Compositions and methods for treating Alzheimer's disease

Abstract

Disclosed is a method of treating, preventing, or delaying the onset of Alzheimer's disease (AD) in a subject by targeting a novel pathway, STAT1-Ch25H, in AD development, specifically by administering to the subject a pharmaceutically effective amount of a STAT1 inhibitor, a CH25H inhibitor, or a 25-OHC inhibitor, such as a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, e.g., simvastatin. [Selected Figure] Figure 6B

Description

[Technical field]

[0001] The present invention relates generally to agents and their use for the treatment of disease, specifically agents that target and inhibit signal transducer and activator of transcription 1 (STAT1), cholesterol 25-hydroxylase (CH25H) and 25-hydroxylated cholesterol (25-OHC); and their use in managing physiological conditions, specifically in the treatment and / or prevention of various diseases, including central nervous system diseases, neurodegenerative diseases such as Alzheimer's disease, and other diseases. FIELD OF THEINVENTION

[0002] As human life spans increase, Alzheimer's disease (AD) is becoming a widespread health problem worldwide. AD patients suffer from cognitive decline in their memory, adaptation, judgment, and reasoning (Tanzi & Bertram, 2005). Typically, the disease takes about 10 years from onset to the final stage leading to death. To date, no curative or preventive therapy is available.

[0003] The deposition of Aβ proteins in the brain, a metabolite of the amyloid precursor protein (hereafter referred to as APP), together with their toxicity, is considered to be the main causative factor in the pathogenesis of AD (Selkoe 2001). Aβ is a protein component identified in senile plaques found in AD patients (Ikeda, Wong et al. 1987; Cole, Masliah et al. 1991). Biologically, Aβ is generated through sequential cleavage of the amyloid beta precursor protein (APP) that produces fragments of different sizes. Pharmacological studies have identified Aβ42 as the most pathogenic form that contributes to the pathogenesis of AD (Roher, Chaney et al. 1996; Morelli, Prat et al. 1999). The production of Aβ42 results from beta cleavage followed by gamma cleavage of APP (Citron 2010, De Strooper, Vassar et al. 2010). These insoluble Aβ42 fragments aggregate in the extracellular space to form plaques. Several lines of evidence suggest that soluble oligomers of Aβ42 are much more toxic. The presence of Aβ42 affects synaptic activity. Accumulation of Aβ42 can potentially induce abnormal network activity and synaptic depression (Palop & Mucke 2009).

[0004] Preclinical studies in transgenic mouse models have shown that immunotherapy can be highly effective in preventing both AD and prion disease (Wisniewski 2012). Since Aβ is known to be the central molecule in AD, several strategies have been developed aiming at eradicating Aβ by small molecules or immunotherapy (Tabira 2011;Huang & Mucke, 2012;Ozudogru & Lippa, 2012;Delrieu, Ousset et al., 2014). Although Aβ-directed immunization has given promising results in mouse models of AD, the translation of effective therapies to humans remains elusive.

[0005] In active vaccination trials, actively immunized individuals showed a significant reduction in plaque deposition and a marked reduction in Aβ compared to non-immunized controls. Despite the promising results, treatment groups showed no improvement in long-term survival, time to develop severe dementia, or cognitive function (Holmes, Boche et al. 2008). Two recent large-scale Phase III clinical trials of passive immunization targeting Aβ were terminated without evidence of clinical benefit (Doody, Thomas et al. 2014; Salloway, Sperling et al. 2014). It is therefore desirable to find new and novel therapeutic or preventative therapies for AD. BRIEF DESCRIPTION OF THE DISCLOSURE

[0006] In one aspect, the invention is a method of treating Alzheimer's disease (AD) in a subject by administering to the subject a pharma- ceutically effective amount of a STAT1 inhibitor. In one embodiment, the STAT1 inhibitor is an antibody against IFN or an IFN receptor. In another embodiment, the STAT1 inhibitor is an antibody against IL-6 or an IL-6 receptor. In yet another embodiment, the STAT1 inhibitor is an agent that inhibits JAK1 and JAK3. In yet another embodiment, the STAT1 inhibitor is an agent that reduces STAT1 phosphorylation. In a further embodiment, the STAT1 inhibitor is an agent that prevents nuclear translocation of STAT1. In a further embodiment, the STAT1 inhibitor is an RNAi agent against STAT1.

[0007] In another aspect, the present invention is a method of treating Alzheimer's disease (AD) in a subject by administering a pharma- ceutically effective amount of CH25H to the subject. In some embodiments, the CH25H inhibitor is a STAT1 inhibitor. In another embodiment, the CH25H inhibitor is a STAT1 inhibitor that is an antibody to IFN or IFN receptor. In yet another embodiment, the CH25H inhibitor is a STAT1 inhibitor that is an antibody to IL-6 or IL6 receptor. In yet another embodiment, the CH25H inhibitor is a STAT1 inhibitor that is an agent that reduces STAT1 phosphorylation. In yet another embodiment, the CH25 inhibitor is an agent that prevents STAT1 nuclear translocation. In yet another embodiment, the CH25H inhibitor is a STAT1 inhibitor that is an RNAi agent against STAT1. In yet another embodiment, the CH25H inhibitor is a STAT1 inhibitor that is an RNAi agent against STAT1.

[0008] In yet another aspect, the present invention is a method for treating Alzheimer's disease (AD) in a subject, by administering a 25-OHC inhibitor in a pharmacologic effective amount to said subject.In one embodiment, the 25-OHC inhibitor is a CH25H inhibitor or a STAT1 inhibitor.In another embodiment, the 25-OHC inhibitor is a derivative of 25-OHC.In yet another embodiment, the 25-OHC inhibitor is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, such as simvastatin.

[0009] In another aspect, the invention is a method of preventing or delaying the onset of Alzheimer's disease (AD) in a subject by administering to said subject a pharma- ceutically effective amount of a STAT1 inhibitor. In one embodiment, the STAT1 inhibitor is an antibody against IFN or an IFN receptor. In another embodiment, the STAT1 inhibitor is an antibody against IL-6 or an IL-6 receptor. In yet another embodiment, the STAT1 inhibitor is an agent that inhibits JAK1 and JAK3. In yet another embodiment, the STAT1 inhibitor is an agent that reduces STAT1 phosphorylation. In a further embodiment, the STAT1 inhibitor is an agent that prevents nuclear translocation of STAT1. In a further embodiment, the STAT1 inhibitor is an RNAi agent against STAT1.

[0010] In yet another aspect, the invention is a method of preventing or delaying the onset of Alzheimer's disease (AD) in a subject by administering to said subject a pharma- ceutically effective amount of a CH25H inhibitor. In some embodiments, the CH25H inhibitor is a STAT1 inhibitor. In one embodiment, the STAT1 inhibitor is an antibody against IFN or an IFN receptor. In another embodiment, the STAT1 inhibitor is an antibody against IL-6 or an IL-6 receptor. In yet another embodiment, the STAT1 inhibitor is an agent that inhibits JAK1 and JAK3. In yet another embodiment, the STAT1 inhibitor is an agent that reduces STAT1 phosphorylation. In a further embodiment, the STAT1 inhibitor is an agent that inhibits STAT1 nuclear translocation. In a further embodiment, the STAT1 inhibitor is an RNAi agent against STAT1.

[0011] In yet another aspect, the present invention is a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject, by administering a 25-OHC inhibitor in a pharmacologic effective amount to said subject.In one embodiment, the 25-OHC inhibitor is a CH25H inhibitor or a STAT1 inhibitor.In another embodiment, the 25-OHC inhibitor is a derivative of 25-OHC.In yet another embodiment, the 25-OHC inhibitor is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, such as simvastatin.

[0012] In yet another aspect, the present invention provides a method for treating Alzheimer's disease (AD) in a subject, comprising: providing a genome editing tool; delivering the genome editing tool to a neuronal cell; and editing the STAT1 gene by deleting the entire STAT1 gene, the phosphorylation site of the STAT1 gene, the promoter region of the STAT1 gene, or the SH2 domain of the STAT1 gene. In some embodiments, the genome editing tool is a CRISPR-CAS9 system.

[0013] In yet another aspect, the present invention provides a method for treating Alzheimer's disease (AD) in a subject, comprising: providing a genome editing tool; delivering the genome editing tool to a neuronal cell; and editing the STAT1 gene by deleting the entire STAT1 gene, the phosphorylation site of the STAT1 gene, the promoter region of the STAT1 gene, or the SH2 domain of the STAT1 gene. In some embodiments, the genome editing tool is a CRISPR-CAS9 system.

[0014] In yet another aspect, the present invention provides a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject, comprising: providing a genome editing tool; delivering the genome editing tool to a neuronal cell; and editing the STAT1 gene by deleting the entire STAT1 gene, the phosphorylation site of the STAT1 gene, the promoter region of the STAT1 gene, or the SH2 domain of the STAT1 gene. In some embodiments, the genome editing tool is a CRISPR-CAS9 system.

[0015] In yet another aspect, the present invention provides a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject, comprising: providing a genome editing tool; delivering the genome editing tool to a neuronal cell; and editing the STAT1 gene by deleting the entire STAT1 gene, the phosphorylation site of the STAT1 gene, the promoter region of the STAT1 gene, or the SH2 domain of the STAT1 gene. In some embodiments, the genome editing tool is a CRISPR-CAS9 system.

[0016] The patent or patent application file contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0017] [Figure 1A] Figures 1A and 1B are images showing that elevated levels of phosphorylated STAT1 are detected in Alzheimer's disease patients. Figure 1A shows four images showing staining of phosphorylated STAT1 (pSTAT1) in brain tissue from three AD patients (cases 1, 2, and 3) and one healthy individual (control). [Figure 1B] FIG. 1B is a magnified image revealing the morphology of pSTAT1 staining, which is indicative of accumulation of pSTAT1 in the nucleus (see staining within the rectangular box).

[0018] [Figure 2A] Figures 2A-2E are images and graphs showing that STAT1 gene deficiency attenuates amyloid-β deposition in the brain. Figure 2A shows images of amyloid-β (Aβ) staining in a mouse model of AD (APP / PS1 mice) and AD mice with STAT1 deficiency (APP / PS / STAT1- / -). Brain sections were obtained from 3-month-old APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. Images show Aβ staining in the hippocampal region in addition to whole brain sections at high magnification. [Figure 2B] FIG. 2B is a graph depicting the number of Aβ plaques in five serial sections each from 3-month-old APP / PS1 and APP / PS1 / STAT1− / − mice. [Figure 2C] FIG. 2C is a graph depicting Aβ plaque area in five serial sections from 3-month-old APP / PS1 and APP / PS1 / STAT1− / − mice, respectively. [Figure 2D] FIG. 2D is a graph showing ELISA measurements of Aβ42 in TBS extracts of brain tissue obtained from 3-month-old wild-type, APP / PS1, and APP / PS1 / STAT1− / − mice, respectively. [Figure 2E] FIG. 2E is a graph showing ELISA measurements of Aβ42 in formic acid extracts of brain tissue obtained from 3-month-old wild-type, APP / PS1, and APP / PS1 / STAT1− / − mice, respectively.

[0019] [Figure 3A] Figures 3A and 3B are graphs depicting the identification of CH25H as a STAT1 downstream target. Specifically, Figure 3A shows a heatmap depicting the ontology analysis of differentially expressed genes with fold change >1.5 and p-value <0.05, using two biological repeats for each genotype. [Figure 3B] FIG. 3B is a pie chart depicting pathway analysis of differentially expressed genes by David functional annotation clustering.

[0020] [Figure 4A] Figure 4A and Figure 4B show a graph and an image, respectively, demonstrating that CH25H is reduced in STAT1-deficient mice. Specifically, Figure 4A shows a graph depicting the results of real-time PCR quantification of CH25H mRNA levels in the brains of APP / PS1 and APP / PS1 / STAT1- / - mice. Data are representative of three independent experiments. [Figure 4B] FIG. 4B shows images depicting the results of Western blots of CH25H, STAT1, and α-tubulin in brain homogenates from three pairs of APP / PS1 and APP / PS1 / STAT1− / − mice.

[0021] [Figure 5A] Figures 5A and 5B show graphs depicting the physical binding of STAT1 to the CH25H promoter. Specifically, Figure 5A shows a schematic diagram of the CH25H gene and its promoter. [Figure 5B] Figure 5B shows a graph depicting the results of a chromatin histone immunoprecipitation (ChIP) experiment in which a DNA fragment containing the CH25H promoter was enriched by STAT1 antibody pull-down and this enrichment was reduced in samples from STAT1 KO (STAT1- / -) mice compared to those from wild-type (WT) mice.

[0022] [Figure 6A] Figures 6A and 6B show graphs illustrating that STAT1 deficiency causes a decrease in 25-OHC in the brain. Specifically, Figure 6A shows a graph illustrating the measurement of total brain cholesterol levels in APP / PS1 and APP / PS1 / STAT1- / - mice. n=3 for each group. [Figure 6B] FIG. 6B shows a graph depicting the results of measuring 25-OHC levels in the same mice used for the cholesterol measurements in FIG. 6A.

[0023] [Figure 7]Figure 7 shows images demonstrating that full-length APP protein is reduced in the exosome fraction of STAT1- / - mice. Brain tissues from APP / PS1 and APP / PS1 / STAT1- / - mice were homogenized and the postnuclear fraction was subjected to 5%-45% sucrose density gradient centrifugation. Ten fractions were collected and solubilized with Triton-100. Each fraction was used for Western blot and blotted for APP and the exosome marker Flotillin 1.

[0024] [Figure 8A] Figures 8A-8C show graphs and images showing that treatment with 25-OHC reduces exosomal APP but increases intracellular APP. SH-SY5Y cells overexpressing APP were treated with different doses of 25-OHC. Figure 8A shows images showing the results of Western blot with APP from samples generated by harvesting culture medium for exosome purification and lysing the purified exosomes. [Figure 8B] FIG. 8B shows an image depicting the results of a Western blot with APP from samples generated by harvesting cell lysates to assess intracellular APP levels. [Figure 8C] FIG. 8C shows a graph depicting the results of FACS analysis of surface APP in control and 25-OHC treated cells.

[0025] [Figure 9] Figure 9 shows a graph depicting the increase of Aβ42 in exosomes after 25-OHC treatment. ELISA measurement of Aβ42 in exosomes harvested from cell culture medium treated with different concentrations of 25-OHC. Data are representative of three independent experiments. *p<0.05.

[0026] [Figure 10A]Figures 10A-10C show images depicting the increased retention time of intracellular APP after 25-OHC treatment. Figure 10A shows images depicting the transport of APP molecules, depicted by surface APP labeled with FITC-conjugated antibodies. Cells were fixed at various time points to examine the distribution of APP at specific time points. [Figure 10B] FIG. 10B shows images of snapshots from a time-lapse video demonstrating the extended duration of APP transport in control cells. [Figure 10C] FIG. 10C shows images of snapshots from a time-lapse video showing APP transport in 25-OHC treated cells.

[0027] [Figure 11A] Figures 11A, 11B and 11C show images and graphs demonstrating that infusion of 25-OHC promoted Aβ deposition in APP / PS1 mice. Figure 11A shows images of brain tissue sections stained for amyloid-β. Two-month-old mice were administered 25-OHC or saline as a control every other day for one month. Brains from the mice were sectioned and stained for amyloid-β. Data are representative of triplicate pairs of saline or 25-OHC-injected mice. [Figure 11B] 11B and 11C show graphs depicting the number of Aβ plaques in five serial sections of the whole brain (FIG. 11B) and hippocampus (FIG. 11C) from saline control and 25-OHC treated mice, respectively. [Figure 11C] 11B and 11C show graphs depicting the number of Aβ plaques in five serial sections of the whole brain (FIG. 11B) and hippocampus (FIG. 11C) from saline control and 25-OHC treated mice, respectively.

[0028] [Figure 12A] Figures 12A-12E are graphs and images depicting the creation of CH25H KO mice. Figure 12A is a schematic diagram of the CH25H gene and two targeting sgRNAs. [Figure 12B]Figure 12B shows the DNA sequences of the two targeting sgRNAs. [Figure 12C] FIG. 12C depicts sequence analysis results showing deletion of the CH25H gene in CH25H knockout mice. [Figure 12D] FIG. 12D shows an image depicting the genotyping results of the WT (534 bp) and KO (488 bp) bands. [Figure 12E] FIG. 12E shows a graph depicting the results of real-time PCR of CH25H RNA levels in WT and CH25H KO mice.

[0029] [Figure 13A] Figures 13A and 13B show images and graphs showing that Aβ plaque deposition is reduced in CH25H KO mice. Figure 13A shows images showing amyloid-β staining in a mouse model of AD (APP / PS1 mice) and AD mice with CH25H deficiency (APP / PS / CH25H- / -). Brain sections were obtained from 3-month-old APP / PS1 and APP / PS1 / CH25H- / - mice, respectively. Higher magnifications of whole brain sections as well as amyloid-β staining in the hippocampal region are shown. [Figure 13B] Figures 13B and 13C are graphs depicting the number of Aβ plaques in five serial sections of the whole brain (Figure 13B) and hippocampus (Figure 13C) from 3-month-old APP / PS1 and APP / PS1 / CH25H- / - mice, respectively. [Figure 13C] Figures 13B and 13C are graphs depicting the number of Aβ plaques in five serial sections of the whole brain (Figure 13B) and hippocampus (Figure 13C) from 3-month-old APP / PS1 and APP / PS1 / CH25H- / - mice, respectively.

[0030] [Figure 14]Figure 14 shows a graph illustrating that CH25H gene deletion improves learning and memory in APP / PS1 mice. APP / PS1 (n=11) and APP / PS1 / CH25H- / - (n=8) mice were trained in the water maze. The average time taken to reach the hidden platform during 5 days of training is recorded. The x-axis is the number of days, and the y-axis is the time taken for the mice to find the platform in the water maze.

[0031] [Figure 15] Figure 15 shows images of Western blot results of APP in cells treated with 25-OHC together with various doses of prednisolone trimethylacetate or simvastatin. Simvastatin acts as a potent 25-OHC inhibitor. SH-SY5Y cells were treated with 25-OHC together with various doses of prednisolone trimethylacetate or simvastatin. Cells were lysed after 24 hours and blotted for APP.

[0032] [Figure 16A] Figures 16A, 16B, 16C, and 16D show graphs demonstrating that the differences in Aβ deposition are not due to differences in APP expression levels or secretases that cleave APP. Figure 16A shows graphs depicting the results of real-time PCR quantification of APP mRNA levels in the brains of APP / PS1 and APP / PS1 / STAT1- / - mice. [Figure 16B] Figures 16B, 16C and 16D show three graphs depicting the results of real-time PCR quantification of Adam10 (Figure 16B), BACE1 (Figure 16C) and Nicastrin (Figure 16D), the α, β and γ secretases of APP, respectively. [Figure 16C] Figures 16B, 16C and 16D show three graphs depicting the results of real-time PCR quantification of Adam10 (Figure 16B), BACE1 (Figure 16C) and Nicastrin (Figure 16D), the α, β and γ secretases of APP, respectively. [Figure 16D]Figures 16B, 16C and 16D show three graphs depicting the results of real-time PCR quantification of Adam10 (Figure 16B), BACE1 (Figure 16C) and Nicastrin (Figure 16D), the α, β and γ secretases of APP, respectively.

[0033] [Figure 17A] Figures 17A and 17B show images and graphs demonstrating the similar phagocytic capacity of microglial cells between WT and STAT1-deficient mice. Figure 17A shows images of microglial cells from WT or STAT1-deficient mice incubated with fluorescent beads. After 5 minutes of incubation, cells were fixed and internalized beads were imaged. WT and KO media are media from WT or KO microglial cells, respectively. [Figure 17B] FIG. 17B shows a graph depicting the quantification of internalized beads. [Figure 17C] FIG. 17C is a chart showing real-time PCR measurements of SV2A in WT and STAT1-deficient mice. [Figure 17D] FIG. 17D is a chart showing real-time PCR measurements of CCR2 in WT and STAT1-deficient mice.

[0034] [Figure 18A] Figures 18A, 18B, 18C and 18D show four graphs depicting the results of real-time PCR quantification of CH25H (Figure 18A) and other known cholesterol hydroxylases Cyp46al (Figure 18B), Cyp7b1 (Figure 18C) and Cyp7a1 (Figure 18D) in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. These results indicate that other cholesterol hydroxylases Cyp46al, Cyp7b1 and Cyp7a1 were not affected by STAT1 deficiency. [Figure 18B]Figures 18A, 18B, 18C and 18D show four graphs depicting the results of real-time PCR quantification of CH25H (Figure 18A) and other known cholesterol hydroxylases Cyp46al (Figure 18B), Cyp7b1 (Figure 18C) and Cyp7a1 (Figure 18D) in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. These results indicate that other cholesterol hydroxylases Cyp46al, Cyp7b1 and Cyp7a1 were not affected by STAT1 deficiency. [Figure 18C] Figures 18A, 18B, 18C and 18D show four graphs depicting the results of real-time PCR quantification of CH25H (Figure 18A) and other known cholesterol hydroxylases Cyp46al (Figure 18B), Cyp7b1 (Figure 18C) and Cyp7a1 (Figure 18D) in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. These results indicate that other cholesterol hydroxylases Cyp46al, Cyp7b1 and Cyp7a1 were not affected by STAT1 deficiency. [Figure 18D] Figures 18A, 18B, 18C and 18D show four graphs depicting the results of real-time PCR quantification of CH25H (Figure 18A) and other known cholesterol hydroxylases Cyp46al (Figure 18B), Cyp7b1 (Figure 18C) and Cyp7a1 (Figure 18D) in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. These results indicate that other cholesterol hydroxylases Cyp46al, Cyp7b1 and Cyp7a1 were not affected by STAT1 deficiency.

[0035] [Figure 19A] Figures 19A-19F show graphs and images showing that STAT1 deficiency did not affect general lipid metabolism. Figure 19A shows a graph showing the body weight of 3-month-old APP / PS1 and APP / PS1 / STAT1- / - mice (n=5 per group). [Figure 19B]Figure 19B shows images of Oil-RedO staining of liver sections from APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. Images are representative of three pairs of mice. [Figure 19C] Figures 19C, 19D, 19E and 19F show four graphs depicting the results of real-time PCR quantification of LPL, ABCA1, APOE, HMGCR in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. [Figure 19D] Figures 19C, 19D, 19E and 19F show four graphs depicting the results of real-time PCR quantification of LPL, ABCA1, APOE, HMGCR in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. [Figure 19E] Figures 19C, 19D, 19E and 19F show four graphs depicting the results of real-time PCR quantification of LPL, ABCA1, APOE, HMGCR in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. [Figure 19F] Figures 19C, 19D, 19E and 19F show four graphs depicting the results of real-time PCR quantification of LPL, ABCA1, APOE, HMGCR in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively.

[0036] [Figure 20A] Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. [Figure 20B]Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. [Figure 20C] Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. [Figure 20D] Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. [Figure 20E]Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. [Figure 20F] Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. [Figure 20G] Figures 20A, 20B, 20C, 20D, 20E, 20F and 20G show graphs depicting the results of real-time PCR quantification of c-fos (Figure 20A), zif268 (Figure 20B), BDNF-IV (Figure 20C), BDNF-IX (Figure 20D), Gadd45b (Figure 20E), Npas4 (Figure 20F) and CH25H (Figure 20G) in primary neuronal cultures obtained from WT or STAT1 KO mice after 30 min treatment with 50 mM KCl to measure induction of immediate early genes after KCl treatment. The results show that STAT1 deficiency did not alter basal neuronal activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure is based in part on the novel and surprising discovery that STAT1-regulated CH25H expression influences the pathogenesis of Alzheimer's disease (AD). The inventors discovered that CH25H is a downstream target of STAT1, and that STAT1 and its target CH25H promote Aβ deposition during AD development. Genetic deficiency of STAT1 delayed Aβ deposition in the brain, and thus the high amount of phosphorylated STAT1 (pSTAT1) detected in AD patients was a causative event in AD development. CH25H is an enzyme that converts cholesterol to 25-OHC. One unique feature of 25-OHC is that it can cross the blood-brain barrier (BBB), a feature that may be beneficial for pharmaceutical development, since many drugs currently in clinical trials have problems penetrating the BBB. The inventors demonstrated the effect of CH25H or 25-OHC on AD development. CH25H knockout mice had reduced Aβ deposition to an extent comparable to STAT1 knockout mice. In contrast, 25-OHC administration increased Aβ deposition in the brain.

[0038] Described herein is a means for treating or preventing diseases or medical conditions that are at least partially related to the STAT1-CH25H axis, which generates 25-OHC as an effector that regulates Aβ deposition. In such diseases or medical conditions, the enhancement of STAT1-CH25H activity may be due to, but not limited to: 1) increased STAT1 phosphorylation; 2) increased expression of STAT1 or CH25H; 3) increased cholesterol as a substrate for CH25H. The antagonist described herein for such treatment or prevention can be a chemical substance, a small molecule, a drug, a non-coding RNA, an antisense nucleotide, a peptide, a protein or an antibody, or a part thereof. In certain embodiments, the antagonist is administered in a therapeutically effective amount.

[0039] The present invention provides a method for treating a disease or medical condition associated with Aβ deposition in brain cells of a subject.In one embodiment, the disease or medical condition is caused by abnormal production, transport or clearance of Aβ.In another embodiment, the disease or medical condition is caused by increased activity of STAT1-CH25H pathway caused by factors such as, but not limited to, IFN-α, IFN-β, IFN-γ, IL-6 family cytokines, or acute or chronic inflammatory conditions.In a further embodiment, the disease or medical condition is caused by increased 25-OHC amount caused by high cholesterol amount in the subject.

[0040] The present inventors have found that blocking STAT1 signaling, abolishing STAT1 expression or depleting CH25H reduces Aβ plaques and attenuates the onset of AD. Chemical analogs of 25-OHC also reduce APP in cultured cells. The therapeutic method of the present invention includes administering an agent that alters the signaling, expression or activity of STAT1 or CH25H, such as an agent that blocks 25-OHC activity or promotes 25-OHC degradation. In particular, the present invention provides a method and means for treating Alzheimer's disease.

[0041] Thus, the present invention is directed in one aspect to a method of treating a disease such as AD by inhibiting STAT1, its downstream target CH25H, or 25-hydroxylated cholesterol (25-OHC) in the brain of a subject.

[0042] In one aspect, the present invention is directed to a method of preventing or delaying the onset of a disease such as AD by inhibiting STAT1, its downstream target CH25H, or 25-hydroxylated cholesterol (25-OHC) in the brain of a subject.

[0043] In another aspect, the present invention is directed to a method of reducing amyloid-β (Aβ) deposition in the brain of a subject by inhibiting STAT1, its downstream target CH25H, or 25-hydroxylated cholesterol (25-OHC).

[0044] In another aspect, the present invention is directed to a method of preventing or slowing amyloid beta (Aβ) deposition by inhibiting STAT1, its downstream target CH25H, or 25-hydroxylated cholesterol (25-OHC) in the brain of a subject.

[0045] In one embodiment, the inhibition of STAT1 in the above method is by administering to the subject a pharma- ceutically effective amount of a STAT1 inhibitor.

[0046] The term "subject" as used herein means an animal, preferably a mammal, most preferably a human. In some cases, subjects may be classified into different subgroups according to the presence or absence of a certain biomarker. In other cases, subjects may be classified into different subgroups based on whether the amount of a certain biomarker is below or above a threshold value. In either case, the subgroups may be included or excluded from the treatment of the present invention.

[0047] In some embodiments, the STAT1 inhibitor is a composition containing a pharma- ceutically effective amount of an active ingredient, such as a compound, a peptide, an antibody or fragment thereof, or a nucleic acid, and a pharma- ceutically acceptable excipient.

[0048] "Excipient," "carrier," "pharmaceutical excipient," "pharmaceutical acceptable carrier" or similar terms refer to one or more components or ingredients that are acceptable in the sense of being compatible with the other ingredients of a composition or formulation of the invention and not unduly deleterious to the patient, animal, tissue or cell to which the STAT1 inhibitor composition or formulation is to be administered.

[0049] The terms "pharmacologically effective amount", "effective amount" and the like with respect to STAT1 refer to an amount of a STAT1 inhibitor that is sufficient to elicit a desired response, e.g., an amount sufficient to reduce Aβ deposition in a subject, e.g., a human, to which it is administered, or an amount sufficient to detectably alter or alleviate a molecular or cellular parameter of AD or a clinical state or symptom of AD. An effective amount, e.g., an effective amount for human therapeutic use, can be a single dose or two or more divided doses of a STAT1 inhibitor administered daily, or it can be administered as multiple doses over a period of time, e.g., 1, 2, 3, 4, or about 7 days to about 1 year.

[0050] Determining therapeutically effective amount is well within the capabilities of those skilled in the art in light of the detailed disclosure provided herein.For example, therapeutically effective amount or dose can be initially estimated from in vitro and cell culture assays.In another example, effective amount can be dosed in animal models to achieve desired concentration or titer.Using such information, effective amount in humans can be more accurately determined.

[0051] The toxicity of the active ingredients described herein can be determined by standard formulation processes in vitro, cell culture or experimental animals.The data obtained from the above in vitro and cell culture assays can be used to determine the dosage range for human use.Dosage can vary depending on the dosage form used and the administration route used.The exact formulation, administration route and dosage can be selected by individual physicians taking into account the patient's condition.

[0052] Dosage and intervals can be tailored to blood levels of the active ingredient sufficient to induce or inhibit the biological effect (minimal effective concentration, MEC). The MEC may vary for each formulation, but can be estimated from in vitro data. The dosage required to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure or attenuation of the condition is achieved. The amount of the composition to be administered will, of course, depend on, for example, the subject to be treated, the severity of the affliction, the method of administration, and the judgment of the prescribing physician.

[0053] For example, in some embodiments, a pharma- tically effective amount of a STAT1 inhibitor may be as little as about 0.02 mg / kg / day to about 0.03 mg / kg / day in humans, or as much as about 2 mg / kg / day to about 3 mg / kg / day. Moreover, in some embodiments, when Aβ deposition becomes more severe, the effective amount may be greater than 3 mg / kg / day. In other embodiments, the effective amount may be greater when a STAT1 inhibitor is used therapeutically than when a STAT1 inhibitor is used to prevent or delay the onset of Aβ deposition.

[0054] In some embodiments, the dosage and treatment schedule will also depend on the gender of the human subject to be treated. In some cases, female patients are more likely to respond to STAT1 treatment and therefore may require a lower dose and a less frequent dosing schedule than male patients. In other cases, female patients are more sensitive to STAT1 treatment and therefore may require a lower dose and a less frequent dosing schedule than male patients.

[0055] In some embodiments, administration can be started preferably much earlier than Aβ deposits are detected. In other embodiments, administration can be started after Aβ deposits are detected but before any clinical symptoms of AD are observed. In yet other embodiments, administration can be started after clinical symptoms of AD are observed. Administration can be for a single dose in some cases. In other cases, administration can be for multiple doses, for example 2, 3, 4, 5 or 6 doses in other cases. Administration can continue as many times as the physician deems necessary.

[0056] In another embodiment, the administration schedule and the dosage are related. In one example, if the first administration is started before the clinical symptoms of AD are recognized, the dosage of each administration can be smaller than if the first administration is administered after the clinical symptoms of AD are recognized. In another example, when the administration is frequent (e.g., once every 12 hours), the dosage of each administration can be smaller than when the administration is infrequent (e.g., once every 24 hours).

[0057] In the context of using a STAT1 inhibitor in a treatment method or other method disclosed herein, the terms "use", "treat", "treatment", "address" and the like mean that the STAT1 inhibitor is administered to a subject, delivered to a tissue of a subject, or contacted with a tissue, cell, or cell-free system in vivo or in vitro, e.g., as described herein or as described in the references cited herein. Typically, such use or treatment results in, for example: (1) a detectable improvement or alleviation of the condition or symptoms being treated, (2) a detectable change in the activity, level, or number of a relevant biomolecule, therapeutic cell population, or pathological cell population, (3) a delay in the progression of the condition, a delay in the onset of symptoms, or a reduction in the severity of symptoms, or (4) another detectable response as described herein. Such relief may last, for example, at least a few hours or days, e.g., about 1-24 hours or days, e.g., about 1, 2, 3, 4, 5, 6, or 7 days, or the relief may last for a longer period, e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 35, 42, 49, 56 days to about 60 days or more, or the relief may be permanent. Treatment may slow the progression of the disease or condition or reduce its severity, e.g., delay the onset of the disease or condition in at least some subjects by about 1-24 hours, about 2-10 days, about 2-30 days, or about 1-5 years, as compared to subjects not treated with a sufficient amount of a STAT1 inhibitor. Thus, use or treatment with a STAT1 inhibitor typically results in a detectable change in a relevant immune parameter, such as a change in the level, activity or relative amount of a target effector or suppressor cell population, interleukin, cytokine, chemokine, immunoglobulin, compared to an appropriate control (e.g., untreated). STAT1 inhibitor treatment may also result in a change in the level or activity of associated transcription factors, enzymes, cellular biological activities, or the level or activity of disease pathogenic factors.Treatment with a STAT1 inhibitor may be used to delay or prevent the onset of a disease, condition, or complication (e.g., Aβ deposition in AD), to alleviate or slow the progression of an existing disease, condition, symptom, or complication, or to promote the elimination of said disease, condition, or complication.

[0058] "Alleviate," "reduction," "improvement," and the like refer to a detectable improvement or a detectable change consistent with improvement occurring in a subject or at least a minority of subjects, e.g., at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, or within a range between any two of these figures. Such an improvement or change may be observed in a treated subject compared to a subject not treated with a STAT1 inhibitor, where the untreated subject would have or develop the same or a similar disease, condition, symptom, or the like. The alleviation of a disease, condition, symptom or assay parameter may be determined passively or actively, for example by self-assessment by the subject, by a clinician's assessment, or by performing a suitable assay or measurement, for example by evaluating the quality of life, the delay in the progression of the disease(s) or symptom(s), the reduction in the severity of the disease(s) or symptom(s), or by suitable assays for the level or activity of biomolecules, cells, or by detecting cell migration in the subject. The alleviation may be temporary, long-term, or permanent, or may vary at a relevant time during or after the STAT1 inhibitor is administered to the subject or used in an assay or other method described herein or in the cited references, for example, from within about one hour of administration or use of the STAT1 inhibitor to about 3, 6, 9 months or more after the subject has received the STAT1 inhibitor.

[0059] "Modulation" of a molecule, cellular response, cellular activity, or other indicia, level, or biological activity means that the cell, level, or activity, etc. is detectably increased or decreased. Such an increase or decrease may be observed in a treated subject compared to a subject not treated with a STAT1 inhibitor, where the untreated subject has or suffers from the same or similar disease, condition, symptom, etc. Such an increase or decrease may be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more, or may be an approximate number within any range between any two of these values. The alteration may be determined passively or actively, for example by subject self-assessment, by clinician's assessment, or by performing a suitable assay or measurement, for example by evaluating the quality of life, the delay in progression of disease(s) or condition(s), the reduction in severity of disease(s) or condition(s), or by suitable assays for the level or activity of biomolecules, cells, or by detecting cell migration in the subject. The alteration may be temporary, long-term, or permanent, or may be variable at a relevant time during or after the STAT1 inhibitor is administered to the subject or used in an assay or other method described herein or in the cited references, for example, from within about one hour of administration or use of the STAT1 inhibitor to about 3, 6, 9 months or more after the subject has received the STAT1 inhibitor.

[0060] At various points in this disclosure, for example in any disclosed embodiment or claim, reference is made to compounds, compositions, formulations, or methods that "comprise" one or more particular components, elements, or steps. The embodiments of the invention also specifically include those compounds, compositions, formulations, or methods that are, consist of, or consist essentially of the specified components, elements, or steps. The terms "comprise," "consist," and "consist essentially of" have their ordinary meanings under U.S. patent law. For example, components or methods of the present disclosure that "comprise" a component or step are open-ended and they can include or be read as including the composition or method plus the additional component or step. Similarly, compositions or methods of the present disclosure that "consist" of a component or step can include or be read as including those compositions or methods with the appropriate amount of the additional component or additional step.

[0061] In some cases, the STAT1 inhibitor may be an antibody against interferon, because interferon is required for the activation of STAT1, and the inhibition of interferon will prevent or reduce the activation of STAT1. One example of an interferon antibody is Fontolizumab (proposed trade name HuZAF), a humanized monoclonal antibody against interferon gamma. Methods for producing antibodies and assaying antibody activity are generally known in the art, so that similar antibodies against interferon can also be produced by those skilled in the art and screened for their inhibitory effect against interferon.

[0062] In some cases, the STAT1 inhibitor may be an antibody against the interferon receptor, because the interferon receptor is required for the activation of STAT1 and the inhibition of the interferon receptor will prevent or reduce the activation of STAT1. One example of an interferon receptor antibody is Anifrolumab, a human monoclonal antibody that targets type I interferon (IFN) receptor 1. Methods for producing antibodies and assaying antibody activity are generally known in the art, so that similar antibodies against the interferon receptor can also be produced by those skilled in the art and screened for their inhibitory effect on the interferon receptor.

[0063] In some cases, the STAT1 inhibitor may be an antibody against IL-6, since IL-6 is required for the activation of STAT1 and the inhibition of IL-6 will prevent or reduce the activation of STAT1. One example of an IL-6 antibody is Siltuximab, a chimeric (made from human and mouse proteins) monoclonal antibody against IL-6. Methods for producing antibodies and assaying antibody activity are generally known in the art, so that similar antibodies against IL-6 can also be produced by those skilled in the art and screened for their inhibitory effects on IL-6.

[0064] In some cases, the STAT1 inhibitor may be an antibody against the IL-6 receptor (IL-6R), because the interferon receptor is required for the activation of STAT1 and the inhibition of the IL-6 receptor will prevent or reduce the activation of STAT1. One example of an IL-6 receptor antibody is Tocilizumab, a humanized monoclonal antibody against the interleukin 6 receptor. Methods for producing antibodies and assaying antibody activity are generally known in the art, so that a similar antibody against IL-6R can also be produced by one skilled in the art and screened for its inhibitory effect on IL-6R.

[0065] In yet another case, the STAT1 inhibitor may be an agent that inhibits JAK1 or JAK3, because JAK1 and JAK3 are well-known upstream kinases that activate STAT1 signaling. The agent that inhibits JAK1 and JAK3 may be a small molecule, such as Filgotinib, which is used as a JAK1 inhibitor, and Tofacitinib, which is a JAK3 inhibitor.

[0066] In yet another embodiment, the STAT1 inhibitor may be an agent that reduces STAT1 phosphorylation. Such an agent that reduces STAT1 phosphorylation may be FLLL32, which has been reported to inhibit STAT1 phosphorylation.

[0067] In yet another embodiment, the STAT1 inhibitor may be an agent that inhibits the nuclear translocation of STAT1. Such an agent that inhibits the nuclear translocation of STAT1 may be the measles virus P protein and V protein.

[0068] In another case, STAT1 inhibitor is an RNAi agent for STAT1.Such RNAi agent can be any siRNA that targets STAT1, for example, product 105153 from Thermo Fisher Scientific.The method of making siRNA for specific target is generally known in the art, so that the skilled person can make other siRNA agent that targets STAT1.

[0069] In yet another case, a genome editing tool may be used to delete the entire STAT1 gene, the phosphorylation site of the STAT1 gene, the promoter region of the STAT1 gene, or the SH2 domain of the STAT1 gene in a neuronal cell as a method of treating, preventing, or delaying the onset of AD. The genome editing tool can be any genome editing tool as long as it can be used to target a specific region of the STAT1 gene and deliver it to a cell of interest. One example of such a genome editing tool is the CRISPR-CAS9 system described in Ran, F. et al., "In vivo genome editing using Staphylococcus aureus Cas9", Nature (2015). Another example of such a genome editing tool is the CRISPR-Cpfl system described in Zetsche et al., CPfl1 is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell (2015).

[0070] In another embodiment, the inhibition of CH25H in the above method is by administering to the subject a pharma- ceutically effective amount of a CH25H inhibitor. In some embodiments, the CH25H inhibitor is a STAT1 inhibitor. This is because STAT1 activates CH25H, and inhibiting STAT1 abolishes the ability of STAT1 to activate CH25H. The STAT1 inhibitor can be any STAT1 inhibitor disclosed herein.

[0071] In another embodiment, the method of treating, preventing or delaying the onset of AD includes administering to a neuron the entire CH25H gene, HistidineA genome editing tool may be used to delete the raster region or the promoter region of the CH25H gene. The genome editing tool can be any genome editing tool as long as it can be used to target a specific region of the CH25H gene and deliver it to a cell of interest. One example of such a genome editing tool is the CRISPR-CAS9 system described in Ran, F. et al., "In vivo genome editing using Staphylococcus aureus Cas9", Nature (2015). Another example of such a genome editing tool is the CRISPR-Cpfl system described in Zetsche et al., "Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System", Cell (2015).

[0072] In another embodiment, the inhibition of 25-OHC in the above method is by administering to the subject a pharma- ceutically effective amount of a 25-OHC inhibitor.

[0073] In some cases, the 25-OHC inhibitor can be a CH25H inhibitor or a STAT1 inhibitor, since CH25H and STAT1 are upstream of 25-OHC, and inhibition of either CH25H or STAT1 abolishes the ability of CH25H to generate 25-OHC. In other cases, the 25-OHC inhibitor can be an analog of 25-OHC. In yet other cases, the 25-OHC inhibitor can be a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor. One example of an HMG-CoA reductase inhibitor is simvastatin.

[0074] Throughout this specification, various embodiments of the present invention may be expressed in a range format. The description in range format is understood to be merely for convenience and brevity and should not be construed as a limitation on the scope of the present invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose each individual number within that range, e.g., 1, 2, 3, 4, 5, and 6, in addition to subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0075] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or in any other description of the invention, as appropriate. Certain features described within the scope of various embodiments are not to be regarded as essential features of those embodiments, unless the embodiment would be ineffective without those elements.

[0076] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

[0077] Examples of the disclosed subject matter are provided below. Other features, objects, and advantages of the disclosed subject matter will be apparent from the detailed description, drawings, examples, and claims. Methods and materials substantially similar or equivalent to those described herein can be used in the practice or testing of the subject matter disclosed herein. Exemplary methods and materials are described below. EXAMPLES

[0078] Working Example

[0079] method

[0080] mouse

[0081] APP / PS1 5XFAD mice were purchased from the Jackson lab, and STAT1-deficient mice were provided by Daved Levy. CH25H KO mice were generated by CRISPR / Cas9 method. The pST1374-Cas9-N-NLS-flag-linker plasmid (Addgene ID44758) used for Cas9 protein expression was previously described (Zhou et al., 2014). The Ch25h gene sequence was downloaded from the UCSC genome browser website (http: / / genome.ucsc.edu / ). Two sgRNA oligos were synthesized and annealed to the pUC57-sgRNA construct. In vitro transcription was performed as previously described (Zhou et al., 2014). Cas9 mRNA and sgRNA were introduced into a C57BL6 / J background. APP / PS1 mice were bred with CH25H(- / -) mice to generate offspring carrying the APP / PS1 gene and one of the WT or KO alleles of CH25H. All mice were on a CH57BL / 6 genetic background and were housed under special pathogen-free conditions at the National University of Singapore. All experiments were performed with 3-4 month-old mice and were approved by the NUS Laboratory Animal Care and Use Committee.

[0082] Preparation of tissue lysates and sucrose gradients

[0083] Mouse brains were weighed and homogenized in 9 volumes of TBS. The homogenate was centrifuged at 1000×g for 15 min at 4°C. The supernatant was collected as the post-nuclear fraction. The post-nuclear fraction was carefully placed on a 5%-45% sucrose gradient and centrifuged at 147,000×g for 16 h at 4°C. After centrifugation, 1 mL was collected from the top to the bottom as each fraction. To dissolve membrane-associated proteins, Triton was added to each fraction to a final concentration of 0.1%. The obtained samples were used for Western blot analysis.

[0084] Preparation of exosomes

[0085] SH-SY5Y cells overexpressing human APP were cultured in DMEM containing 10% FBS. One day before exosome preparation, the medium was replaced with blank DMEM to avoid serum exosome contamination. 24 hours after medium replacement, the medium was collected and spun at 200×g for 5 min to pellet floating cells. The cell-free medium was further centrifuged at 100,000×g for 1 h at 4°C, and the pellet containing exosomes was dissolved in RIPA buffer.

[0086] Microarray analysis

[0087] Microarray analysis was performed using the affimetrix microarray system provided by Molecular Genomics (Affymetrix). Data analysis was performed using GeneSpring software. Differentially expressed genes were used as input in the David Functional Annotation Clustering website (http: / / david.abcc.ncifcrf.gov / home.jsp), using the medium classification stringency default. Clustering results were replotted in pie charts.

[0088] Histological analysis

[0089] Tissues were fixed in 4% paraformaldehyde, dehydrated, infiltrated, and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin (H&E) to assess general morphology. For immunofluorescence (IF) or immunohistochemistry (IHC), sections were rehydrated and stained with primary antibodies against Aβ (Cell Signaling Tec.) and incubated with fluorescently labeled secondary antibodies (Invitrogen). For Oil Red O staining, livers were embedded in Tissue Tek (Electron Microscopy Science) and frozen at -80°C. Sections 10 μm thick were cut and hydrated in dH2O, followed by incubation in Oil Red O staining solution (0.3% Oil Red O (Sigma) in 60% isopropanol) for 1 h. Slides were then quickly washed three times with 60% isopropanol until the droplets of isopropanol falling off the slide were clear. Slides were then washed with H2O and counterstained with hematoxylin.

[0090] Real-time PCR

[0091] Total RNA was extracted from cells using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using Superscript reverse transcriptase (Invitrogen). Gene expression was measured by 7500 Real-Time PCR System (Applied Biosystems) with SYBR qPCR Kit (KAPA). Actib, Gapdh or Rn18S were used as internal controls. Primer sequences are available upon request.

[0092] ELISA

[0093] Aβ42 was quantified using an ELISA kit (Millipore) according to the manufacturer's instructions.

[0094] Chromatin immunoprecipitation assay

[0095] Brain homogenates were obtained by homogenizing half of the brain hemispheres of APP / PS1 or APP / PS1 / STAT1(- / -) mice in 9 volumes of TBS. Crosslinks were formed by adding formaldehyde to a final concentration of 1% for 10 min and quenching with glycine. The homogenates were treated with hypotonic buffer and then treated with nuclear lysis buffer to release chromatin. Chromatin was fragmented by sonication, precleared with protein G beads, and precipitated with anti-STAT1 antibody (Santa Cruz) or normal rabbit IgG (Santa Cruz) overnight at 4°C. After washing and elution, decrosslinking was performed by incubation at 65°C for 8 h. The eluted DNA was purified and analyzed by RT-PCR using primers specific for the CH25H promoter as described above.

[0096] statistics

[0097] Statistical significance was determined by Student's t-test using GraphPad Prism 6.01. A p-value <0.05 was considered significant. p-values ​​for clinical scores were determined by one-way multiple range analysis of variance (ANOVA) for multiple comparisons. Data are expressed as the mean and standard error of the mean (mean ± SEM) unless otherwise stated.

[0098] Example 1: STAT1 knockout mice have reduced Aβ deposition

[0099] Higher expression of STAT1 was demonstrated in AD cases than in age-matched control cases (Figures 1A and 1B). In Figure 1A, no staining for phosphorylated STAT1 (pSTAT1) was observed in control cells. In contrast, there was significantly more staining for pSTAT1 in each of the three cases, as shown in the enlarged snapshots of the photographs of case 1 in the same Figures 1A and 1B.

[0100] To elucidate whether activation of the STAT1 pathway is the cause of AD pathogenesis or the consequence of neuroinflammation in the later stages of AD, we crossed STAT1- / - mice with APP / PS1 mice to generate AD mice with a STAT1- / - background. APP / PS1 / STAT1- / - mice and their littermate control mice of the APP / PS1 genotype were kept for 3-4 months and then sacrificed for histological examination of Aβ deposition. Surprisingly, we found that Aβ numbers as well as the area occupied by Aβ were consistently decreased in STAT1- / - mice (Figures 2A and 2B). Similar results were observed when Aβ42 was measured by TBS or formic acid extraction (Figures 2C and 2D). Moreover, the decrease in Aβ was not due to changes in APP expression levels or secretase-cleaved APP (Figures 16A and 16B).

[0101] Example 2: Reduction of Aβ deposition in STAT1-KO mice was due to a reduction in its downstream target CH25H

[0102] Our microarray data identified CH25H as a STAT1-regulated gene in AD conditions (Figures 3A and 3B). Furthermore, real-time PCR, Western blot, and ChIP assays confirmed the regulatory effect of STAT1 on CH25H (Figures 4A, 4B, 5A, and 5B). As shown in Figure 4A, which shows the results of real-time PCR, CH25H mRNA expression is significantly reduced in STAT1-deficient mice. As shown in Figure 4B, which shows the results of Western blot, CH25H protein levels were also significantly reduced in STAT1-deficient mice. The results of ChIP assays in Figures 5A and 5B also showed that only a small amount of STAT1 was bound to the CH25H gene promoter sequence in STAT1-deficient mice. The result that the levels of 25-OHC were reduced in STAT1-deficient mice further supported the notion that the STAT1-CH25H axis regulates 25-OHC levels in the brain (Figures 6A-6B).

[0103] Example 3: STAT1-CH25H-regulated 25-OHC affects APP secretion in exosomes

[0104] Mouse brain homogenates were fractionated by sucrose gradient to examine the subcellular localization of APP protein. Notably, the distribution pattern of APP was similar between 5XFAD and 5XFAD / STAT1- / - mice, except for the highest density fraction, which was enriched in the exosome marker Flotillin 1. In STAT1- / - mice, this fraction contained significantly more APP (Figure 7).

[0105] We further confirmed whether the difference in exosomal APP was due to 25-OHC using APP-overexpressing SH-SY5Y cell line. Cells were treated with various doses of 25-OHC, and both cell lysates and exosomes from the medium were collected. 25-OHC showed a dose-dependent effect, increasing cytoplasmic APP while decreasing exosomal APP (Figures 8A and 8B). As shown in Figure 8A, as the dose of 25-OHC increased, less APP was detected in the exosomal fraction. In contrast, as the dose of 25-OHC increased, more APP was found in the cell lysate.

[0106] FACS analysis of surface APP also revealed that 25-OHC increased the amount of APP on the cell surface (Figure 8C). After 25-OHC treatment, higher amounts of APP were detected on the cell surface of cells overexpressing APP.

[0107] Furthermore, we detected an increase in Aβ42 in exosomes after treatment with 25-OHC. Cell culture media treated with various concentrations of 25-OHC were collected, and Aβ42 in exosomes recovered from the media was measured by ELISA. As shown in Figure 9, increased Aβ42 was detected in the media of cells treated with increasing concentrations of 25-OHC. Data are representative of three independent experiments.

[0108] Example 4: 25-OHC increased the retention time of APP in cells

[0109] Multiple mechanisms may contribute to the increase in cellular APP after 25-OHC treatment. However, our mouse data suggest that both APP synthesis and degradation were normal: APP expression was similar, as were the enzyme levels of APP cleavage (Figures 16A and 16B). As shown in Figure 16A, the expression levels of APP were similar regardless of STAT1 deficiency. As shown in Figure 16B, the expression levels of Adam10, BACE1 and Nicastrin, i.e., α, β and γ secretase levels of APP, respectively, were also similar regardless of STAT1 deficiency.

[0110] We next set out to examine the trafficking of APP protein. Surface APP was labeled with antibodies, and cells were returned to the incubator and fixed at various time points for staining. In untreated cells, APP was translocated to specific compartments within the cell, and the signal disappeared within 6 hours. However, in 25-OHC-treated cells, APP trafficking was slowed down (Figure 10A). Time-lapse movies clearly showed that in untreated cells, APP was clustered in specific compartments, whereas in 25-OHC-treated cells, APP was distributed evenly throughout the study period (Figure 10B).

[0111] Example 5: CH25H KO reconstituted the STAT1 KO phenotype and delayed the onset of AD

[0112] The present inventors studied the effect of CH25H KO on the pathogenesis of AD. sgRNAs targeting CH25H were designed such that sgRNA1 was SEQ ID NO: 1 and sgRNA2 was SEQ ID NO: 2. See Figures 12A and 12B. Using the two sgRNAs, the CH25H gene was knocked out by CRISPR / Cas9 method to delete 46 base pairs (bp) in the exon of the CH25H gene, generating CH25H knockout (KO) mice.

[0113] In CH25H KO mice, a deletion of a 46 bp fragment of the CH25H gene was detected, the 488 bp band was the deleted CH25H gene, and the 534 bp was the wild-type gene. The expression of CH25H mRNA was significantly reduced in CH25H KO mice (Figure 12E).

[0114] When crossed with 5XFAD mice, CH25H KO presented a similar phenotype to STAT1 KO: Aβ was significantly reduced by both immunostaining and ELISA quantification (Figures 13A, 13B, and 13C, respectively). In contrast, mice injected with 25-OHC had significantly higher amounts of Aβ (Figures 11A, 11B, and 11C).

[0115] To test the effect of reduced Aβ on cognitive ability, mice were tested in a water maze. 5XFAD mice gradually learned to reach the underwater platform, whereas CH25H KO mice took significantly (p<0.05) less time to find the platform, indicating that they performed better in learning and memory tasks (Figure 14).

[0116] Example 6: Simvastatin inhibits 25-OHC-induced APP accumulation

[0117] We screened a number of small molecules for potential 25-OHC inhibitors and found that simvastatin blocked the 25-OHC-induced increase in cellular APP, an effect that could be observed at concentrations of simvastatin as low as 10 nM, well below doses that could cause cytotoxic effects.

[0118] As shown in Figure 15, 25-OHC treatment caused an increase in APP levels. However, simvastatin treatment was able to reduce the increase in APP levels induced by 25-OHC treatment. In contrast, prednisolone trimethylacetate treatment had no effect on the increase in APP levels induced by 25-OHC. Thus, simvastatin was a potent inhibitor of 25-OHC.

[0119] Example 7: Phagocytosis between WT and STAT1-deficient microglial cells was similar

[0120] WT or STAT1- / - microglial cells were incubated with fluorescent beads and their phagocytic ability was tested. To examine the effect of factors secreted into the medium, both cells were kept in medium from WT cells or medium from STAT1- / - cells. Imaging and quantification showed that phagocytosis was similar in all conditions tested (Figures 17A and 17B). We also measured the mRNA of SV2a and CCR2, key genes involved in the phagocytic process. Their expression levels were comparable between APP / PS1 and APP / PS1 / STAT1- / - (Figures 17C and 17D).

[0121] Example 8: CH25H was a STAT1-dependent cholesterol hydroxylase

[0122] We tested several known cholesterol hydroxylases, including CH25H, Cyp46a1, Cyp7b1 and Cyp7a1, which add hydroxy groups to cholesterol at different positions. Of the enzymes we tested, only CH25H showed a STAT1-dependent expression pattern, as only its expression was significantly decreased (Figures 18A, 18B, 18C and 18D).

[0123] Example 9: STAT1 deficiency did not cause common metabolic disorders

[0124] CH25H is known to regulate cholesterol metabolism in the first place. However, when comparing body weight, lipid deposition in the liver, and key enzymes involved in lipid metabolism, no significant changes were found between WT and STAT1- / - mice (Figures 19A-19F). As shown in Figure 19A, there was no significant difference in body weight between APP / PS1 and APP / PS1 / STAT1- / - mice. As shown in Figure 19B, there was no significant difference in lipid deposition in hepatocytes between APP / PS1 and APP / PS1 / STAT1- / - mice. Furthermore, as shown in Figures 19C-19F, there was no significant difference in the expression of key enzymes, including LPL, ABCA1, APOE, and HMGCR, between APP / PS1 and APP / PS1 / STAT1- / - mice.

[0125] Example 10: STAT1 deficiency did not alter neuronal activity as measured by induction of immediate early genes

[0126] Primary hippocampal neurons from WT or STAT1- / - mice were cultured and then treated with 50 mM KCl. The induction of several immediate early genes, including cfos, Zif268, BDNF-IX, Gadd45b, Npas4 and Ch25h, was measured by RT-PCR as markers of neuronal activity. As shown in Figures 20A-20G, both WT and STAT1- / - neurons were able to induce these genes to the same extent.

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Claims

1. 1. A kit for treating Alzheimer's disease (AD) in a subject, comprising a genome editing tool, wherein the genome editing tool is a CRISPR-CAS9 system; wherein the genome editing tool is delivered to a neuronal cell; and Editing the CH25H gene by deleting the entire CH25H gene, the histidine cluster region of the CH25H gene, or the promoter region of the CH25H gene. kit.

2. A kit for preventing or delaying the onset of Alzheimer's disease (AD) in a subject, said kit comprising a genome editing tool, wherein said genome editing tool is a CRISPR-CAS9 system; wherein the genome editing tool is delivered to a neuronal cell; and Editing the CH25H gene by deleting the entire CH25H gene, the histidine cluster region of the CH25H gene, or the promoter region of the CH25H gene. kit.

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