CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to US 62/508,662, filed May 19, 2017, the entire contents of which is hereby incorporated by references.

FIELD

[0002] The present disclosure relates generally to the treatment of a prion disease.

BACKGROUND

[0003] Prions are proteinaceous infectious particles that cause fatal spongiform encephalopathies in humans and animals [1]. They are transmissible within and between species under natural conditions, and can even be zoonotic, exemplified by bovine spongiform encephalopathy (BSE) which has been transmitted to humans and gave rise to a new variant of Creutzfeldt-Jakob disease (vCJD).

[0004] In humans, there are sporadic (Creutzfeldt-Jakob disease), familial (e.g.

Gerstmann-Straeussler-Scheinker syndrome) or infectiously acquired (e.g. Kuru, variant CJD) forms [2].

[0005] Prions consist solely of a misfolded and aggregation-prone isoform of the cellular prion protein (PrPc), termed PrPSc, and replicate without using genetic information by forcing PrPc into the pathological conformation of PrPSc [3].

[0006] Despite intensive research no therapy is available for prion diseases [4,5]. SUMMARY

[0007] In one aspect there is described a method of treating a subject having, suspected or having, or at risk of having, a prion disease, comprising: administering a cholesterol 24-hydroxylase (CH240H)-activating compound.

[0008] In one example, said compound comprises efavirenz and/or mirtazapine.

[0009] In one example, said prion disease is CJD, iCJD, vCJD, BSE, CWD, scrapie, GSS (Gerstmann-Straeussler-Scheinker syndrome), fatal familial insomnia (FFI), or familial CJD.

[0010] In one example, said subject is human. [0011] In one aspect there is described use of a cholesterol 24-hydroxylase

(CH240H)-activating compound for treating a subject having, suspected or having, or at risk of having, a prion disease.

[0012] In one aspect there is described use of a cholesterol 24-hydroxylase (CH240H)-activating compound in the manufacture of a medicament for treating a subject having, suspected or having, or at risk of having, a prion disease.

[0013] In one example, said compound comprises efavirenz and/or mirtazapine.

[0014] In one example, said prion disease is CJD, iCJD, vCJD, BSE, CWD, scrapie, GSS (Gerstmann-Straeussler-Scheinker syndrome), fatal familial insomnia (FFI), or familial CJD.

[0015] In one example, said subject is human.

[0016] In one aspect there is described a cholesterol 24-hydroxylase (CH240H)- activating compound for treating a subject having, suspected or having, or at risk of having, a prion disease.

[0017] In one example, the cholesterol 24-hydroxylase (CH240H)-activating compound of claim 10 which is efavirenz and/or mirtazapine.

[0018] In one example, the cholesterol 24-hydroxylase (CH240H)-activating compound of claiml O or 1 1 , wherein said prion disease is CJD, iCJD, vCJD, BSE, CWD, scrapie, GSS (Gerstmann-Straeussler-Scheinker syndrome), fatal familial insomnia (FFI), or familial CJD.

[0019] In one example, the cholesterol 24-hydroxylase (CH240H)-activating compound of any one of claims 10 to 12, wherein said subject is human.

[0020] In one aspect there is described a kit for treating a subject having, suspected or having, or at risk of having, a prion disease, comprising: a cholesterol 24- hydroxylase (CH240H)-activating compound or composition, a container, and optionally instructions for the use thereof.

[0021] In one example, said compound comprises efavirenz and/or mirtazapine.

[0022] In one example, said prion disease is CJD, iCJD, vCJD, BSE, CWD, scrapie, GSS (Gerstmann-Straeussler-Scheinker syndrome), fatal familial insomnia (FFI), or familial CJD.

[0023] In one example, said subject is human.

BRIEF DESCRIPTION OF THE FIGURES

[0024] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures. [0025] Fig. 1A-D depicts increased hydroxycholesterol (HC) levels reduce PrPSc levels in persistently prion-infected neuroblastoma cells.

[0026] Fig. 2 depicts 24-hydroxycholesterol treatment down-regulates cholesterogenic gene (SC4MOL) mRNA expression in prion-infected and non-infected neuroblastoma cells.

[0027] Fig. 3 depicts Efavirenz treatment reduces PrPSc levels in persistently prion-infected neuroblastoma cells.

[0028] Fig. 4A-B depicts Efavirenz treatment reduces PrPSc levels independent of prion strain in persistently infected neuronal cells.

[0029] Fig. 5A-C depicts five days treatment of efavirenz efficiently reduces

PrPSc levels in persistently infected neuroblastoma and neuronal cells in prion strain independent manner.

[0030] Fig. 6 depicts prolonged treatment with efavirenz clears PrPSc in RML

N2a cells.

[0031] Fig. 7A-B depicts Efavirenz induced 24-hydroxycholesterol production down-regulates SC4MOL mRNA expression in prion-infected and non-infected neuroblastoma cells.

[0032] Fig. 8 depicts PrPSc levels in non-neuronal prion infected cells are not affected by efavirenz treatment.

[0033] Fig. 9 depicts PrPC levels are not significantly reduced by efavirenz treatment in non-infected neuroblastoma cells.

[0034] Fig. 10A-C depicts Efavirenz treatment stimulates autophagy in prion- infected and non-infected neuroblastoma cells.

[0035] Fig. 1 1 A-B depicts Autophagy and lysosomal degradation pathways are not the main mechanisms to explain reduced PrPSc levels upon efavirenz treatment.

[0036] Fig. 12 depicts Efavirenz treatment likely inhibits de novo synthesis of

PrPSc in prion infected neuroblastoma cells.

[0037] Fig. 13 depicts Mirtazapine treatment reduces PrPSc levels in persistently prion-infected neuroblastoma cells and neuronal cells.

[0038] Fig. 14 depicts autophagy is not a main degradation pathways in

Mirtazapine mediated PrPSc reduction.

[0039] Fig. 15 depicts in vivo treatment with efavirenz. DETAILED DESCRIPTION

[0040] Generally, the present disclosure relates to the treatment of prion disease in a subject.

[0041] In one example, there is described compounds, compositions, methods and use, for the treatment of a subject having a prion disease, or a subject suspected of having a prion disease, or a subject at risk of having a prion disease.

[0042] The term "subject", as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, cervids, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human.

[0043] The term "treatment" or "treat" as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.

[0044] "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0045] "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject in the early stage of disease can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent progression.

[0046] Accordingly, in one example, the phrase "to treat the prion disease" as used herein means to prevent or slow the development of, slow the progression of, halt the progression of, or eliminate the prion disease.

[0047] In one example the treatment is in vitro treatment. In one example the treatment is in vivo treatment.

[0048] As used herein, a "subject having a prion disease" is a subject known or diagnosed to have a prion disease. Generally a subject having a prion disease will have some objective manifestation of the prion disease, such as a sign, symptom, or result of a suitable diagnostic test that indicates the presence of a prion disease. [0049] In some examples, objective manifestations can include dementia, ataxia, myoclonus, tremor, presence of protease-resistant prion protein in brain extract, and typical or characteristic abnormalities on brain CT, brain MRI, and/or EEC

[0050] A subject having a prion disease may also include any subject having a test result which specifically indicates the presence in that subject of any amount of prion protein that is associated with a prion.

[0051] In some examples, a subject having a prion disease may have an identifiable risk factor for having a prion disease. An identifiable risk factor for having a prion disease may include a family history of prion disease, a history of consuming known or suspected prion- diseased tissue, or a history of exposure to a prion protein that is associated with a prion disease or to a product derived from a known or suspected prion- diseased tissue (e.g., through administration of pituitary extract).

[0052] In some example, a subject at risk of developing a prion disease is a subject with a known or suspected exposure to prion-diseased tissue, a known or suspected exposure to prion protein that is associated with a prion disease, or a known or suspected predisposition to develop a prion disease.

[0053] Prion disease includes, but is not limited to, the prion disease is scrapie,

BSE, CDD or a form of CJD and other genetic prion diseases. CJD in one example is iatrogenic CJD (iCJD). In another example the CJD is variant CJD (vCJD). In yet another example the CJD is sporadic CJD. In another example the prion disease is familial CJD, GSS, or FFI. In another example, the prion disease is CWD.

[0054] In one example, a subject is treated with a cholesterol 24-hydroxylase

(CH240H)-activating compound.

[0055] In a specific example, a subject is treated with efavirenz.

[0056] In a specific example, a subject is treated with mirtazapine.

[0057] In a specific example, a subject is treated with efavirenz and mirtazpine.

[0058] In one example, infected cells treated with efavirenz show a concentration dependent reduction of prion protein (PrPSc) accumulation.

[0059] Described herein the antiretroviral drug efavirenz activates a neuronal enzyme (cholesterol 24-hydroxylase) which modifies cholesterol and thereby enables its export from neurons and the brain. Drug-induced activation of cholesterol 24-hydroxylase e.g. by efavirenz is a new strategy for the treatment of prion diseases.

[0060] In one example, a subject having, suspected of having, or at risk of having a prion disease is treated by administration with efavirenz. [0061] In one example, a subject having, suspected of having, or at risk of having a prion disease is treated by administration with mirtazapine.

[0062] In one example, a subject having, suspected of having, or at risk of having a prion disease is treated by administration with efavirenz and mirtazapine.

[0063] In one example, there is described the use of a cholesterol 24-hydroxylase

(CH240H)-activating compound for the treatment of a prion disease. In one example, the compound is efavirenz. In one example, the compound is a mirtazapine.

[0064] The term "administration" typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0065] In one example, treatment comprises administration of a therapeutically effective amount of a pharmaceutical composition comprising efavirenz or mirtazapine, or both efavirenz and mirtazapine, to the central nervous system (CNS) of a subject.

[0066] The compounds and compositions may be provided in a pharmaceutically acceptable form.

[0067] The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions, and/or dosage forms (such as unit dosages) which are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. is also "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0068] Pharmaceutically acceptable carriers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Compositions as described herein may be sterilized by conventional methods and/or lyophilized.

[0069] In one example, treatment provides efavirenz or mirtazapine, or both efavirenz and mirtazapine, to the tissues of the CNS by administration directly into the cerebrospinal fluid (CSF).

[0070] In some examples, delivery to the CSF and brain include, but are not limited to, intrathecal (IT), intracerebroventricular (ICV), and intraparenchymal administration. Intrathecal and intracerebroventricular administration may be carried out through the use of surgically implanted pumps that infuse the therapeutic agent into the cerebrospinal fluid. Intraparenchymal delivery may be carried out by the surgical placement of a catheter into the brain.

[0071] As used herein, "delivery to the CSF" and "administration to the CSF" encompass the IT infusion or ICV infusion of efavirenz and/or mirtazapine through the use of an infusion pump. In some embodiments, IT infusion is a suitable means for delivery to the CSF. In other examples, efavirenz and/or mirtazapine is continuously infused into the CSF for the entire course of treatment; such administration is referred to as "continuous infusion" or, in the case of IT infusion, "continuous IT infusion." Also contemplated is continuous intraparenchymal infusion using a pump.

[0072] In some examples, an infusion pump is employed to deliver efavirenz and/or mirtazapine to the CNS. Such infusion pumps and their method of implantation and use are known to the skilled worker. In a specific example, the Medtronic

SyncroMed® II pump, is employed to deliver efavirenz and/or mirtazapine to the CNS. The SyncroMed® II pump is surgically implanted according the procedures set forth by the manufacturer. The pump contains a reservoir for retaining a drug solution, which is pumped at a programmed dose into a catheter that is surgically implanted.

[0073] Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[0074] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[0075] EXAMPLES

[0076] Prions are proteinaceous infectious particles that cause fatal spongiform encephalopathies in humans and animals [1]. They are transmissible within and between species under natural conditions, and can even be zoonotic, exemplified by bovine spongiform encephalopathy (BSE) which has been transmitted to humans and gave rise to a new variant of Creutzfeldt-Jakob disease (vCJD).

[0077] In humans, there are sporadic (Creutzfeldt-Jakob disease), familial (e.g. Gerstmann-Straeussler-Scheinker syndrome) or infectiously acquired (e.g. Kuru, variant CJD) forms [2].

[0078] Prions consist solely of a misfolded and aggregation-prone isoform of the cellular prion protein (PrPc), termed PrPSc, and replicate without using genetic information by forcing PrPc into the pathological conformation of PrPSc [3].

[0079] Despite intensive research no therapy is available for prion diseases [4,5].

[0080] Neuronal cell lines persistently infected with mouse-adapted scrapie prions, confirmed by the presence of proteinase K (PK) resistant PrPSc signals in immunoblot are the most frequently used tools to screen drugs for anti-prion properties. Here, measuring a reduction of PrPSc levels in cells by immunoblot or other assays serves as an indicator of anti-prion action.

[0081] Cholesterol is ubiquitous in the central nervous system (CNS) and vital to normal brain function including signaling, synaptic plasticity, and learning and memory. Cholesterol is so important to brain function that it is generated independently of cholesterol metabolism in the rest of the body and is sequestered from the body by the blood brain barrier (BBB) ¹ .

[0082] Cholesterol in the CNS is converted to 24S-hydroxycholesterol by the enzyme 24S- hydroxylase (CYP46A1 , a member of the cytochrome P450 family) and can cross the BBB to be excreted from the brain (Bjorkhem et al., 2009; Russell et al., 2009). Cholesterol in the rest of the body can be converted to 27-hydroxycholesterol by 27- hydroxylase (CYP27A1) and cross the BBB into the CNS (Heverin et al., 2005) to act as a ligand at specific receptors (e.g., LXR) and regulate enzymatic activity.

[0083] Literature on the effect of cholesterol levels on learning and memory and contradictory, with some studies showing that elevated cholesterol levels are a risk factor for mild cognitive impairment while others show that increased cholesterol improves learning and memory. [0084] Here, we describe that pharmacological induction of the activity of cholesterol 24-hydroxylase using previously described compounds [15] exhibits anti-prion activity in persistently infected neuroblastoma cell lines (N2a and CAD5). The reduction of PrPSc signals is dose- and time-dependent, and upon prolonged treatment cells can be cured from prion infection. Mechanistically, we demonstrate that activation of cholesterol 24-hydroxylase results in increased levels of 24-hydroxycholesterol, which induces a down-regulation of cholesterogenic gene expression and presumably a reduction of cellular cholesterol levels.

[0085] Reagents and antibodies

[0086] Proteinase K (PK), Pefabloc, NH4CI, Bafilomycin A1 , 22- hydroxycholesterol (22-HC), 25-hydroxycholesterol (25-HC), mouse monoclonal anti-β actin antibody, goat anti-mouse secondary antibody, deionized water, and mirtazapine were obtained from Sigma. All cell culture media and solutions were from Invitrogen. Random primer 6 was purchased from New England Biolabs, the RNA isolation kit (RNeasy mini kit) was from Qiagen and the Perfecta SYBR GREEN super mix was from Quanta Bio. Oligonucleotide primers were synthesized by the University of Calgary DNA services. The ELISA kit for quantification of 24(S) hydroxycholesterol and 24 (S)- hydroxycholesterol (24-HC) were from Enzo life sciences. The XTT cell proliferation assay kit was obtained from Trevigen. Luminata Forte HRP substrate was from Millipore. The mouse monoclonal anti-LC3 antibody was from Nanotools, the monoclonal anti-prion antibody 3F4 was from Bio Legend. Mouse anti-PrP (4H1 1) was prepeared in-house. 24- HC, 25-HC were dissolved in ethanol, 22-HC was dissolved in chloroform, stocks of 1 , 5 10, 20 mM were prepared of each compound and stored at -20° C. (S) Efavirenz and mirtazapine were dissolved in methanol and stocks of 10, 15, 20, 25 and 40 mM for evavirenz and 10, 20, 30 and 60 mM for mirtazapine were prepared and stored at -20°C.

[0087] Cell culture

[0088] Cells lines used in this study were murine neuroblastoma cells N2a and neuronal CAD5 cells, respectively, not infected or permanently infected with 22L or RML prions, and mouse embryonic fibroblast (MEF) cells uninfected and persistently infected with 22L prions. RML-N2a cells stably overexpress murine PrP containing the epitope for mAb 3F4 and are persistently infected with RML scrapie prions. 22LN2a5-K21 ATG5-KO cells were prepared by knocking out the Atg5 gene using Crispr/Cas9 technology. All N2a cells were cultured in Opti-MEM (Invitrogen) containing 10% (v/v) fetal bovine serum (PAA) and penicillin/streptomycin. CAD5 cells were grown in OptiMEM with 10% bovine growth serum (Hyclone). MEF cells were kept in MEM (Invitrogen) with the addition of 10% (v/v) fetal bovine serum and penicillin/streptomycin. All cells were grown in at 37° C in a 5% C0 ₂ atmosphere.

[0089] Cell lysis, PK digestion and immunoblot

[0090] Briefly, cell cultures were washed with ice cold PBS and then incubate for 10 minutes with 1 ml ice cold lysis buffer (10 mM Tris-HCL pH 7.5, 100 mM NaCI, 10 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate). Half volume of the supernatant obtained upon 1 min centrifugation at 14,000 rpm were used for PK digestion (20 μg/ml PK; 30 min; 37° C), the second half was immediately supplemented with Pefabloc proteinase inhibitor. PK digestion was stopped by addition of Pefabloc protease inhibitor, all samples were precipitated by adding 5 volumes of methanol and overnight incubation at -20° C. Protein precipitates were resuspended in TNE buffer and aliquots subjected to SDS-PAGE and immunoblot using mAb 4H1 1 or 3F4for detection of PrP, β-actin was detected using an anti-actin mAb (Sigma) as a loading control.

[0091] qRT-PCR

[0092] 22L N2a5 or N2a5 cells were seeded and 4 days after seeding, 24-HC treatment was applied for 4 h (hydroxycholesterol) or two days (EFV). Total RNA was isolated according to RNeasy mini kit manual, concentration and purity of RNA was measured by using nanovue spectrophotometer to determine OD at 260 nm and 280 nm. One μg of total RNA was used to prepare cDNA by using Random primer 6 and

Superscript II Reverse Transcriptase kit according to the manufacturer's recommendation and incubated at 25° C for 10 mins, 42° C for 50 min and 70°C for 15 mins. Amplification of cDNA was done by employing real-time PCR (Bio Rad, CFX 96) using SYBR green super mix (Quantum Biosciences). The following PCR conditions were used: 95° C for 2 min, then 45 cycles of 95° C for 15 sec, 60° C for 15 sec, 72° C for 1 min), followed by 95° C for 10 sec and 4° C for 30 min. RNA polymerase II was used as a housekeeping gene. Differnces were evaluated using the 2 ^~ΔΔα method.

[0093] Table 1 : Sequences of oligodeoxynucleotides used in this study.

[0094] Quantification of 24-hydroxycholesterol

[0095] N2a5 cells or 22LN2a5 cells were seeded and treated for two days with efavirenz (20 μΜ; MeOH treated cells used as control). Cell culture media was collected centrifuged at 1 ,000 rpm for 2 min to remove dead cells. Standards of 24- hydroxycholesterol were prepared by dissolving obtained 24-hydroxycholesterol in the fresh cell culture media. 24-hydroxycholesterol ELISA was performed according to the instructions provided in the ELISA kit. Signals were measured at 450 nm. Plots were prepared by using 24-HC standard values. Concentrations of samples were determined using the standard curve. Each sample was run in triplicates. Three independent experiments were performed. Statistical evaluation was done using unpaired t-test,

(GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p-value < 0,001 , ns= non-significant). Bars represent standard error of mean.

[0096] Results

[0097] Fig. 1 : Increased hydroxycholesterol (HC) levels reduce PrPSc levels in persistently prion-infected neuroblastoma cells.

[0098] 2.5x10 ⁵ of 22LN2a5 cells were seeded and after two days of treatment with (A) 24-hydroxycholesterol (1 , 5 or 10 μΜ, Ethanol (EtOH) treated cells used as control), (B) 25-hydroxycholesterol (5, 10, or 15 μΜ, Ethanol (EtOH) treated cells used as control), (C) 22-hydroxycholesterol (5, 10, or 15 μΜ, chloroform treated cells used as control), cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as a loading control. Three independent experiments were performed. Signals of PK-resistant PrPSc of three independent experiments were quantified by Quantity One (Bio-Rad) and statistical evaluation using one-way ANOVA and post-hoc analysis (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p-value < 0.001) was performed. Bars represent standard error of mean. (D) Highest concentrations of 24-hydroxycholesterol (24-HC), 25-Hydroxycholesterol (25-HC), 22- Hydroxycholesterol (22-HC) and Efavirenz (EFV) used in this study are not toxic to the used cell lines as indicated, tested by XTT assay. Viability of solvent-treated cells was expressed as 100 %, viability of cells treated with the different compounds is shown as percentage thereof. Statistical analysis was performed using unpaired t- test (GraphPad Prism software; ns = not significant)

[0099] Fig. 2: 24-hydroxycholesterol treatment down-regulates

cholesterogenic gene (SC4MOL) mRNA expression in prion-infected and non- infected neuroblastoma cells. [00100] 2.5x10 ⁵ of 22LN2a5 or N2a5 cells were seeded and after 4 hrs of treatment with 24-HC 5 μΜ (24-HC, cholesterol biosynthesis down-regulator, and ethanol (control)), total RNA was isolated using Qiagen RNA isolation kit. Random primers were used to synthesise cDNA from total RNA and qRT-PCR was performed using SYBR GREEN master mix and gene specific primers. Three independent experiments were done and sterol-C4-methyl oxidase-like protein (SC4MOL) mRNA levels normalized to RNA polymerase II gene expression were compared between treated and mock-treated cells. Statistical evaluation using unpaired t-test, (GraphPad Prism software; ns=non- significant) was performed. According to AACt method the cut off-for significance is a difference of >2 fold up- or down-regulation of mRNA levels, in relation to controls. Bars represent standard error of mean.

[00101] Fig. 3: Efavirenz treatment reduces PrPSc levels in persistently prion- infected neuroblastoma cells.

[00102] 2.5x10 ⁵ 22LN2a5 cells were seeded and treated for two days with efavirenz (15, 20, or 25 μΜ), MeOH treated cells used as control). Then cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as loading control. Three independent experiments were performed. PrPSc signals (see +PK lanes) of three independent experiments were quantified by Quantity One (Bio- Rad) and statistical evaluation using one-way ANOVA test and post-hoc analysis was performed (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p- value < 0,001 , ns= non-significant). Bars represent standard error of mean.

[00103] Fig. 4: Efavirenz treatment reduces PrPSc levels independent of prion strain in persistently infected neuronal cells.

[00104] 2.5x10 ⁵ of (A) RML CAD5 cells or (B) 22L CAD5 cells were seeded and treated for two days with efavirenz (15, 20, or 25 μΜ), MeOH treated cells used as control). Then cells were lysed and subjected to PK digestion (+PK) or not (-PK).

Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as a loading control. Three independent experiments were performed. PrPSc signals of three independent experiments were quantified by Quantity One (Bio-Rad) and statistical evaluation using one-way ANOVA test and post-hoc analysis was performed (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p-value < 0,001 , ns= non-significant). Bars represent standard error of mean. [00105] Fig. 5: Five days treatment of efavirenz efficiently reduces PrPSc levels in persistently infected neuroblastoma and neuronal cells in prion strain independent manner.

[00106] 2.5x10 ⁵ of (A) 22L N2a5 cells were treated for five days and seven days, respectively, with efavirenz (15 μΜ), MeOH treated cells used as control), (B) RML CAD5 cells (C) 22L CAD5 cells were treated for five days with efavirenz (15, 20, or 25 μΜ), MeOH treated cells used as control). Then cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as a loading control.

[00107] Fig. 6: Prolonged treatment with efavirenz clears PrPSc in RML N2a cells.

[00108] 2.5x10 ⁵ of N2a cells stably overexpressing 3F4-PrP and persistently infection with RML scrapie prions (RML N2a) were seeded and one batch of cells was lysed after 7, 10, or 14 days of treatment with efavirenz (15 μηι, methanol treated cells used as control), and subjected to PK digestion (+PK) or not (-PK). After treatment another batch of cells was grown without drug for another week, lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as a loading control. Three independent experiments were performed. After 14 days of efavirenz treatment, PrPSc signal did not re-appear one week after drug treatment was withdrawn (red box), indicating clearance of PrPSc.

[00109] Fig. 7: Efavirenz induced 24-hydroxycholesterol production down- regulates SC4MOL mRNA expression in prion-infected and non-infected neuroblastoma cells.

[00110] 2.5x10 ⁵ of (A) N2a5 cells or 22L N2a5 cells were seeded and treated for two days with efavirenz (20 μΜ), MeOH treated cells used as control) and cell culture media was collected. Samples were analyzed by 24-HC Elisa kit for quantitative measurement of 24-HC levels in the media. Three independent experiments were performed. Statistical evaluation was done using unpaired t-test, (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p-value < 0,001 , ns= nonsignificant). Bars represent standard error mean. (B) 2.5x105 of 22LN2a5 or N2a5 cells were seeded and treated with EFV 20 μΜ, or methanol (control) for two days. Total RNA was isolated using Qiagen RNA isolation kit. Random primers were used to synthesise cDNA from total RNA and qRT-PCR was performed using SYBR GREEN master mix and gene specific primers. Three independent experiments were done and cholesterogenic gene, sterol-C4-methyl oxidase-like protein (SC4MOL) mRNA levels normalized to RNA polymerase II gene expression, were compared and statistical evaluation using unpaired t-test, (GraphPad Prism software; ns=non-significant). According to AACt method the cut off-for significance is a difference of >2 fold in mRNA levels. Bars represent standard error of mean.

[00111] Fig. 8: PrPSc levels in non-neuronal prion infected cells are not affected by efavirenz treatment.

[00112] 2.5x10 ⁵ 22L MEF (Mouse embryonic fibroblasts) cells were seeded and treated for two days with efavirenz (10, 15 or 20μΜ), MeOH treated cells used as control). Then cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as a loading control. Three independent experiments were performed. PrPSc signals of three independent experiments were quantified by Quantity One (Bio-Rad) and statistical evaluation using one-way ANOVA test and post-hoc analysis was performed (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p-value < 0,001 , ns= non-significant). Bars represent standard error of mean.

[00113] Fig. 9: PrPC levels are not significantly reduced by efavirenz treatment in non-infected neuroblastoma cells.

[00114] 2.5x10 ⁵ of N2a5 cells were seeded and treated for two days with efavirenz (15, 20, or 25 μΜ, MeOH treated cells used as control). Then cells were lysed and subjected to +PK, -PK digestion. Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1000), anti β-actin antibody (1 :50000) was used as loading control. Three independent experiments were performed. PrPC signals of three independent experiments were quantified by Quantity One (Bio-Rad) and statistical evaluation using one-way ANOVA test and post-hoc analysis was performed (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p-value < 0,001 , ns= nonsignificant). Bars represent standard error of mean.

[00115] Fig. 10: Efavirenz treatment stimulates autophagy in prion-infected and non-infected neuroblastoma cells.

[00116] 2.5x10 ⁵ of (A) 22L N2a5 cells were seeded and treated for 30 min with efavirenz 20 μΜ), MeOH treated cells used as control), (B) N2a5, 22L N2a5 cells were treated for 4 hrs with efavirenz 20 μΜ; MeOH treated cells used as control) along with Bafilomycin A1 , (100 nM) (C) N2a5, 22LN2a5 cells were treated for two days with efavirenz 20 μΜ; MeOH treated cells used as control) along with NH4CI 20 mM. For analysis of autophagy induction cells were lysed and samples were analyzed by immunoblot for LC3 I and LC3 II content using anti-LC3 mAb (1 :5,000) or for p62 levels using anti-p62 antibody (1 :10,000). Anti β-actin antibody (1 :50,000) was used as a loading control.

[00117] Fig. 11 : Autophagy and lysosomal degradation pathways are not the main mechanisms to explain reduced PrPSc levels upon efavirenz treatment.

[00118] 2.5x10 ⁵ of (A) 22L N2a-K21 ATG5 KO (Atg5 knock-out cells) were seeded and treated for two days with efavirenz (15, 20, or 25 μηι), MeOH treated cells used as control), (B) 22L N2a K21 ATG5 KO cells treated for two days with efavirenz (15, 20, or 25 μΜ; MeOH treated cells used as control) in combination with NH4CI 20 mM. Then cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as loading control. Three independent experiments were performed.

[00119] Fig. 12: Efavirenz treatment likely inhibits de novo synthesis of PrPSc in prion infected neuroblastoma cells.

[00120] 22L N2a5 cells were transfected with pcDNA3.1 encoding mouse PrP with 3F4 epitope. After 48 h of transfection, cells were treated for two days with efavirenz (20 μΜ, MeOH treated cells used as control) Then cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for 3F4-PrP content using the monoclonal anti-PrP antibody 3F4 (1 :5,000). Anti β-actin antibody (1 :50,000) was used as a loading control. No de novo synthesized PrPSc signal was detected in cells treated with EFV (red box).

[00121] Fig. 13: Mirtazapine treatment reduces PrPSc levels in persistently prion-infected neuroblastoma cells and neuronal cells.

[00122] 2.5x10 ⁵ of (A) 22LN2a5 cells, (B) 22L CAD5 cells were treated for two days with Mirtazapine (10, 20, or 30 μΜ), MeOH treated cells used as control), cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as loading control. Three independent experiments were performed. PrPSc signals of three independent experiments were quantified by Quantity One (Bio- Rad) and statistical evaluation using one-way ANOVA test and post-hoc analysis was performed, (GraphPad Prism software; * = p-value < 0.05 ** = p-value < 0.01 ; *** = p- value < 0,001 , ns= non-significant). Bars represent standard error of mean. [00123] Fig. 14: Autophagy is not a main degradation pathways in Mirtazapine mediated PrPSc reduction.

[00124] 2.5x10 ⁵ of 22L N2a5 K21 ATG5 KO (Atg-5 knock-out cells) were treated for two days with mirtazapine (10, 20, or 30 μΜ), MeOH treated cells used as control. Cells were lysed and subjected to PK digestion (+PK) or not (-PK). Samples were analyzed by immunoblot for PrP content using anti-PrP mAb 4H1 1 (1 :1 ,000), anti β-actin antibody (1 :50,000) was used as loading control. Three independent experiments were performed.

[00125] Fig. 15 Efavirenz treatment in vivo

[00126] 10 mice/group (strain FVB) were inoculated intracerebral^ with 30 μΙ of a 1 % prion-infected mouse brain homogenate (strain RML). 30 days post infection, for one group treatment with efavirenz was started and continued until the experimental end point. Efavirenz was applied orally and dissolved in the drinking water (1.68 mg/l).

According to the drinking volume per day, the mice received a dose of 0.22 mg/kg/day. The onset and progression of clinical prion disease was monitored and animals were euthanized when they reached terminal disease. In the graph, the days post infection are plotted against the treatment groups. Each dot represents a single mouse. One mouse from each group died shortly after intracerebral prion inoculation. A statistically significant (p-value = 0.0147) extension of incubation time was recorded for the efavirenz treatment group.

[00127] First, we have analysed how treatment with hydroxycholesterols (HCs) of neuroblastoma cells (N2a5) persistently infected with mouse-adapted scrapie prion strain 22L (22LN2a5) affects PrPSc propagation (Fig. 1). 22LN2a cells were treated for 2 days with 24-HC (Fig. 1A), 25-HC (Fig. 1 B) or 22-HC (Fig. 1C) at various concentrations as indicated. Immunoblot analysis of cell lysates revealed a reproducible dose-dependent reduction of PrPSc signals (see lanes +PK) when compared to the mock-treated control cells (EtOH). PrPSc levels were quantified a statistically significant difference is shown in the graphs. To exclude toxicity of the various HCs used for treatment, toxicity assays were performed using the highest concentration used of each HC and the anti-retroviral drug efavirenz (EFV). No statistically significant reduction of cell viability was observed compared to the control cells which were treated with the solvents of the compounds (Fig. 1 D). HCs are known to down-regulate cholesterogenic gene expression. To verify whether this was the case in 24-HC treated N2a5 and 22LN2a5, respectively, we analysed the mRNA levels of Sc4Mol (sterol-C4-methyl oxidase-like gene) gene expression by reverse transcription quantitative PCR (RT-qPCR). In both N2a5 and 22LN2a5 cells, a more than 3fold down-regulation of Sc4MOL expression was found compared to mock-treated cells. The extent of down-regulation did not significantly differ between non-infected and infected cells (Fig. 2).

[00128] These experiments provide evidence that increasing the levels of HC results in a reduction of PrPSc in persistently prion-infected neuronal cells. In order to translate these findings into a potential treatment for prion disease, we tested the effects of drug-induced activation of the neuronal enzyme cholesterol 24-hydroxylase (CH240H) which converts unesterified cholesterol into 24-HC which is the cholesterol derivative that is exported from the brain as it can cross cellular membranes and the blood brain barrier by diffusion. The drugs efavirenz (EFV) and mirtazipine were selected since they have been shown to activate CH240H in vitro and in vivo. Both drugs have been approved for application in humans and cross the blood brain barrier. Since anti-prion effects can be cell- and prion strain-specific, we tested the effects of EFV on different neuronal (N2a5, CAD5) and non-neuronal (mouse embryonic fibroblasts MEF) cell lines infected with various mouse-adapted scrapie prion strains (22L, RML, ME7). 22LN2a5 (Fig. 3), RML CAD5 and 22L CAD5 (Fig. 4A, Fig. 4B) were treated for 2 days with various

concentrations as indicated of EFV. In all cell lines, a dose-dependent and statistically significant reduction of PrPSc was achieved (Fig. 3 and 4). Upon prolonged treatment of the same cell lines for 5 or 7 days, a time-dependent reduction of PrPSc levels was observed (Fig. 5A-C).

[00129] Next we were interested in whether upon prolonged treatment with EFV prion infection was completely cured, or whether PrPSc levels were only reduced to levels below the detection limit of immunoblot and propagation would start again if the drug treatment is withdrawn, resulting in a re-appearance of PrPSc signals. Thus, we treated RML-infected N2a cells (RML N2a) for 7, 10 or 14 days with EFV. Cultures of one set of cells for each treatment duration were continued for a further week without EFV treatment, another set was lysed immediately after treatment. Fig. 6 demonstrates that upon 14 days of EFV treatment and continued culture for another week PrPSc signal does not re-appear (box). This result shows that prion infection can be cured by treatment of prion-infected neuronal cells with a low dose of EFV.

[00130] Since our previous work has shown that up-regulation of cholesterol synthesis and amount upon prion infection is specific for neuronal cells, and CH240H is a neuron-specific enzyme, we used prion-infected non-neuronal cells (22L MEF) for EFV treatment in order to delineate whether the EFV effect on PrPSc levels was specific for the induction of CH240H activity. Immunoblot analysis and quantification of PrPSc signals from 22L MEF cells upon 2 days of treatment with different concentrations of EFV revealed that in contrast to the effects observed in neuronal cells, EFV treatment does not significantly reduce the amounts of PrPSc in 22LMEF cells (Fig. 8).

[00131] In summary, the experiments described above show that EFV treatment of prion-infected cells reduces PrPSc in neuronal, but not in non-neuronal cell lines. This reduction is independent of the type of neuronal cell line, and is observed for different prion strains. The next set of experiments was designed to get more insights into the exact molecular mechanisms of the effects of CH240H activation on PrPSc propagation.

[00132] PrPC expression is essential for prion propagation, and PrP gene knock out mice are resistant to prion infection. Therefore, a reduction of PrPC due to drug treatment can add to the anti-prion effects of EFV treatment. To test this, we treated non- infected N2a cells with different concentrations of EFV for 2 days and quantified the PrPC content in cell lysates. No statistically significant alterations of cellular PrPC content were found (Fig. 9).

[00133] Other possible targets for interference with prion propagation are an activation of PrPSc degradation by enhancing autophagy and/or lysosomal degradation, or the inhibition of PrPSc de novo conversion. First, we analysed the impact of EFV treatment on autophagy in 22LN2a cells (Fig. 10A). As markers for autophagy activation we employed p62 and LC3II. The latter is a lipidated form of LC3I which is formed upon autophagy activation; therefore, an increase of LC3II signals upon short-term treatment indicates induction of autophagy, whereas p62 levels are reduced in this case. As can be seen in Fig. 10A, EFV treatment increased levels of LC3II and reduced p62 protein, indicating that EFV increases the formation of autophagosomes and autophagic degradation. In order to rule out that LC3II levels are increased because of a block of autophagosome-lysosome fusion, N2a5 and 22LN2a5 cells were treated for 4 h with vehicle, EFV or EFV in combination with bafilomycin A, an inhibitor of autophagic degradation. In both cell lines, EFV combined with bafilomycin A treatment caused an increase of LC3II levels as compared to vehicle treated cells and cells treated only with EFV (Fig. 10B). Similarly, LC3II levels were increased in N2a5 and 22LN2a5 cells treated with a combination of EFV and NH4CI, a more general inhibitor of degradation in acidic vesicles (Fig. 10C). Altogether, these data indicate that EFV treatment enhances autophagy, which has been described previously by our group as a method to reduce PrPSc propagation. In order to determine whether EFV-mediated induction of autophagy and/or lysosomal degradation plays a major role in PrPSc reduction, we utilized an autophagy-deficient and prion-infected N2a cell line (22LN2a5-K21 ATG5-KO; Fig. 1 1) which was established by knocking out the Atg5-encoding gene using Crispr/Cas9 technology (Schaetzl and Gilch, personal communication). If autophagy had a prominent role in EFV-mediated PrPSc reduction, we would expect that EFV treatment of 22LN2a5- K21 ATG5-KO cells would not result in a PrPSc reduction. However, this was not the case, and also in these autophagy-incompetent cells EFV treatment resulted in a dose- dependent reduction of PrPSc (Fig. 1 1 A). Furthermore, we analysed the role of lysosomal degradation by co-treatment of the same cell line with EFV and NH4CI. Whereas in the vehicle-treated cells (MeOH) a strong signal of PrPSc was visible upon NH4CI treatment, cells incubated in addition with EFV had a much weaker PrPSc signal (Fig. 1 1 B), which demonstrates that neither autophagy induction nor lysosomal degradation are

prominently involved in PrPSc reduction upon EFV treatment.

[00134] Given the findings that degradative pathways only play a minor role in EFV-mediated PrPSc reduction, we were interested in investigating whether EFV treatment blocks the new formation of PrPSc. For this purpose, we employed

overexpression of a tagged version of murine PrP containing the epitope for recognition by the monoclonal antibody 3F4 (3F4 PrP). This epitope allows discriminating between PrPSc converted from the N2a cell's endogenous mouse PrP and PrPSc produced from the exogenously introduced 3F4-PrP, as only the latter would be recognised by the 3F4 antibody. Therefore, transient overexpression of 3F4-PrP in persistently prion-infected cells and subsequent detection of 3F4-positive PrPSc is a commonly used method to determine de novo conversion of PrPSc under specified conditions such as drug treatment. We transiently transfected 22LN2a5 cells with a plasmid encoding 3F4-PrP. Transfected cells were either left untreated, treated with solvent (MeOH), or treated with EFV. Then lysates were digested with PK or not and analysed by immunoblot using 3F4 antibody. Under these conditions, a weak signal for 3F4-positive (de novo synthesized) PrPSc is visible in control and MeOH treated cells, but not in EFV treated cells (Fig. 12). Therefore, we conclude that EFV inhibits the de novo conversion of PrPC into PrPSc.

[00135] In order to further examine the hypothesis that CH240H activation provides a strategy to reduce PrPSc and to further rule out possible Off-target' effects of EFV treatment, we tested the effects of another previously published CH240H-activating drug, namely mirtazapine. In line with the EFV treatment, we incubated 22LN2a5 or 22L CAD5 cells with different concentrations of mirtazapine for 2 days. As with EFV, we observed a dose-dependent and statistically significant reduction of PrPSc levels in 22LN2a5 cells (Fig. 13A), and a dose-dependent reduction of PrPSc in 22L CAD 5 (Fig. 13B). Notably, treatment with 22L CAD5 has been performed only once and needs to be replicated to enable statistical analysis. Similarly to EFV, autophagy is not a major pathway mediating reduction of PrPSc by mirtazapine treatment. When 22LN2a5-K21 ATG5-KO cells were treated with different concentrations of mirtazapine, a strong reduction of PrPSc was observed (Fig. 14).

[00136] As shown in Fig. 15, mice inoculated intracerebrally with prion-infected mouse brain homogenate that were treated with efavirenz displayed a statistically significant extension of incubation time.

[00137] In summary, we describe herein that activation of CH240H is a novel strategy for treatment of prion infection. This conclusion is supported by the finding that two unrelated drugs (EFV and mirtazapine) which both activate CH240H in vitro have similar effects on PrPSc levels in neuronal cells. Furthermore, EFV treatment suppresses cholesterogenic gene expression, indicating increased production of 24-HC, a negative regulator of cholesterol synthesis. Mechanistically, CH240H activation results in an inhibition of PrPSc de novo synthesis, and activation of autophagy and other degradative pathways does not play a major role in the anti-prion effect. Of note, both drugs are approved for application to humans and cross the blood brain barrier, which is an important pre-requisite for successful human therapy.

[00138] References

[00139] 1 . Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216: 136-144.

[00140] 2. Watts JC, Balachandran A, Westaway D (2006) The expanding universe of prion diseases. PLoS Pathog 2: e26. 10.1371/journal.ppat.0020026 [doi].

[00141] 3. Wang F, Wang X, Yuan CG, Ma J (2010) Generating a prion with bacterially expressed recombinant prion protein. Science 327: 1 132-1 135.

science.1 183748 [pii]; 10.1 126/science.1 183748 [doi].

[00142] 4. Gilch S, Krammer C, Schatzl HM (2008) Targeting prion proteins in neurodegenerative disease. Expert Opin Biol Ther 8: 923-940.

10.1517/14712598.8.7.923 [doi].

[00143] 5. Krammer C, Vorberg I, Schatzl HM, Gilch S (2009) Therapy in prion diseases: from molecular and cellular biology to therapeutic targets. Infect Disord Drug Targets 9: 3-14.

[00144] 6. Taraboulos A, Scott M, Semenov A, Avrahami D, Laszlo L, Prusiner SB (1995) Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform. J Cell Biol 129: 121 - 132. [00145] 7. Gilch S, Kehler C, Schatzl HM (2006) The prion protein requires cholesterol for cell surface localization. Mol Cell Neurosci 31 : 346-353. S1044- 7431 (05)00250-2 [pii]; 10.1016/j.mcn.2005.10.008 [doi].

[00146] 8. Hannaoui S, Shim SY, Cheng YC, Corda E, Gilch S (2014) Cholesterol balance in prion diseases and Alzheimer's disease. Viruses 6: 4505-4535. v61 14505 [pii]; 10.3390/V61 14505 [doi].

[00147] 9. Bach C, Gilch S, Rost R, Greenwood AD, Horsch M, Hajj GN, Brodesser S, Facius A, Schadler S, Sandhoff K, Beckers J, Leib-Mosch C, Schatzl HM, Vorberg I (2009) Prion-induced activation of cholesterogenic gene expression by Srebp2 in neuronal cells. J Biol Chem 284: 31260-31269. M 109.004382

[pii]; 10.1074/jbc.M109.004382 [doi].

[00148] 10. Brown AR, Rebus S, McKimmie CS, Robertson K, Williams A, Fazakerley JK (2005) Gene expression profiling of the preclinical scrapie-infected hippocampus. Biochem Biophys Res Commun 334: 86-95. S0006-291X(05)01280-5 [pii]; 10.1016/j.bbrc.2005.06.060 [doi].

[00149] 1 1 . Montag J, Brameier M, Schmadicke AC, Gilch S, Schatzl HM, Motzkus D (2012) A genome-wide survey for prion-regulated miRNAs associated with cholesterol homeostasis. BMC Genomics 13: 486. 1471 -2164-13-486 [pii]; 10.1 186/1471 - 2164-13-486 [doi].

[00150] 12. Cui HL, Guo B, Scicluna B, Coleman BM, Lawson VA, Ellett L, Meikle PJ, Bukrinsky M, Mukhamedova N, Sviridov D, Hill AF (2014) Prion infection impairs cholesterol metabolism in neuronal cells. J Biol Chem 289: 789-802.

M 1 13.535807 [pii]; 10.1074/jbc.M 1 13.535807 [doi].

[00151] 13. Kumar R, McClain D, Young R, Carlson GA (2008) Cholesterol transporter ATP-binding cassette A1 (ABCA1) is elevated in prion disease and affects PrPC and PrPSc concentrations in cultured cells. J Gen Virol 89: 1525-1532. 89/6/1525 [pii]; 10.1099/vir.0.83358-0 [doi].

[00152] 14. Bate C, Tayebi M, Williams A (2008) Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A2 activation. BMC Biol 6: 8. 1741 -7007-6-8 [pii]; 10.1 186/1741 -7007-6-8 [doi].

[00153] 15. Mast N, Li Y, Linger M, Clark M, Wiseman J, Pikuleva lA (2014) Pharmacologic stimulation of cytochrome P450 46A1 and cerebral cholesterol turnover in mice. J Biol Chem 289: 3529-3538. M 1 13.532846 [pii]; 10.1074/jbc.M 1 13.532846 [doi].

[00154] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00155] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00156] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.