised using a scalpel and sterilized with 70% ethanol. 3% hydrogen peroxide was used to better visualize the cranial sutures. After identifying the bregma, a 0.5 mm microburr (Fine Science Tools) was used to drill holes in the unilateral stereotaxic site using coordinates X=+1.5, Y=-2.1, Z=-2.1 (where Z is measured from the surface of the brain). Mice were injected with 2 μL of the corresponding virus (AAV2 (Y444F) -smCBA-human_P301S_Tau-WPRE, 2.10E+13 vg / mL, Virovek; AAV2-synaptophysin-GFP, 1.0E+13 vg / mL, SignaGen) or ISF fraction at a flow rate of 500 nL / min and allowed to diffuse for 3 minutes. After surgery, mice were sutured with nylon monofilament non-absorbable 6-0 sutures (Henry Schein) and analgesics buprenorphine (intraperitoneal 0.0375 mg / kg), ketophen (subcutaneous 5 mg / kg) and saline (intraperitoneal 500 μL). Mice were monitored on a heating pad until they were ambulatory and provided with hydrogel for hydration.

[0305] Brain slice electrophysiological recording and data analysis

[0306] For electrophysiological recording studies, 8-month-old PS19-fE3 mice and PS19-fE4 mice without Cre, with Syn1-Cre or with GFAP-Cre were anesthetized with isoflurane and decapitated. The brain was quickly removed from the skull and placed in an ice-cold (2 to 5 ° C) slice solution. The slice solution contained (in mM): 110 choline chloride, 2.5 KCl, 26 NaHCO3, 10 MgCl2, 1.25 NaH2PO4, 0.5 CaCl2, 10 glucose, 3 pyruvic acid Na, 1 L-ascorbic acid, pH 7.4. 350 μm thick sagittal sections were cut from both hemispheres using a vibratome (VT1200, Leica) and transferred to a 95% O2-CO2 vapor interface holding chamber (BSK5, Scientific Systems Design) containing artificial cerebrospinal fluid (ACSF), where they were allowed to recover for one hour at 34°C and then kept at room temperature (20 to 22°C). ACSF contained (in mM): 126 NaCl, 2.5 KCl, 1.5 CaCl2, 1.5 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, and 1.5 L-ascorbic acid, pH 7.4.

[0307] For input / output recording studies, a concentric bipolar stimulating electrode (FHC) connected to a constant voltage separation and placed in the CA2 stratum radiatum was used to induce local field postsynaptic potentials (fPSPs) by stimulating Schaffer collaterals in the forward direction. fPSPs were recorded with a glass borosilicate microelectrode filled with ACSF and placed in the CA1 stratum radiatum. The signal was collected and digitized by a MultiClamp 700B amplifier and a Digidata 1550B1 acquisition system (Molecular Devices) with pClamp10 software, and analyzed by running a custom macro using IgorPro6 software (Wavemetrics). The fPSP slope was analyzed as a linear fit slope value of 10% to 90% of the fPSP peak. The input-output relationship was recorded as the fPSP slope value in response to the increased stimulation intensity (20 to 60 μA), and the fPSP slope gain was calculated as the linear slope of the resulting input-output curve.

[0308] Microdialysis of mouse hippocampus.Use in vivo microdialysis of hippocampus to collect brain tissue interstitial fluid.Surgical operation (including preoperative and postoperative care) is performed for stereotaxic surgery as described above.During the operation, a 1.2mm bone drill (BASi) is used to drill a unilateral stereotaxic site, and an AtmosLM guide cannula PEG-4 (Amuza) is stereotaxically implanted above the right hippocampus at coordinates X=+1.5, Y=-2.1, and Z=-1.1. The cannula is fixed in place using dental cement (GC America), and a temporary PEG-4AtmosLM dummy probe (dummy probe) (Amuza) is inserted and fixed with an AC-5 cap nut screw (Eicom).Two days after the operation, mice are placed in a microdialysis independent system (BASi) overnight to assimilate, and the 1000kDa AtmosLM collection probe (Eicom) is inserted into the hippocampus through the guide cannula in the afternoon of the second day, which further extends 1mm downward to Z=-2.1 to target the dentate gyrus. Artificial CSF (Harvard Apparatus) prepared with 0.15% BSA (Thermo Scientific) was circulated through the system at a flow rate of 0.5 μL / min using a push-pull method, and interstitial fluid (ISF) was collected every hour in a refrigerated fraction collector (BASI) for approximately 24 hours. In order to prevent clogging of the tube, the pump was operated at 10× collection speed for the first two hours before the pump was adjusted to a flow rate of 0.5 μL / min. After completing the ISF collection, the mice were euthanized as described above and perfused with 0.9% saline. The brain was dissected into hemispheres, the right hemisphere was post-fixed in 4% PFA for 48 hours and the left hemisphere was freshly frozen. The ISF fractions were frozen at -80°C for further analysis.

[0309] Treated with HMGB1 inhibitors.At 6.5 months of age, male and female PS19-fE4 and PS19-fE3 mice were randomly assigned to a control group or a treatment group. Mice were injected intraperitoneally with a mixture of sterile 0.9% saline (Fisher Scientific) or the following HMGB1 inhibitors: ethyl pyruvate (80mg / kg) (Sigma-Aldrich) and glycyrrhizic acid (20mg / kg) (Sigma-Aldrich) dissolved in 0.9% saline. Mice were injected three times a week for 12 weeks, starting at 6.5 months of age until they arrived at 9.5 months of age. The weight changes, hair licking changes and postures of all mice were monitored during the experiment and no changes were observed. After treatment, animals were perfused and their brain tissues were processed for histopathological analysis as described above.

[0310] 10× loaded single nucleus preparation. Mouse hippocampus was dissected on ice and placed in a pre-cooled 2 mL Dounce containing 1 mL cold 1× homogenization buffer (1×HB) (250 mM sucrose, 25 mM KCL, 5 mM MgCl2, 20 mM Tricine-KOH pH7.8, 1 mM DTT, 0.5 mM Sermidine, 0.15 mM Sermine, 0.3% NP40, 0.2 units / μL RNase inhibitor, 0.2 units / μL protease inhibitor). Pestle loosely with Dounce "A" (about 10 pestles) and then tightly with "B" (about 15 pestles). The homogenate was filtered using a 70 μM Flowmi filter (Eppendorf) and transferred to a pre-cooled 2 mL LoBind tube (Fischer Scientific). The nuclei were precipitated by centrifugation at 350 RCF for 5 minutes at 4°C. The supernatant was removed and the nuclei were resuspended in 400 μL of 1×HB. Next, 400 μL of 50% iodixanol solution was added to the nuclei and then slowly layered with 600 μL of 30% iodixanol solution at 25% mixture, and then layered with 600 μL of 40% iodixanol solution at 30% mixture. The nuclei were then centrifuged at 3,000 g for 20 minutes at 4°C in a pre-cooled swing bucket centrifuge. The 200 μL nuclear band at the 30% to 40% interface was collected and transferred to a new tube. Then, 800 μL of 2.5% BSA in PBS plus 0.2 units / μL of RNase inhibitor was added to the nuclei and then centrifuged at 500 RCF for 10 minutes at 4°C. The nuclei were resuspended with 2% BSA in PBS plus 0.2 units / μL of RNase inhibitor to reach approximately 500 nuclei / μL. The nuclei were then filtered with a 40 μM Flowmi filter. Nuclei were counted and approximately 13,000 nuclei / sample were subsequently loaded onto a 10× GenomicsNext GEM Chip G. snRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 (10× Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq6000 sequencer at the UCSF CAT Core.

[0311] Custom reference genome. The PS19 tau mutant floxed APOE knock-in mouse model was used for single-cell nuclear RNA sequencing (snRNA-seq). Homo sapiens microtubule associated protein tau (MAPT) (NCBI reference sequence: NM_001123066.4) and Homo sapiens APOE were the target genes of this study. These genes are not expected to be part of the mouse reference genome, so in order to quantify the reads aligned to these target genes, a custom mouse reference genome was made using the following: the reference mouse genome sequence (GRCm38) from Ensembl (release 98) and the GENCODE (release M23) from GENCODE (release M23). 79 Mouse gene annotation files were prepared similar to those used in the 10×Genomics Cell Ranger mouse reference package mm102020-A. The headers of the Ensembl reference mouse genome sequence fasta files with chromosome names were modified to match the chromosome names in the fasta files from GENCODE. The annotation GTF files contained entries from non-polyA transcripts that overlapped with protein-coding genes. These reads were marked as multiply mapped and not counted by the 10×Genomics Cell Ranger v6.1.1 counting pipeline. To avoid this, the GTF files were modified to (1) remove the version suffixes from the transcript, gene, and exon ids to match the Cell Ranger reference package, and (2) remove non-polyA transcripts. The Homo sapiens MAPT sequence and Homo sapiens APOE sequence were appended to the end of the mouse reference genome sequence as separate chromosomes and the corresponding gene annotations were appended to the filtered mouse reference gene annotation GTF file. The modified fasta files and GTF files were used to build a custom reference genome using the 10×Genomics CellRanger v6.1.1mkref pipeline.

[0312] Pretreatment and clustering of mouse snRNA-seq samples. snRNA-seq samples include a total of 16 samples, of which four genotype groups (PS19-fE4, PS19-fE4 Syn1-Cre, PS19-fE4GFAP-Cre and PS19-fE3) each have four mice. There are two male mice and two female mice in each group of four mice. As described in the Cell Ranger document, the demultiplexing fastq files of these samples are compared with the custom mouse reference genome (see the other description of the custom reference genome method) using 10×Genomics Cell Ranger v6.1.1 counting process. The flag containing introns of the counting process is set to true to count the reads mapped to the intron region. The Cell Ranger counting website summarizes the "Low Fraction Reads in Cells" error of two samples-one from the PS19-fE4 GFAP-Cre group and one from the PS19-fE3 group. These two samples had only about 40% of the reads assigned to cell-associated barcodes and <80% of the reads mapped to the genome. For the other 14 samples, these metrics were much higher. Inspection of the experimental records showed that these two samples had problems in the nuclear isolation step and that lower cDNA was recovered due to the use of expired old batches of sample preparation reagents. All other 14 samples were prepared with new batches of sample preparation reagents. Therefore, these two samples were excluded and only the remaining 14 samples were used for downstream analysis with Seurat.

[0313] Single cell nucleus analysis using Seurat v4.0.5 81 The filtered count matrices generated by the Cell Ranger counting pipeline for the 14 samples were processed using the R package sctransform. Each sample was preprocessed into a Seurat object, and the top 1% of cells of each sample with a large number of unique genes, cells with <=200 unique genes, and cells with >=0.25% mitochondrial genes were filtered out for each sample. The 14 samples were merged into a single Seurat object and normalized and variance stabilized using sctransform with the "glmGamPoi" (Bioconductor package version 1.6.0) method for initial parameter estimation.

[0314] Graph-based clustering was performed using the Seurat v4.0.5 functions FindNeighbors and FindClusters. First, cells were built into a k-nearest neighbor (KNN) graph based on the Euclidean distance in PCA space. The edge weights between two cells were further modified using Jaccard similarity. Next, clustering was performed using the Louvain algorithm implementation in the FindClusters Seurat function. All combinations of 10, 15, and 20 PCs with 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 resolutions were clustered. Clustering with 15 PCs and 0.7 resolution produced 33 different biologically relevant clusters, which were used for further analysis.

[0315] Cell type assignment. Data visualization of 14 samples in UMAP space using Seurat v4.0.5 showed no batch effects for age, sex, genotype, date of birth, or date of nuclear isolation. Marker genes for each cluster were identified for SCT assay data using the FindAllMarkers Seurat function. The algorithm used the Wilcoxon rank sum test to iteratively identify differentially expressed genes for one cluster against all other clusters. Marker genes were filtered to retain only positively expressed genes that were detected in at least 25% of cells in any population and had at least a 0.5log2 fold change. Cell clusters were assigned identities by matching them to known cell types with expression of typical cell type-specific genes, expression of genes identified in a publicly available mouse hippocampal single-cell RNA-seq dataset, and expression of marker genes for each cluster in a publicly available whole-brain in situ hybridization image resource.

[0316] Subclustering of astrocyte and microglia sn-RNA-seq data. Annotate hippocampal cluster 10 as astrocytes and hippocampal clusters 11, 21, and 29 as microglia. Both cell types are subclustered. Use sctransform 82Normalization and variance stabilization were performed using the "glmGamPoi" (Bioconductor package version 1.6.0) method for initial parameter estimation. Graph-based clustering was performed using the Seurat v4.0.5 functions FindNeighbors and FindClusters. First, cells were built into a k-nearest neighbor (KNN) graph based on the Euclidean distance in PCA space. The edge weights between two cells were further modified using Jaccard similarity. Next, clustering was performed using the Louvain algorithm implemented in the FindClusters Seurat function. All combinations of 10, 15, 20, 25, and 30 PCs with resolutions of 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 were clustered. Subclustering with 15 PCs and 0.9 resolution produced 18 different biologically relevant subclusters of astrocytes. Subclustering with 15 PCs and 0.9 resolution produced 18 different biologically relevant subclusters of microglia.

[0317] Gene set enrichment analysis. The FindMarkers Seurat function was used to identify differentially expressed genes between the target clusters for SCT assay data. The algorithm used the Wilcoxon rank sum test to identify differentially expressed genes between the two populations. Differentially expressed genes were limited to genes detected in at least 10% of cells in either population and had at least a 0.1log2 fold change. Overexpression (or enrichment) analysis was performed using clusterProfiler v4.2.1 to find gene sets for mice associated with differentially expressed genes in the KEGG database. The p value was based on a hypergeometric test and adjusted for multiple testing using the Benjamini-Hochberg method. Significantly enriched gene sets were filtered to have an adjusted p value of less than 0.8, and there were at least 10 differentially expressed genes in the gene set. The same method was used to perform gene set enrichment analysis of astrocyte subclusters and microglia subclusters.

[0318] The correlation between cluster and genotype.In lme4 (v1.1-27.1) R package, implement the generalized linear mixed effects model (GLMM_AM) for assessing the correlation with animal model, and use this generalized linear mixed effects model to estimate the correlation between cluster members and mouse model.These models are run respectively for each cell cluster.When the family parameter is set to binomial probability distribution and the bobyqa control optimizer is used for maximum likelihood estimation, the GLM model is run.According to whether the cell belongs to the cluster under consideration, the cluster member of each cell is simulated as a 0-1 response variable.The corresponding mouse id from which the cell is derived is a random effect variable, and the animal model of the mouse id is included as a fixed variable.The reference animal model is set to PS19 fE4.The obtained p value of the logarithmic odds ratio estimated in three animal models (relative to PS19 fE4) and clusters is adjusted for multiple tests using the Benjamini-Hochberg method.The inter-cluster correlation of the genotype with astrocyte subcluster and microglia subcluster is estimated using the same method.

[0319] Correlation between the proportion of cell types and histopathological parameters. A generalized linear mixed effects model (GLMM_histopathology) was implemented in the lme4 (v1.1-27.1) R package to assess the correlation with histopathology and was used to identify cell types whose proportions were significantly correlated with histopathological changes in the samples. These models were run separately for each cell cluster combination and the following eight histological parameters: hippocampal volume (mm 3 ), AT8 coverage area percentage, GFAP coverage area percentage, S100β coverage area percentage, IBA1 coverage area percentage, CD68 coverage area percentage, MBP coverage area percentage and OPC coverage area percentage. GLM model is performed when the family parameter is set to the binomial probability distribution family and the 'bobyqa' control optimizer is used for maximum likelihood estimation. According to whether the cell belongs to the cluster under consideration, the cluster member of each cell is simulated as a 0-1 response variable. The corresponding mouse model from which the cell is derived is included as a random effect, and the mouse id in a given mouse model is also further simulated as a random effect. Note that this represents the hierarchical nature of this data for GLMM, and first assumes that the mouse model is sampled from a "universal" mouse model, and then mice are sampled in each mouse model. In contrast to fixed effects, the modeling selection including the mouse model as a random effect means improving degrees of freedom (or maximizing statistical power) to detect the relevance of the purpose, particularly considering the relatively small amount of repetitions (3 to 4 times) of each animal model. The histological parameters considered are simulated as the fixed effects in the model.

[0320] After adjusting the distribution of p-values ​​in the histopathological parameters across cell types with the Benjamini-Hochberg multiple testing correction, a subset of the cell types of interest was selected and the log odds ratio estimates (derived from the GLMM fit) were visualized in a heatmap using the pheatmap package 1.0.12. The process was applied to astrocyte and microglia subtypes and the correlation between the astrocyte and microglia subtypes of interest and the eight histopathological parameters in Figures 4h and 4i were visualized, respectively. The first five principal component coordinates were estimated using the eight log odds ratios of unit changes in the histopathological parameters for each cell type and the astrocyte and microglia subtypes of interest. This was implemented using prcomp(scale=T, center=T) in the stats R package. We visualized the first two PCs using fviz_pca_ind() implemented in the factoextra1.0.7 R package.

[0321] Mouse snRNA-seq dataset from GEO: GSE164507. Mouse snRNAseq data (accession number GSE164507) available on the Gene Expression Omnibus database at ncbi.nlm.nih.gov / geo were reanalyzed. Download the fully publicly available dataset, which contains, for each sample, a filtered gene matrix based on cellular expression, a file with barcodes, and a file with expressed genes. Briefly, this study examined P301S mutant Tau transgenic mice carrying floxed APOE-ε4 or APOE-ε3 alleles. These mice were crossed with mice expressing Cre recombinase under the regulation of tamoxifen-inducible ER elements and the Aldh1l1 astrocyte-specific promoter. Tamoxifen was administered to these Aldh1l1-CreERT2 mice at 5.5 months of age after the onset of tau pathology to induce Cre recombinase expression, or vehicle was administered. Single nuclei isolated from the hippocampus of these mice were sequenced using 10×Genomics Chromium single-cell sequencing, and the data were processed using the Cell Ranger single-cell software suite (v3.0.2). The filtered count matrices generated by the Cell Ranger counting process for all 8 samples were processed using Seurat v4.0.4. The samples were filtered to contain only cells with 500 to 2,000 genes detected and <5% mitochondrial reads. The filtered samples were merged into a single Seurat object, which contained a matrix of 33,457 genes by 63,248 nuclei. Normalization and variance stabilization were performed using sctransform. Clustering was determined as implemented in Seurat v4.0.4 using the RunPCA(), FindNeighbors(), and FindClusters() functions. The nearest neighbor distances were calculated using up to the first 15 PCs. The algorithm builds cells into a k-nearest neighbor graph based on the Euclidean distance in the PCA space. Jaccard similarity was used to further refine the edge weights between any two cells. Clustering was performed using the Louvain algorithm with default settings and a resolution of 0.7, resulting in a set of 22 different clusters. Differential gene expression was detected using the Wilcoxon rank sum test in Seurat, which was implemented by the FindMarkers() function with min.pct=0.1, test.use="wilcox", and logfc.threshold=0.05.

[0322] General statistical analysis. The differences between genotype groups were evaluated by common one-way ANOVA and Tukey's post hoc multiple comparison test, where the mean of each column was compared to the mean of every other column. All plotted data are shown as mean ± SEM. Simple linear regression was used to analyze the correlation between two data in the same genotype group and plotted as mean ± SEM. GraphPad Prism version 9.2.0 was used for analysis and to create the graph.

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[0411] All patents and publications cited or mentioned herein are indicative of the level of skill of those skilled in the art to which the invention pertains, and each such cited patent or publication is expressly incorporated by reference herein to the same extent as if individually incorporated by reference in its entirety or set forth herein in its entirety. Applicants reserve the right to actually incorporate into this specification any and all materials and information from any such cited patent or publication.

[0412] The following statement summarizes some aspects and features of the invention.

[0413] statement:

[0414] 1. A method comprising administering one or more inhibitors of high mobility group box protein 1 (HMGB1) to a subject having at least one genomic APOE4 allele.

[0415] 2. The method of statement 1, wherein the subject has two genomic APOE4 alleles.

[0416] 3. The method of statement 1 or 2, wherein the subject expresses detectable levels of APOE4 protein.

[0417] 4. The method of statement 1, 2, or 3, wherein the subject's cerebrospinal fluid has elevated levels of high mobility group box protein 1 (HMGB1), dipeptidyl peptidase 10 (DPP10), or elevated levels of both HMGB1 and DPP10 relative to a control.

[0418] 5. The method of any one of statements 1 to 4, wherein the level of HMGB1 or DPP10 in the subject's cerebrospinal fluid is increased by at least 25%, or at least 50%, or at least 90% relative to a control.

[0419] 6. The method of any one of statements 1 to 5, wherein the one or more inhibitors of HMGB1 are glycyrrhizic acid, ethyl pyruvate, nicotine, (-) epigallocatechin gallate (EGCG), tanshinone, chlorogenic acid, emodin-6-O-β-D-glucoside, rosmarinic acid, isorhamnetin-3-O-galactoside, polygonum hydropiper, forsythiaside B, chloroquine, actinomycin, shikonin, carbenoxolone, quercetin, lycopene, nafamostat mesylate, gabexate mesylate, sivelestat sodium, HMGB1 monoclonal antibodies such as m2G7 or #10-22, recombinant HMGB1 box A protein, acetylcholine, nicotinic acetylcholine receptor subtype α7 agonist GTS-21, peptide P5779, resveratrol, metformin, or a combination thereof.

[0420] 7. The method of any one of statements 1 to 6, wherein the subject exhibits symptoms of: HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, tau pathology, or myelin defects.

[0421] 8. The method of any one of statements 1 to 7, wherein the subject exhibits at least one symptom of tauopathy.

[0422] 9. The method of any one of Statements 7 or 8, wherein the tauopathy is a neurodegenerative disorder characterized by deposition of abnormal tau protein in the brain.

[0423] 10. The method of any one of statements 7 to 9, wherein the tauopathy is Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, primary age-related tauopathy, chronic traumatic encephalopathy, or frontotemporal dementia.

[0424] 11. A method comprising measuring the levels of high mobility group box protein 1 (HMGB1) and dipeptidyl peptidase 10 (DPP10) in the cerebrospinal fluid of at least one subject and administering one or more HMGB1 inhibitors to any subject having elevated levels of HMGB1 or DPP10 relative to a control.

[0425] 12. The method of statement 11, wherein at least one of the subjects has at least one genomic APOE4 allele.

[0426] 13. The method of statement 11 or 12, wherein the subject has two genomic APOE4 alleles.

[0427] 14. The method of any one of statements 11 to 13, wherein the subject expresses detectable levels of APOE4 protein.

[0428] 15. The method of any one of statements 11 to 14, wherein the increase in the level of HMGB1 or DPP10 relative to a control is an increase in the level of HMGB1 or DPP10 relative to a control of at least 25%, or at least 50%, or at least 90%.

[0429] 16. The method of any one of statements 11 to 15, wherein the one or more inhibitors of HMGB1 are glycyrrhizic acid, ethyl pyruvate, nicotine, (-) epigallocatechin gallate (EGCG), tanshinone, chlorogenic acid, emodin-6-O-β-D-glucoside, rosmarinic acid, isorhamnetin-3-O-galactoside, polygonum hydropiper, forsythiaside B, chloroquine, actinomycin, shikonin, carbenoxolone, quercetin, lycopene, nafamostat mesylate, gabexate mesylate, sivelestat sodium, HMGB1 monoclonal antibodies such as m2G7 or #10-22, recombinant HMGB1 box A protein, acetylcholine, nicotinic acetylcholine receptor subtype α7 agonist GTS-21, peptide P5779, resveratrol, metformin, or a combination thereof.

[0430] 17. The method of any one of statements 11 to 16, wherein the subject exhibits symptoms of: HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, tau pathology, or myelin defects.

[0431] 18. The method of any one of statements 11 to 17, wherein the subject exhibits at least one symptom of tauopathy.

[0432] 19. The method of any one of Statements 17 or 18, wherein the tauopathy is a neurodegenerative disorder characterized by deposition of abnormal tau protein in the brain.

[0433] 20. The method of any one of statements 18 to 19, wherein the tauopathy is Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, primary age-related tauopathy, chronic traumatic encephalopathy, or frontotemporal dementia.

[0434] The specific methods and compositions described herein represent some preferred embodiments, and are exemplary and are not intended to limit the scope of the invention. Those skilled in the art will think of other purposes, aspects and embodiments after considering this specification, and the other purposes, aspects and embodiments are all encompassed in the spirit of the present invention as defined by the scope of the claims. It will be apparent to those skilled in the art that, without departing from the scope and spirit of the present invention, various replacements and modifications may be made to the present invention disclosed herein.

[0435] The present invention illustratively described herein can be appropriately practiced in the absence of any one or more elements or one or more limitations that are not specifically disclosed as essential elements herein. The methods and processes illustratively described herein can be appropriately practiced in different step orders, and the methods and processes are not necessarily limited to the step orders indicated in this article or in the claims.

[0436] Unless the context clearly dictates otherwise, nouns as used herein and in the appended claims without quantifiers refer to both one and more. Thus, for example, reference to "nucleic acid" or "protein" or "cell" includes a plurality of such nucleic acids, proteins or cells (e.g., solutions or dried preparations of nucleic acids or expression cassettes, solutions or cell populations of proteins), etc. In this document, unless otherwise indicated, the term "or / or" is used to refer to a non-exclusive or / or, such that "A or B" includes "A but not B", "B but not A", and "A and B".

[0437] In no event shall this patent be construed as limited to the particular examples or embodiments or methods specifically disclosed herein. In no event shall this patent be construed as limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office unless such statement is expressly and unconditionally or without reservation expressly adopted in the applicant's responsive written submission.

[0438] The terms and expressions that have been used are used as descriptive rather than limiting terms, and it is not intended that such terms and expressions be used to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications may be made within the scope of the invention claimed. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims and statements of the present invention.

[0439] The present invention has been described broadly and generally herein. Each narrower substance and subclass grouping falling within the general disclosure also forms a part of the present invention. This includes the general description of the invention with a proviso or negative limitation (removing any subject matter from the genus), regardless of whether the removed content is specifically described herein. In addition, where features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will recognize that the present invention is also described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A method comprising administering one or more inhibitors of high mobility group box protein 1 (HMGB1) to a subject having at least one genomic APOE4 allele.

2. The method of claim 1, wherein the subject has two genomic APOE4 alleles.

3. The method of claim 1 or 2, wherein the subject expresses detectable levels of APOE4 protein.

4. The method of claim 1, 2 or 3, wherein the subject's cerebrospinal fluid has elevated levels of high mobility group box protein 1 (HMGB1), dipeptidyl peptidase 10 (DPP10), or elevated levels of both HMGB1 and DPP10 relative to a control.

5. The method of any one of claims 1 to 4, wherein the level of HMGB1 or DPP10 in the subject's cerebrospinal fluid is increased by at least 25%, or at least 50%, or at least 90% relative to a control.

6. The method of any one of claims 1 to 5, wherein the one or more inhibitors of HMGB1 are glycyrrhizic acid, ethyl pyruvate, nicotine, (-) epigallocatechin gallate (EGCG), tanshinone, chlorogenic acid, emodin-6-O-β-D-glucoside, rosmarinic acid, isorhamnetin-3-O-galactoside, polygonum hydropiper, forsythiaside B, chloroquine, actinomycin, shikonin, carbenoxolone, quercetin, lycopene, nafamostat mesylate, gabexate mesylate, sivelestat sodium, HMGB1 monoclonal antibodies such as m2G7 or #10-22, recombinant HMGB1 box A protein, acetylcholine, nicotinic acetylcholine receptor subtype α7 agonist GTS-21, peptide P5779, resveratrol, metformin, or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the subject exhibits symptoms of: HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, tau pathology, or myelin defects.

8. The method of any one of claims 1 to 7, wherein the subject exhibits at least one symptom of tauopathy.

9. The method of any one of claims 7 or 8, wherein the tauopathy is a neurodegenerative disorder characterized in that Deposits of abnormal tau protein in the brain.

10. The method of any one of claims 7 to 9, wherein the tauopathy is Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, primary age-related tauopathy, chronic traumatic encephalopathy, or frontotemporal dementia.

11. A method comprising measuring the levels of high mobility group box protein 1 (HMGB1) and dipeptidyl peptidase 10 (DPP10) in the cerebrospinal fluid of at least one subject and administering one or more HMGB1 inhibitors to any subject having elevated levels of HMGB1 or DPP10 relative to a control.

12. The method of claim 11, wherein the at least one subject has at least one genomic APOE4 allele.

13. The method of claim 11 or 12, wherein the subject has two genomic APOE4 alleles.

14. The method of any one of claims 11 to 13, wherein the subject expresses detectable levels of APOE4 protein.

15. The method of any one of claims 11 to 14, wherein the increase in the level of HMGB1 or DPP10 relative to a control is an increase in the level of HMGB1 or DPP10 relative to a control of at least 25%, or at least 50%, or at least 90%.

16. The method of any one of claims 11 to 15, wherein the one or more inhibitors of HMGB1 are glycyrrhizic acid, ethyl pyruvate, nicotine, (-) epigallocatechin gallate (EGCG), tanshinone, chlorogenic acid, emodin-6-O-β-D-glucoside, rosmarinic acid, isorhamnetin-3-O-galactoside, polygonum hydropiper, forsythiaside B, chloroquine, actinomycin, shikonin, carbenoxolone, quercetin, lycopene, nafamostat mesylate, gabexate mesylate, sivelestat sodium, HMGB1 monoclonal antibodies such as m2G7 or #10-22, recombinant HMGB1 box A protein, acetylcholine, nicotinic acetylcholine receptor subtype α7 agonist GTS-21, peptide P5779, resveratrol, metformin, or a combination thereof.

17. The method of any one of claims 11 to 16, wherein the subject exhibits symptoms of HMGB1 nuclear-cytoplasmic translocation, gliosis, neurodegeneration, tau pathology, or myelin defects.

18. The method of any one of claims 11 to 17, wherein the subject exhibits at least one symptom of tauopathy.

19. The method of any one of claims 17 or 18, wherein the tauopathy is a neurodegenerative disorder characterized in that Deposits of abnormal tau protein in the brain.

20. The method of any one of claims 18 to 19, wherein the tauopathy is Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, primary age-related tauopathy, chronic traumatic encephalopathy, or frontotemporal dementia.

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