Compositions and methods for treating alzheimer's disease

Abstract

Disclosed are methods of treating or preventing or delaying the onset of Alzheimer's disease (AD) in a subject by targeting a new STAT1-CH25H pathway in the pathogenesis of AD, in particular 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-CoA (HMG-CoA) reductase inhibitor, such as simvastatin.

Description

[0001] This application is a divisional application of a patent application with a priority date of May 16, 2017, application number 201880045403.4, and invention name “Compositions and methods for treating Alzheimer’s disease”. Technical Field

[0002] The present invention generally relates to the field of pharmaceutical agents and their use in the treatment of diseases, particularly agents that target and inhibit signal transducer and activator of transcription 1 (STAT1), such as cholesterol 25-hydroxylase (CH25H) and 25-hydroxylated cholesterol (25-OHC); and the use of such agents in the management of physiological conditions, particularly by reducing amyloid beta deposits to treat and / or prevent various diseases, including central nervous system diseases, neurodegenerative diseases (e.g., Alzheimer's disease) and other diseases. Background Art

[0003] As the life expectancy of the population increases, Alzheimer's disease (AD) has become a widespread health problem worldwide. AD patients suffer cognitive decline in memory, sense of direction, judgment, and reasoning ability (Tanzi and Bertram 2005). Usually, it takes about 10 years for the disease to develop to the final stage leading to death. So far, there is no cure or preventive treatment.

[0004] The deposition of Aβ protein, a metabolite of amyloid precursor protein (hereinafter referred to as APP), and its toxicity in the brain are considered to be the main causes of AD development (Selkoe 2001). Aβ is a protein component identified in senile plaques of AD patients (Ikeda, Wong et al., 1987; Cole, Masliah et al., 1991). Biologically, Aβ is produced by continuous cleavage of amyloid-β precursor protein (APP), which forms fragments of different sizes. Drug studies have identified Aβ42 as the most pathogenic form that contributes to the development of AD (Roher, Chaney et al., 1996; Morelli, Prat et al., 1999). The production of Aβ42 is the result of β- and subsequent γ-cleavage of APP (Citron 2010, De Strooper, Vassar et al. 2010). This insoluble Aβ42 fragment aggregates in the extracellular space to form plaques. Several lines of evidence suggest that soluble oligomers of Aβ42 are more toxic. The presence of Aβ42 has an impact on synaptic activity. The accumulation of Aβ42 can potentially cause abnormal neural network activity and synaptic inhibition (Palop and Mucke 2009).

[0005] Preclinical studies in transgenic mouse models have shown that immunotherapy has great efficacy in preventing AD and prion diseases (Wisniewski 2012). Based on the fact that Aβ is a core molecule in AD, several strategies have been developed to eliminate Aβ through small molecules or immunotherapy (Tabira 2011, Huang and Mucke 2012, Ozudogru and Lippa 2012, Delrieu, Ousset et al. 2014). Although Aβ-directed immunity has shown promising results in mouse models of AD, translation to effective treatment for humans remains challenging.

[0006] In active vaccination trials, individuals who have received active immunization showed a significant reduction in plaque burden and a significant reduction in Aβ relative to non-immunized controls. No matter how encouraging the results were, the treatment group showed no improvement in long-term survival, time to severe dementia, and cognitive function (Holmes, Boche et al., 2008). Two recent large Phase III trials of passive immunization against Aβ also failed to obtain evidence of clinical benefit (Doody, Thomas et al., 2014, Salloway, Sperling et al., 2014). Therefore, the industry expects to find a completely new treatment or prevention therapy for AD. Summary of the invention

[0007] On the one hand, the present invention is a method for treating Alzheimer's disease (AD) in a subject by administering a pharmaceutically effective amount of a STAT1 inhibitor to the subject. In one embodiment, the STAT1 inhibitor is an antibody against IFN or IFN receptor. In another embodiment, the STAT1 inhibitor is an antibody against IL-6 or IL6 receptor. In another embodiment, the STAT1 inhibitor is a preparation that inhibits JAK1 and JAK3. In yet another embodiment, the STAT1 inhibitor is a preparation that reduces STAT1 phosphorylation. In another embodiment, the STAT1 inhibitor is a preparation that prevents STAT1 translocation from entering the nucleus. In another embodiment, the STAT1 inhibitor is an RNAi agent for STAT1.

[0008] On the other hand, the present invention is a method for treating Alzheimer's disease (AD) in a subject by administering a pharmaceutically effective amount of a CH25H inhibitor to the subject. In some embodiments, the CH25H inhibitor is a STAT1 inhibitor. In another embodiment, the CH25H inhibitor is a STAT1 inhibitor, which is an antibody for IFN or IFN receptor. In another embodiment, the CH25H inhibitor is a STAT1 inhibitor, which is an antibody for IL-6 or IL6 receptor. In another embodiment, the CH25H inhibitor is a STAT1 inhibitor, which is a preparation for inhibiting JAK1 and JAK3. In another embodiment, the CH25H inhibitor is a STAT1 inhibitor, which is a preparation for reducing STAT1 phosphorylation. In another embodiment, the CH25H inhibitor is a STAT1 inhibitor, which is a preparation for preventing STAT1 translocation from entering the nucleus. In yet another embodiment, the CH25H inhibitor is a STAT1 inhibitor, which is an RNAi agent for STAT1.

[0009] On the other hand, the present invention is a method for treating Alzheimer's disease (AD) in a subject by administering a pharmaceutically effective amount of a 25-OHC inhibitor to the subject. In one embodiment, the 25-OHC inhibitor is a CH25H inhibitor or a STAT1 inhibitor. In another embodiment, the 25-OHC inhibitor is an analog of 25-OHC. In another embodiment, the 25-OHC inhibitor is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, such as simvastatin.

[0010] On the other hand, the present invention is a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject by administering a pharmaceutically effective amount of a STAT1 inhibitor to the subject. In one embodiment, the STAT1 inhibitor is an antibody against IFN or IFN receptor. In another embodiment, the STAT1 inhibitor is an antibody against IL-6 or IL-6 receptor. In another embodiment, the STAT1 inhibitor is a preparation that inhibits JAK1 and JAK3. In another embodiment, the STAT1 inhibitor is a preparation that reduces STAT1 phosphorylation. In another embodiment, the STAT1 inhibitor is a preparation that prevents STAT1 translocation from entering the nucleus. In another embodiment, the STAT1 inhibitor is an RNAi agent for STAT1.

[0011] On the other hand, the present invention is a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject by administering a pharmaceutically effective amount of a CH25H inhibitor to the subject. In some embodiments, the CH25H inhibitor is a STAT1 inhibitor. In one embodiment, the STAT1 inhibitor is an antibody against IFN or IFN receptor. In another embodiment, the STAT1 inhibitor is an antibody against IL-6 or IL-6 receptor. In another embodiment, the STAT1 inhibitor is a preparation that inhibits JAK1 and JAK3. In another embodiment, the STAT1 inhibitor is a preparation that reduces STAT1 phosphorylation. In another embodiment, the STAT1 inhibitor is a preparation that prevents STAT1 translocation from entering the nucleus. In yet another embodiment, the STAT1 inhibitor is an RNAi agent for STAT1.

[0012] On the other hand, the present invention is a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject by administering a pharmaceutically effective amount of a 25-OHC inhibitor to the subject. In some embodiments, the 25-OHC inhibitor is a CH25H inhibitor or a STAT1 inhibitor. In other embodiments, the 25-OHC inhibitor is an analog of 25-OHC. In other embodiments, the 25-OHC inhibitor is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, such as simvastatin.

[0013] In another embodiment, the present invention is a method for treating Alzheimer's disease (AD) in a subject by providing a genome editing tool; providing the genome editing tool to a neural 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] On the other hand, the present invention is a method for treating Alzheimer's disease (AD) in a subject by providing a genome editing tool; providing the genome editing tool to a neural cell; and then 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. In some embodiments, the genome editing tool is a CRISPR-CAS9 system.

[0015] On the other hand, the present invention is a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject by providing a genome editing tool; providing the genome editing tool to a neural cell; and then 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] On the other hand, the present invention is a method for preventing or delaying the onset of Alzheimer's disease (AD) in a subject by providing a genome editing tool; providing the genome editing tool to a neural cell; and then 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. In some embodiments, the genome editing tool is a CRISPR-CAS9 system. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] Figure 1A and 1B showed that elevated levels of phosphorylated STAT1 were detected in Alzheimer's disease patients. Figure 1A Four images showing the staining of phosphorylated STAT1 (pSTAT1) in brain tissues from three AD patients (Case-1, Case-2, and Case-3) and one healthy person (Control) are shown. Figure 1B Magnified images are shown to illustrate the morphology of pSTAT1 staining, which indicates that pSTAT1 accumulates in the nucleus. See the staining spots within the rectangular box.

[0019] Figure 2A-2E showed that STAT1 gene deficiency attenuated amyloid-β deposition in the brain. Figure 2A Shown are images of amyloid beta (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 whole brain sections as well as the hippocampus at higher magnifications. Figure 2B Shown are the numbers of Aβ plaques in five consecutive sections obtained from 3-month-old APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. Figure 2CShown are the Aβ plaque areas in 5 consecutive sections obtained from 3-month-old APP / PS1 and APP / PS1 / STAT1- / - mice, respectively. Figure 2D Elisa measurements of Aβ42 in TBS extracts of brain tissues obtained from 3-month-old wild-type, APP / PS1, and APP / PS1 / STAT1- / - mice, respectively, are shown. Figure 2E Elisa measurements of Aβ42 in formic acid extracts of brain tissues obtained from 3-month-old wild-type, APP / PS1, and APP / PS1 / STAT1- / - mice, respectively, are shown. * indicates a P value less than 0.05.

[0020] Figure 3A and 3B The identification of CH25H as a STAT1 downstream target was shown. Specifically, Figure 3A Heatmaps showing gene ontology analysis of differentially expressed genes with fold change >1.5 and p-value <0.05 using two biological replicates per genotype are shown. Figure 3B A pie chart showing pathway analysis of differentially expressed genes clustered by David functional annotation is shown.

[0021] Figure 4A and 4B The results show that CH25H is attenuated in STAT1-deficient mice. Figure 4A Real-time quantitative PCR results of CH25H mRNA levels in the brains of APP / PS1 and APP / PS1 / STAT1- / - mice are shown. Data are representative of 3 independent experiments. Figure 4B Shown are the results of Western blotting of CH25H, STAT1, and α-tubulin in brain homogenates of three pairs of APP / PS1 and APP / PS1 / STAT1- / - mice.

[0022] Figure 5A and 5B STAT1 was shown to physically bind to the CH25H promoter. Specifically, Figure 5A A schematic diagram of the CH25H gene and its promoter is shown. Figure 5B The results of a chromatin histone immunoprecipitation (ChIP) experiment are shown, in which DNA fragments containing the CH25H promoter were enriched by pulldown with a STAT1 antibody. Such enrichment was reduced in samples from STAT1 KO (STAT1- / -) mice compared to wild-type (WT) mice.

[0023] Fig. 6A and 6B It was shown that STAT1 deficiency leads to decreased 25-OHC in the brain. Specifically, Fig. 6AShown are the results of measurements of total brain cholesterol levels in APP / PS1 and APP / PS1 / STAT1- / - mice, with n=3 per group. Figure 6B Displayed in Fig. 6A Results from measuring 25-OHC levels in the same mice as the cholesterol measurements in .

[0024] Figure 7 It was shown that the full-length APP protein was reduced in the exosome fraction of STAT1- / - mice. Brain tissue from APP / PS1 and APP / PS1 / STAT1- / - mice was homogenized, and the post-nuclear fraction was subjected to 5%-45% sucrose gradient centrifugation. Ten fractions were collected and solubilized with Triton-100. These fractions were used for Western blotting and for APP and the exosome marker Flotillin1.

[0025] Figures 8A-8C It is shown that 25-OHC treatment reduces exosomal APP while increasing intracellular APP. APP-overexpressing SH-SY5Y cells were treated with different doses of 25-OHC. Fig. 8A Shown are the results of Western blotting with APP from samples obtained by collecting purified exosomes and lysing them. Figure 8B Shown are Western blot results of APP from samples prepared by collecting cell lysates to assess intracellular APP levels. Figure 8C Shown are the results of FACS analysis depicting surface APP in control and 25-OHC treated cells.

[0026] Fig. 9 Shows increase in Aβ42 in exosomes after treatment with 25-OHC. ELISA measurement of Aβ42 in exosomes collected from cell culture media treated with different concentrations of 25-OHC. Data represent three independent experiments. *p<0.05.

[0027] Figures 10A-10C demonstrated an increase in the intracellular retention time of APP after treatment with 25-OHC. Fig. 10A Tracking of APP molecular trafficking by surface APP labeled with FITC-conjugated antibodies is shown. Cells were fixed at different time points to examine the distribution of APP at a specific time. Fig. 10B Shown are snapshot images of a time-lapse video showing the time-lapse translocation of APP in control cells. Fig. 10C Shown are snapshot images from a time-lapse video showing APP translocation in 25-OHC-treated cells.

[0028] Fig.11A , 11Band 11C showed that 25-OHC injection promoted Aβ deposition in APP / PS1 mice. Fig.11A Shown are brain tissue sections for amyloid beta. Two-month-old mice were injected with 25-OHC or saline as a control every other day for 1 month. The brains of these mice were sliced ​​and stained for amyloid beta. Data are representative of 3 pairs of mice injected with saline or 25-OHC. Fig. 11B and 11C Whole brains obtained from saline control and 25-OHC mice ( Fig. 11B ) and hippocampus ( Fig. 11C ) in 5 consecutive sections.

[0029] FIG. 12A to FIG. 12E Diagrams and images of generations of CH25H KO mice are shown. Fig. 12A is a schematic diagram of the CH25H gene and two targeting sgRNAs. Fig. 12B The DNA sequences of the two targeting sgRNAs are shown. Fig. 12C Shown are sequencing results showing the absence of the CH25H gene in CH25H knockout mice. Fig.12D The genotyping results of WT (534 bp) and KO (488 bp) bands are shown respectively. Fig.12E Real-time PCR results of CH25H RNA levels in WT and CH25H KO mice are shown, respectively.

[0030] Fig.13A and 13B demonstrated that Aβ plaque deposition was reduced in CH25H KO mice. Fig.13A Amyloid beta staining is shown in a mouse model of AD (APP / PS1 mice) and in a CH25H-deficient AD mouse (APP / PS / CH25H- / -). Brain sections were obtained from 3-month-old APP / PS1 and APP / PS1 / CH25H- / - mice, respectively. Images show amyloid beta staining in the whole brain and hippocampus at higher magnifications. Fig. 13B and 13C Shown are whole brains obtained from APP / PS1 and APP / PS1 / CH25H- / 3-month-old mice, respectively. Fig. 13B ) and hippocampus ( Fig. 13C ) in 5 consecutive sections.

[0031] Fig.14It was shown that the deficiency of CH25H gene improved the learning and memory of APP / PS1 mice. APP / PS1 (n=11) mice and APP / PS / CH25H- / - (n=8) mice were trained in water maze. The average time to find the hidden platform was recorded during the 5-day training. The x-axis is the number of days and the y-axis is the time the mouse spent in the water maze to find the platform.

[0032] Fig.15 Western blot results of APP in cells treated with 25-OHC and different doses of prednisolone trimethylacetate or simvastatin are shown. Simvastatin can act as a potent 25-OHC inhibitor. SH-SY5Y cells were treated with 25-OHC and different doses of prednisolone trimethylacetate or simvastatin. After 24 hours, the cells were lysed and blotted for APP.

[0033] Fig.16A , 16B , 16C and 16D demonstrate that the differences in Aβ deposition are not due to differences in APP expression levels or the secretase that cleaves APP. Fig.16A Real-time PCR quantification of APP mRNA levels in the brains of APP / PS1 and APP / PS1 / STAT1- / - mice is shown. Fig. 16B , 16C The three figures shown in 16D respectively show the Adam 10 ( Fig. 16B )、BACE1( Fig. 16C ) and Nicastrin( Fig.16D ) Real-time PCR quantitative results of α, β and γ secretase of APP. Data represent 3 independent experiments.

[0034] Fig.17A and 17B These results suggest that the phagocytic capacity of microglia is similar between WT and STAT1-deficient mice. Fig.17A Microglia from WT or STAT1-deficient mice were incubated with fluorescent microbeads. Cells were fixed after 5 minutes of incubation and internalized microbeads were imaged. WT and KO media are media from WT or KO microglia, respectively. Fig. 17B Quantification results of internalized beads are shown. Fig. 17C Real-time PCR measurements of SV2A in WT and STAT1-deficient mice are shown. Fig.17D Real-time PCR measurement of CCR2 in WT and STAT1-deficient mice is shown.

[0035] Fig.18A , 18B, 18C and 18D show the expression of CH25H in the brain tissues of APP / PS1 and APP / PS1 / STAT1- / - mice. Fig.18A ) and other known cholesterol hydroxylase Cyp46al ( Fig.18B )、Cyp7bl( Fig.18C ) and Cyp7al( Fig.18D The results showed that other cholesterol hydroxylases Cyp46al, Cyp7b1 and Cyp7al were not affected by STAT1 deficiency.

[0036] Figures 19A-19F It was shown that the deficiency of STAT1 did not affect general lipid metabolism. Fig.19A The graph shows the body weight of APP / PS1 and APP / PS1 / STAT1- / - mice at 3 months of age, with n=5 in each group. Fig.19B Oil Red-O stained images of liver sections from APP / PS1 and APP / PSI / STAT1- / - mice are shown, respectively. Images are representative of 3 pairs of mice. Fig.19C , 19D The four figures shown in 19E and 19F show the real-time quantitative PCR results of LPL, ABCA1, APOE and HMGCR in APP / PS1 and APP / PS1 / STAT1- / - mice, respectively.

[0037] Fig. 20A , 20B Figures 20C, 20D, 20E, 20F, and 20G show the measurement of the induction of immediate early genes after potassium chloride treatment. After 30 min of treatment with 50 nM KCl, c-fos ( Fig. 20A )、zif268( Fig. 20B )、BDNF-IV( Fig. 20C )、BDNF-IX( Fig.20D )、Gadd45b( Fig.20E )、Npas4( Fig.20F ) and CH25H( Figure 20G ) The results showed that STAT1 deficiency did not alter basal neural activity. DETAILED DESCRIPTION

[0038] The present disclosure is based in part on the new and surprising discovery that STAT1-regulated CH25H expression affects the pathogenesis of Alzheimer's disease (AD). The inventors found that CH25H is a downstream target of STAT1, and STAT1 and its target CH25H promote Aβ deposition during the development of AD. The higher amount of phosphorylated STAT1 (pSTAT1) detected in AD patients is the cause of AD development because the genetic deletion of STAT1 delays Aβ deposition in the brain. CH25H is an enzyme that converts cholesterol to 25-OHC. One of the unique functions of 25-OHC is that it can cross the blood-brain barrier (BBB), which is very beneficial for drug development because many drugs currently in clinical trials have BBB permeability issues. The inventors demonstrated the effect of CH25H or 25-OHC on the pathogenesis of AD. CH25H knockout mice have reduced Aβ deposition comparable to STAT1 knockout mice. In contrast, the administration of 25-OHC promotes Aβ deposition in the brain.

[0039] Described herein are methods and means for treating or preventing diseases or medical symptoms at least in part associated with the STAT1-CH25H axis, which produces 25-OHC as an effector for regulating Aβ deposition. In such diseases or medical symptoms, the activity of STAT1-CH25H is enhanced, resulting in increased Aβ deposition. 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) an increase in the amount of cholesterol as a CH25H substrate. The antagonists used for treatment and prevention herein may be chemicals, small molecules, drugs, non-coding RNA, antisense nucleotides, peptides, proteins or antibodies or portions thereof. In a specific embodiment, the antagonist is administered in a therapeutically effective amount.

[0040] The present invention provides methods for treating diseases or medical symptoms associated with Aβ deposition in brain cells of a subject. In one embodiment, the disease or medical symptom is attributed to abnormal production, transport or clearance of Aβ. In another embodiment, the disease or medical symptom is due to increased activity of the STAT1-CH25H pathway caused by the following factors, such as but not limited to: IFNα, IFN-β, IFN-γ, IL-6 family cytokines, or acute or chronic symptoms. In a further embodiment, the disease or medical symptom is due to an increase in 25-OHC content caused by high cholesterol content in the subject.

[0041] The inventors have found that blocking STAT1 signaling, abolishing STAT1 expression, or depleting CH25H attenuates the pathogenesis of AD with reduced Aβ plaques. Chemical analogs of 25-OHC can also reduce APP in cultured cells. The treatment methods herein include administering agents that modulate STAT1 or CH25H signaling, expression, or activity, such as agents that block 25-OHC activity or promote 25-OHC degradation. In particular, the present invention provides methods and means for treating Alzheimer's disease.

[0042] Thus, in one aspect, the invention relates to methods of treating a disease such as AD in the brain of a subject by inhibiting STAT1, its downstream target CH25H or 25-hydroxylated cholesterol (25-OHC).

[0043] Thus, in one aspect, the invention relates to a method of preventing or delaying the onset of a disease such as AD in the brain of a subject by inhibiting STAT1, its downstream target CH25H or 25-hydroxylated cholesterol (25-OHC).

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

[0045] In another aspect, the invention relates to a method of preventing or delaying the accumulation of amyloid beta (Aβ) deposits in the brain of a subject by inhibiting STAT1, its downstream target CH25H or 25-hydroxylated cholesterol (25-OHC).

[0046] In one embodiment, the inhibition of STAT1 in the above methods is by administering to the subject a pharmaceutically effective amount of a STAT1 inhibitor.

[0047] As used herein, the term "subject" refers to an animal, preferably a mammal, most preferably a human. In some cases, the subject can be divided into different subgroups based on the presence or absence of certain biomarkers. In other cases, the subject can be divided into different subgroups based on whether the amount of certain biomarkers is below or above a threshold. In either case, the subgroup can be included in the treatment of the present invention or excluded from the treatment.

[0048] In some embodiments, the STAT1 inhibitor is a composition comprising a pharmaceutically effective amount of an active agent (eg, a compound, a peptide, an antibody or fragment thereof, or a nucleic acid) and a pharmaceutically acceptable excipient.

[0049] "Excipient," "carrier," "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier" or similar terms refer to one or more ingredients or components that are acceptable in the sense of being compatible with the other ingredients of the 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 administered.

[0050] The terms "pharmaceutically effective amount", "effective dose", etc., with respect to a STAT1 inhibitor, refer to an amount of a STAT1 inhibitor sufficient to induce a desired response, such as reducing Aβ deposition in a subject (e.g., a human) to which it is administered; or to detectably modulate or improve the molecular or cellular parameters or clinical conditions or symptoms of AD. An effective amount, such as for therapeutic use in humans, can be a single dose or two or more sub-doses of a STAT1 inhibitor administered in one day, or can be administered in multiple doses over a period of time, such as more than 1, 2, 3, 4 or about 7 days to a year.

[0051] Based on the detailed disclosure provided herein, the determination of a therapeutically effective amount is well within the capabilities of those skilled in the art. For example, a therapeutically effective amount or dosage can be first estimated from in vitro and cell culture assays. For another example, an effective dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine an effective dose for humans.

[0052] The toxicity of the active ingredients described herein can be determined by standard pharmaceutical methods in vitro, cell culture or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a series of dosages for humans. The dosage can vary depending on the dosage form used and the route of administration used. The specific formulation, route of administration and dosage can be selected by individual physicians based on the patient's condition.

[0053] Dosage and interval can be adjusted to the blood level (minimum effective concentration, MEC) of the active ingredient sufficient to induce or inhibit biological effects, respectively. The MEC of each preparation will be different, but can be estimated based on in vitro data. The dosage required to reach MEC will depend on individual characteristics and route of administration. Detection and determination can be used to determine the plasma administration concentration. The mode of administration depends on the severity and responsiveness of the disease to be treated, and can be single or multiple administrations, and the course of treatment can last for several days to several weeks or until the disease state is cured or alleviated. Of course, the amount of the composition to be given will depend on, for example, the subject being treated, the severity of the disease, the mode of administration, and the judgment of the prescribing physician.

[0054] For example, in some embodiments, the pharmaceutically effective amount of a STAT1 inhibitor can be as low as about 0.02 mg / kg / day to about 0.03 mg / kg / day in humans, and as high as about 2 mg / kg / day to about 3 mg / kg / day. Even in some embodiments, as Aβ deposition becomes more severe, the effective amount can be higher than 3 mg / kg / day. In other embodiments, the effective amount when using a STAT1 inhibitor for treatment can be higher than the effective amount when using a STAT1 inhibitor to prevent or delay the onset of Aβ deposition.

[0055] In some embodiments, the dosage and dosing schedule may also depend on the sex of the human subject to be treated. In some cases, certain female patients may be more susceptible to STAT1 treatment, and therefore the dosage may be smaller than that of male patients, and the frequency of the dosing schedule may be less frequent. In other cases, certain female patients may be more sensitive to STAT1 treatment, and therefore, the dosage may be smaller, and the frequency of the dosing schedule may be less frequent than that of male patients.

[0056] In some embodiments, administration may preferably begin long before Aβ deposition can be detected. In other embodiments, administration may begin after Aβ deposition is detected, before any clinical symptoms of AD are seen. In other embodiments, administration may begin after clinical symptoms of AD are visible. In some cases, administration may be a single dose. Administration may be multiple doses, such as 2, 3, 4, 5 or 6 doses in other cases. Multiple administrations may be performed in succession as many times as the physician deems necessary.

[0057] In other embodiments, the dosing schedule and the dose can be linked. For example, during the first dosing period that begins long before clinical symptoms of AD are seen, the dose per administration can be less than the dose for the first administration after clinical symptoms of AD can be seen. For another example, when the administration is frequent, such as once every 12 hours, the dose per administration can be less than the dose for less frequent administration, such as once every 24 hours.

[0058] When a STAT1 inhibitor is used in the treatment methods or other methods disclosed herein, terms such as "use", "treatment", "treatment", "treatment", etc. refer to the administration of a STAT1 inhibitor to a subject, as described herein or as described in the references cited herein, to a tissue of a subject or to contact with a tissue, cell or cell-free system in vivo or in vitro. Typically, such use or treatment results in, for example, (1) a detectable improvement or alleviation of the condition or symptom being treated; (2) a detectable modulation of the activity, level or number of related biomolecules, therapeutic cell populations or pathological cells; (3) a slowing of the progression of a disease or a delay in its onset, or a reduction in the severity of a symptom of a disease; or (4) another detectable response described herein. Any such improvement may be transient, for example, lasting for at least a period of time, for example, about 1 to 24 hours, or several days, for example, about 1, 2, 3, 4, 5, 6 or 7 days; or the improvement may be prolonged, for example, lasting for about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 35, 42, 49, 56 to 60 days or longer, or permanent improvement. Treatment may slow the progression of a disease or symptom or reduce the severity of a disease or symptom, for example, the onset of a disease or symptom in at least some subjects may be delayed by about 1-24 hours, about 2-10 days, 2-30 days or about 1-5 years compared to a subject not receiving adequate amounts of a STAT1 inhibitor. Therefore, compared to an appropriate control, such as an untreated condition, the use of a STAT1 inhibitor or treatment generally results in a detectable regulation of relevant immune parameters, such as the regulation of the level, activity or relative amount of target effector or inhibitory cell populations, interleukins, cytokines, chemokines, immunoglobulins. STAT1 inhibitor treatment may also result in the regulation of the level or activity of related transcription factors or enzymes, cell biological activity or levels, and disease pathogen activity. Treatment with a STAT1 inhibitor may be used to delay or prevent the onset of a disease, symptom or complication, or to improve or slow the progression of a pre-existing disease, disorder, symptom or complication, or to help eliminate a disease, disorder, symptom or complication, such as the deposition of Aβ in AD.

[0059] "Relieve", "improve", "improve", etc. refer to a detectable improvement or a detectable change consistent with improvement in a subject or at least a few subjects, such as at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or between any two of these values. Such improvement or change can be observed in treated subjects compared to subjects not treated with a STAT1 inhibitor, wherein the untreated subject suffers from or is developing the same or similar disease, disorder, symptom, etc. The improvement of a disease, disorder, symptom or measured parameter can be determined subjectively or objectively, for example, by self-assessment of the subject, by evaluation by a clinician, or by performing appropriate assays or measurements, including, for example, quality of life assessment, slowing of the progression of a disease or disorder, reduction in the severity of a disease or disorder, appropriate assays of the level or activity of a biological molecule or cell (one or more), or detection of cell migration within a subject. The improvement may be transient, prolonged, or permanent, and may be within a variable relative time period during or after administration of the STAT1 inhibitor to the subject, or after use in an assay or other method described herein or in a reference cited, for example, within 1 hour after the subject receives the STAT1 inhibitor, or about 3, 6, 9 months or more thereafter.

[0060] "Modulation", for example, in the context of a symptom, the level of a molecule or a biological activity, a cellular response, a cellular activity, etc., refers to a detectable increase or decrease in a cell, level or activity, etc. Compared to a subject not treated with a STAT1 inhibitor, such an increase or decrease can be observed in a treated subject who suffers from or is experiencing the same or similar disease, condition, symptom, etc. Such an increase or decrease can be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85% in any range between any two of these values, about 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more. Modulation can be determined subjectively or objectively, for example, by self-assessment of the subject, by an assessment by a clinician, or by performing appropriate assays or measurements, including, for example, quality of life assessments or appropriate assays for the level or activity of migration in a molecule, cell, or subject. Modulation can be transient, prolonged, or permanent, or can be over a variable relative time period during or after administration of the STAT1 inhibitor to a subject, or used in an assay or other method described herein or in a cited reference, such as within about 1 hour of administration, or about 3, 6, 9 months or more after the subject receives the STAT1 inhibitor.

[0061] At various places in the present disclosure, for example, in any disclosed example or claim, reference is made to compounds, compositions, formulations, or methods that are described as "comprising" one or more specific components, elements, or steps. Embodiments of the present invention also specifically include those compounds, compositions, formulations, or methods that consist of or consist essentially of those specified components, elements, or steps. According to U.S. patent law, the terms "comprising," "consisting of," and "consisting essentially of" have their generally accepted meanings. For example, disclosed compositions or methods that "comprise" components or steps are open, and they include or are interpreted as those compositions or methods and one or more additional components or steps. Similarly, disclosed compositions or methods that "consist of components or steps" are closed, and they will not include or be interpreted as those compositions or methods having appreciable amounts of additional components or additional steps.

[0062] In some cases, the STAT1 inhibitor can be an antibody against interferon, because interferon is necessary for activating STAT1, and the inhibition of interferon will prevent or reduce the activation of STAT1. An example of an interferon antibody is Fontolizumab (planned commercial name HuZAF), which is a humanized monoclonal antibody against interferon gamma. Because methods for producing and measuring antibody activity are well known in the art, those skilled in the art can prepare similar anti-interferon antibodies and screen their inhibitory effects on interferon.

[0063] In some cases, STAT1 inhibitors can be antibodies against interferon receptors, because interferon receptors are necessary for activating STAT1, and inhibition of interferon receptors will prevent or reduce the activation of STAT1. An example of an interferon receptor antibody is Anifrolumab, a human monoclonal antibody targeting type I interferon (IFN) receptors. Since the preparation and determination of antibody activity are well known in the art, those skilled in the art can prepare similar antibodies against interferon receptors and screen their inhibitory effects on interferon receptors.

[0064] In some cases, a STAT1 inhibitor may be an antibody against IL6, since IL6 is required for activation of STAT1, and inhibition of interferon will prevent or reduce activation of STAT1. An example of an IL-6 antibody is Siltuximab, which is a chimeric monoclonal antibody (made from human and mouse proteins) against IL-6. Because methods for generating and determining antibody activity are well known in the art, one skilled in the art can prepare similar antibodies against IL-6 and screen them for their inhibitory effects on IL-6.

[0065] In some cases, a STAT1 inhibitor may be an antibody against the IL-6 receptor (IL-6R), because interferon receptors are required for activation of STAT1, and inhibition of the IL-6 receptor will prevent or reduce activation of STAT1. An example of an IL-6 receptor antibody is Tocilizumab, a humanized monoclonal antibody against the interleukin 6 receptor. Because methods for generating and determining antibody activity are well known in the art, one skilled in the art can prepare similar antibodies against IL-6R and screen them for their inhibitory effects on IL-6R.

[0066] In other cases, the STAT1 inhibitor can be an agent that inhibits JAK1 or JAK3, because JAK1 and JAK3 are well-known upstream kinases that activate STAT signaling. The agent that inhibits JAK1 and JAK3 can be a small molecule, for example, Filgotinib as a JAK1 inhibitor, tofacitinib as a JAK3 inhibitor.

[0067] In other cases, the STAT1 inhibitor may be an agent that reduces STAT1 phosphorylation. Such an agent that reduces STAT1 phosphorylation may be FLLL32, which is reported to inhibit STAT phosphorylation.

[0068] In other cases, the STAT1 inhibitor may be an agent that prevents STAT1 from translocating into the nucleus. Such agents that prevent STAT1 from translocating into the nucleus may be Meales virus proteins P and V.

[0069] In other cases, the STAT1 inhibitor is an RNAi agent for STAT1. This RNAi agent can be any siRNA targeting STAT1, such as the product 105153 of ThermoSisher Scientific. Other siRNA agents targeting STAT1 can be prepared by those skilled in the art, because the method for preparing siRNA for a specific target is well known in the art.

[0070] In other cases, as a method for treating, preventing or delaying the onset of AD, a genome editing tool can 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 neural cells. 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 can be delivered to the target cell. An example of such a genome editing tool is the CRISPR-CAS9 system described by Ran, F et al. In vivo genome editing using Staphylococcus Cas9, Nature (2015). Another example of such a genome editing tool is the CRISPR-Cpf1 system, as described by Zetsche et al., Cpf1 is a single RNA-guided nuclease of the class 2 CRISPR-Cas system, Cell (2015).

[0071] In other embodiments, the inhibition of CH25H in the above method is by administering a pharmaceutically effective amount of a CH25H inhibitor to the subject. In some embodiments, the CH25H inhibitor is a STAT1 inhibitor, because STAT1 activates CH25H and the inhibition of STAT1 results in STAT1 being unable to activate CH25H. The STAT1 inhibitor can be any STAT1 inhibitor disclosed in the present application.

[0072] In other embodiments, as a method for treating, preventing or delaying the onset of AD, a genome editing tool can be used to delete the entire CH25H gene, the histidine cluster region of the CH25H gene, or the promoter region or the CH25H gene in a neuron. 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 can be delivered to a target cell. An example of such a genome editing tool is the CRISPR-CAS9 system described by Ran, F et al., which uses Staphylococcus Cas9 for in vivo genome editing, Nature (2015). Another example of such a genome editing tool is the CRISPR-Cpf1 system, as described by Zetsche et al., Cpf1 is a single RNA-guided nuclease of a Class 2 CRISPR-Cas system, Cell (2015).

[0073] In other embodiments, the inhibition of 25-OHC in the above methods is by administering to the subject a pharmaceutically effective amount of a 25-OHC inhibitor.

[0074] In some cases, the 25-OHC inhibitor can be a CH25H inhibitor or a STAT1 inhibitor, because CH25H and STAT1 are upstream of 25-OHC, and the inhibition of CH25H or STAT1 will result in the inability of CH25H to produce 25-OHC. In other cases, the 25-OHC inhibitor can be an analog of 25-OHC. In other cases, the 25-OHC inhibitor can be a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor. An example of an HMG-CoA reductase inhibitor is simvastatin.

[0075] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the present invention. Therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and each numerical value within the range. For example, the description of the range from 1 to 6 should be considered to have explicitly disclosed sub-ranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6. This has nothing to do with the amplitude of the range.

[0076] It should be understood that, for the sake of clarity, certain features of the invention described in the context of individual embodiments may also be used in combination in a single embodiment. Conversely, for the sake of brevity, various features of the invention described in the context of a single embodiment may also be used appropriately alone or in any suitable subcombination or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0077] It is to be understood that this invention is not limited to the specific methodology, protocols and reagents described herein, which 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 present invention.

[0078] Embodiments of the disclosed subject matter are set forth 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 the methods and materials described herein can be used to practice or test the presently disclosed subject matter. Exemplary methods and materials are now described as follows.

[0079] Example

[0080] method

[0081] Mouse

[0082] APP / PS1 5XFAD mice were purchased from Jackson Laboratory, while STAT1-deficient mice were provided by Daved Levy. CH25H KO mice were generated by the crisper / cas9 method. The pST1374-Cas9-N-NLS-flag-linker plasmid (Addgene ID44758) for expressing Cas9 protein has been described previously (Zhou et al., 2014). The Ch25h gene sequence was downloaded from the UCSC Genome Browser website (http: / / genome.ucsc.edu / ). Two sgRNA oligonucleotides were synthesized and annealed to the pUC57-sgRNA structure. In vitro transcription was performed as described previously (Zhou et al., 2014). Cas9 mRNA and sgRNA were injected into the background of C57BL6 / J. APP / PS1 mice were mated with CH25H- / - mice and offspring were generated, which carried the APP / PS1 gene and the WT or KO allele of CH25H. APP / PS1 mice were mated with STAT1- / - mice to produce offspring that carried the APP / PS1 gene and either the WT or KO allele of STAT1. All mice had a C57BL / 6 genetic background and were housed under specific pathogen-free conditions at the National University of Singapore. All experiments were performed on 3-4 month old mice and were approved by the Institutional Animal Care and Use Committee of the National University of Singapore.

[0083] Preparation of tissue lysates and sucrose gradients

[0084] The mouse brain was weighed and homogenized in 9 volumes of TBS. The resulting homogenate was centrifuged at 1000 g for 15 minutes at 4 ° C. The supernatant was used as the post-nuclear fraction. The post-nuclear fraction was carefully placed in a 5%-45% sucrose gradient and centrifuged at 147,000 g for 16 hours at 4 ° C. After centrifugation, 1 ml was taken from each fraction from top to bottom. Triton was added to each fraction to reach a final concentration of 0.1% to solubilize membrane-associated proteins. The resulting samples were used for Western blot analysis.

[0085] Preparation of exosomes

[0086] 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 contamination of serum exosomes. 24 hours after the medium replacement, the medium was collected and centrifuged at 200g for 5 minutes to pellet floating cells. The cell-free medium was further centrifuged at 100,000g for 1 hour at 4°C, and the pellet containing exosomes was dissolved in RIPA buffer.

[0087] Microarray analysis

[0088] Microarray analysis was performed using the affymetrix microarray system (Affymetrix) provided by Molecular Genomics. Data analysis was performed using GeneSpring software. Differentially expressed genes were entered into the David Functional Annotation Clustering website (http: / / david.abcc.ncifcrf.gov / home.jsp) using the default settings of medium classification stringency. The clustering results were re-plotted in pie charts.

[0089] Histological analysis

[0090] The tissue was fixed in 4% paraformaldehyde, dehydrated, infiltrated and embedded in paraffin. The sections (5 μm) were stained with hematoxylin and eosin (H&E) to assess the overall morphology. For immunofluorescence (IF) or immunohistochemistry (IHC), the sections were rehydrated and stained with an anti-Aβ primary antibody (Cell Signaling) and then incubated with a fluorescently coupled secondary antibody (Invitrogen). For Oil Red O staining, the liver was embedded in Tissue Tek (Electron Microscopy Science) and frozen at -80 ° C. 10 μm thick sections were cut and hydrated with dH2O, and then incubated in Oil Red O working solution (0.3% Oil Red O (Sigma) dissolved in 60% isopropanol) for 1 h. Afterwards, the slides were quickly washed 3 times in 60% isopropanol until the isopropanol dripped from the slides was clear. Subsequently, the slides were washed with water and counterstained with hematoxylin.

[0091] Real-time PCR

[0092] 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 using a 7500 real-time PCR system (Applied Biosystems) using a SYBR qPCR kit (KAPA). Actib, Gapdh or Rn18S were used as internal controls. Primer sequences are available upon request.

[0093] ELISA

[0094] Aβ42 was quantified using an ELISA kit (Millipore) according to the manufacturer's instructions.

[0095] Chromatin immunoprecipitation assay

[0096] Half of the brain hemisphere from APP / PS1 or APP / PS1 / STAT1- / - mice was homogenized in 9 volumes of TBS to obtain brain homogenates. Formaldehyde was added at a final concentration of 1%, cross-linked for 10 minutes, and then quenched with glycine. The homogenate was treated with hypotonic buffer and then treated with nuclear lysis buffer to release chromatin. Chromatin was disrupted by ultrasound and pre-cleared with Protein G microbeads, then precipitated overnight at 4°C with anti-STAT1 antibody (Santa Cruz) or conventional rabbit IgG (Santa Cruz). After washing and elution, cross-link reversal was completed by incubation at 65°C for 8 hours. As described above, the eluted DNA was purified and RT-PCR analysis was performed with primers specific for the CH25H promoter.

[0097] statistics

[0098] Statistically significant differences were determined by Student's t-test using GraphPad Prism 6.01. p values ​​< 0.05 were considered significant. Clinical p values ​​were determined by multiple comparisons using one-way multiple range analysis of variance (ANOVA). Unless otherwise stated, data are presented as mean ± SEM.

[0099] Example 1: hStat1 knockout mice have reduced Aβ deposition

[0100] We demonstrated that STAT1 expression was higher in AD cases than in elderly matched controls ( Figure 1A and 1B ).exist Figure 1A In the control cells, no phosphorylated STAT1 (pSTAT1) staining was observed. Figure 1AShown and Figure 1B Magnified snapshots of the photograph of case-1 in Figure 2. In each of the three cases, the staining for pSTAT1 was much more abundant.

[0101] To understand whether activation of the STAT1 pathway is the cause of AD pathogenesis or the result of neuroinflammation in the late stage of AD, we crossed STAT1- / - mice with APP / PS1 mice to generate AD mice with a STAT1- / - background. APP / PS1 / STAT1- / - mice and their APP / PS1 genotype littermates were raised for 3-4 months and sacrificed for histological examination of Aβ deposition. Unexpectedly, we found that the amount of Aβ and the area occupied by Aβ in Stat1- / - mice were continuously reduced ( Figure 2A and 2B Similar results were observed when we measured Aβ42 in TBS or formic acid extracts ( Figure 2C and 2D ). In addition, the reduction of Aβ was not caused by changes in APP expression levels or cleavage of APP by secretases ( Fig.16A and 16B ).

[0102] Example 2: Reduction of Aβ deposition in Stat1-KO mice due to reduction of downstream target CH25H

[0103] Our microarray data identified CH25H as a STAT1-regulated gene in AD ( Figure 3A and 3B In addition, we confirmed the regulatory effect of STAT1 on CH25H by real-time PCR, western blot, and ChIP analysis ( Figure 4A , 4B , 5A and 5B). Figure 4A As shown in (showing real-time PCR results), CH25H mRNA expression was significantly reduced in STAT1-deficient mice. Figure 4B Western blot results are shown, indicating that CH25H protein levels are also significantly reduced in STAT1-deficient mice. Figure 5A and 5B The results of ChIP analysis also showed that in STAT1-deficient mice, less STAT1 bound to the CH25H gene promoter sequence. The reduction in 25-OHC levels in STAT1-deficient mice further supports the view that the STAT1-CH25H axis controls brain 25-OHC levels ( Figure 6A-6B ).

[0104] Example 3 STAT1-CH25H-regulated 25-OHC affects the secretion of APP in exosomes

[0105] Mouse brain homogenates were fractionated by sucrose gradient to examine the cellular localization of APP protein. In short, the distribution pattern of APP was similar between 5XFAD and 5XFAD / STAT1- / - mice, except for the exosome-rich, Flotillin1-rich fraction that had the highest density. STAT1- / - mice had significantly higher APP content in this fraction ( Figure 7 ).

[0106] We used the APP-overexpressing SH-SY5Y cell line to further examine whether the differences in exosomal APP were due to 25-OHC. Cells were treated with different doses of 25-OHC, and cell lysates and exosomes were collected from the culture medium. 25-OHC showed a dose-dependent increase in cellular APP, while decreasing exosomal APP ( Fig. 8A and 8B ).like Fig. 8A As shown, less APP was found in the exosome fraction with increasing 25-OHC doses. In contrast, more APP was found in the cell lysate fraction with increasing 25-OHC doses.

[0107] FACS analysis of surface APP also showed that 25-OHC increased the amount of APP on the cell surface ( Figure 8C ). After 25-OHC treatment, more APP was detected on the surface of APP-overexpressing cells.

[0108] In addition, we found that Aβ42 in exosomes increased after treatment with 25-OHC. The culture medium of cells treated with different concentrations of 25-OHC was collected, and Aβ42 in exosomes collected from the culture medium was measured by ELISA. Fig. 9 As shown, an increase in Aβ42 was detected in the cell culture medium after treatment with higher concentrations of 25-OHC. Data are representative of three independent experiments.

[0109] Example 4 25-OHC increases the retention time of APP in cells

[0110] 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 are normal: APP expression is similar, and enzymes that cleave APP are at comparable levels ( Fig.16A and 16B ).like Fig.16A As shown in Figure 2, APP expression levels were similar regardless of STAT1 deficiency. Fig. 16B As shown, the expression levels of Adam10, BACE1, and Nicastrin, the α-, β-, and γ-secretases of APP, respectively, were also similar regardless of STAT1 deficiency.

[0111] We then proceeded to examine the trafficking of APP protein. Surface APP was labeled with antibodies, the cells were returned to the incubator, and fixed for staining at different time points. In untreated cells, APP rapidly entered specific compartments of the cell and the signal disappeared within 6 hours, whereas in cells treated with 25-OHC, APP trafficking was slower ( Fig. 10A The time-lapse video clearly showed that in untreated cells, APP accumulated in specific compartments, whereas in 25-OHC-treated cells, APP remained uniformly distributed over the monitored time ( Fig. 10B ).

[0112] Example 5 CH25H KO reacquires STAT1 KO phenotype to delay the pathogenesis of AD

[0113] We investigated the role of CH25H KO in the pathogenesis of AD. SgRNAs targeting CH25H were designed, where sgRNA1 is SEQ ID NO: 1 and sgRNA2 is SEQ ID NO: 2. Fig. 12A and 12B Using two sgRNAs, the CH25H gene was knocked out by the crisper / cas9 method, thereby deleting 46 base pairs (bp) in the exon of the CH25H gene (see Fig. 12C ) to generate CH25H knockout (KO) mice.

[0114] In CH25H KO mice, a 46 bp fragment of the CH25H gene was detected to be missing, of which the 488 bp band was the missing CH25H gene, while the 534 bp band was the wild-type gene. The expression of CH25H mRNA in CH25H KO mice was significantly reduced ( Fig.12E ).

[0115] Once crossed with 5XFAD mice, CH25H KO showed a phenotype similar to that of STAT1 KO. Aβ was greatly reduced in both immunostaining and ELISA quantification ( Fig.13A , 13B and 13C). In contrast, mice injected with 25-OHC had significantly higher levels of Aβ ( Fig.11A , 11B and 11C).

[0116] To test the effects of Aβ reduction on cognitive abilities, mice were examined using a water maze. XFAD mice gradually learned to locate the platform under water, while CH25H KO mice took significantly less time to find the platform (p<0.05), indicating that they performed better on learning and memory tasks ( Fig.14 ).

[0117] Example 6: Simvastatin inhibits 25-OHC-induced APP accumulation

[0118] We screened a number of potential small molecule 25-OHC inhibitors and found that simvastatin blocked the 25-OHC-induced increase in cellular APP. This effect was seen at simvastatin as low as 10 nM, which is well below the dose that could cause cytotoxic effects.

[0119] like Fig.15 As shown, 25-OHC treatment led to an increase in APP levels. However, simvastatin treatment could reduce the increase in APP levels caused by 25-OHC treatment. In contrast, prednisolone triacetate treatment had no effect on the increase in APP levels induced by 25-OHC. Therefore, simvastatin is an effective inhibitor of 25-OHC.

[0120] Example 7 Similar phagocytosis between WT and STAT1-deficient microglia

[0121] WT or STAT1- / - microglia were incubated with fluorescent microbeads to test their phagocytic capacity. To examine the effects of secreted factors in culture, both cells were kept in culture medium with WT cells or in culture medium with STAT1- / - cells. Imaging and quantification showed that phagocytosis was similar in all conditions tested ( Fig.17A and 17B We also measured the mRNA of SV2a and CCR2, key genes involved in the phagocytic process. Their expression levels were similar between APP / PS1 and APP / PS1 / STAT1- / - ( Fig. 17C and Fig.17D ).

[0122] Example 8 CH25H is a STAT1-dependent cholesterol hydroxylase

[0123] We tested several known cholesterol hydroxylases, including CH25H, Cyp46a1, Cyp7b1, and Cyp7a1, which add hydroxyl groups to cholesterol at different positions. Of the enzymes we tested, only CH25H showed a STAT1-dependent expression pattern, as only CH25H expression was significantly reduced ( Fig.18A , 18B , 18C and 18D).

[0124] Example 9 STAT1 deficiency does not cause general metabolic defects

[0125] Initially, CH25H was known to regulate cholesterol metabolism. However, when we compared body weight, lipid deposition in the liver, and key enzymes involved in lipid metabolism, there were no significant changes between WT and STAT1- / - mice ( Figures 19A-19F ).like Fig.19A As shown, there was no significant difference in body weight between APP / PS1 and APP / PS1 / STAT1- / - mice. Fig.19BAs shown in Figure 2, there was no significant difference in lipid deposition in hepatocytes between APP / PS1 and APP / PS1 / STAT1- / - mice. Figures 19C-19F As shown, 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.

[0126] Example 10 STAT1 deficiency does not alter neuronal activity measured by induction of immediate early genes.

[0127] Primary hippocampal neurons from WT or STAT1- / - mice were cultured and then treated with 50 mM KCl. Some immediate early genes (including cfos, Zif268, BDNF-IV, BDNF-IX, Gadd45b, Npas4, and Ch25h) were measured by RT-PCR as markers of neuronal activity. Figures 20A-20G As shown, both WT and STAT1- / - neurons were able to induce these genes to similar extents.

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Claims

1. A method for treating Alzheimer's disease (AD) in a subject, preventing or delaying the onset of AD in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a STAT1 inhibitor, a CH25H inhibitor or a 25-OHC inhibitor.

2. The method according to claim 1, characterized in that The STAT1 inhibitor is an antibody against IFN or IFN receptor.

3. The method according to claim 1, characterized in that The STAT1 inhibitor is an antibody against IL-6 or IL6 receptor.

4. The method according to claim 1, characterized in that The STAT1 inhibitor is an agent that inhibits JAK1 and JAK3.

5. The method according to claim 1, characterized in that The STAT1 inhibitor is an agent that reduces STAT1 phosphorylation.

6. The method according to claim 1, characterized in that The STAT1 inhibitor is an agent that prevents STAT1 from translocating into the cell nucleus.

7. The method according to claim 1, characterized in that The STAT1 inhibitor is an RNAi agent targeting STAT1.

8. The method according to claim 1, characterized in that The 25-OHC inhibitor is an analog of 25-OHC.

9. The method according to claim 1, characterized in that The 25-OHC inhibitor is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor.

10. The method according to claim 1, characterized in that The 25-OHC inhibitor is simvastatin.

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