Compositions for use in a method of treating a motor neuron disease or condition associated with a SPG11 pathogenic variant

Abstract

Disclosed are methods and compositions for treating or ameliorating a motor neuron disease or condition associated with a spastic paraplegia type 11 pathogenic variant using a Liver X Receptor (LXR) agonist, more particularly to methods and compositions for treating hereditary spastic paraplegias and amyotrophic lateral sclerosis using a LXR agonist.

Description

COMPOSITIONS FOR USE IN A METHOD OFTREATING A MOTOR NEURON DISEASE OR CONDITION ASSOCIATED WITH A SPG11PATHOGENIC VARIANTIntroduction

[0001] This application claims the benefit of priority from U.S. Provisional Application Serial Number 63 / 538,158, filed September 13, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] This invention was made with government support under grant nos. R01 NS118066 and R21 NS089042, awarded by the National Institutes of Health. The government has certain rights in this invention.Reference to an Electronic Sequence Listing

[0003] The contents of the electronic sequence listing (name: UIC0107WO_ST26.xml; size: 6,293 bytes; and date of creation: August 23, 2024) is herein incorporated by reference in its entirety.Background

[0004] Axonal degeneration of human motor neurons underlies debilitating motor neuron diseases including amyotrophic lateral sclerosis and hereditary spastic paraplegias (HSPs). HSPs are a heterogeneous group of neurogenetic diseases characterized by progressive lower limb spasticity due to the length-dependent degeneration of cortical motor neuron axons. Since the identification of the first HSP gene, SPAST, over 80 genetic loci associated with HSP (Blackstone (2018) Curr. Opin. Neurobiol. 51:139-146; Martinuzzi et al. (2021) Front. Neurosci. 15:708642) have been identified. Based on genetic inherence mode, HSPs are further divided into autosomal dominant, autosomal recessive and X-linked inheritance (Finsterer et al. (2012) J. Neurol. Sci. 318:1-18).

[0005] Spastic paraplegia type 11 (SPG11) is one of the most frequent autosomal recessive forms of HSP caused by mutations in the SPG11 gene that encodes spatacsin protein (Denora et al. (2013) Handb. Clin. Neurol. 113:1899-1912; Fink (2013) Acta Neuropathol. 126:307-328; Stevanin et al. (2008) Brain 131:772-784). In addition to typical symptoms of HSP such as lower limb spasticity and weakness, SPG11 patients often exhibit cognitive impairment, mental retardation, early-onset parkinsonism, and thin corpus callosum. The SPG11 protein is a binding partner of the SPG15 protein, another common recessive form of HSP. Though both SPG11 and SPG15 proteins have been shown to regulate autophagy lysosome reformation (Renvoise et al. (2014) Ann, Clin. Transl. Neurol. 1:379- 389), how impaired lysosomal function leads to axonal degeneration remains elusive.

[0006] Cholesterol is highly enriched in the central nerve system, critical for maintaining axonal and synaptic functions. It has been shown that the spatacsin protein can regulate cholesterol trafficking and prevent abnormal cholesterol accumulation in lysosomes (Branchu et al. (2017) Neurobiol. Dis. 102:21-37). Spatacsin protein activates clathrin downstream and recruits dynamin to exocytose gangliosides and lipid from lysosomes (Boutry et al. (2019) Commun, Biol. 2:380). It has been reported that the loss of spatacsin, by knocking down SPG11 in fibroblast cells, inhibits the formation of tubules and causes cholesterol accumulation on the lysosome membrane (Boutry et al. (2019) Commun. Biol. 2:380). Cholesterol, a major component of membranes, plays an important role in maintaining neuronal function. Though these findings suggest that the impaired lipid trafficking caused by lysosomal dysfunction is induced in SPG11 deficient fibroblast cells, whether the impaired cholesterol trafficking underlies axonal defects and whetheraxonal degeneration can be rescued by targeting the cholesterol trafficking defects are unknown,

[0007] It has been demonstrated that treatment with Liver X Receptor (LXR) agonists can reduce axonal disruption and neurodegeneration in an Arl6IPl (ADP-ribosylation factor-like 6 interacting protein 1) knockout model of SPG61 in Drosophila (Byrne et al. (2022) Acta Neuropath. Commun. 10:40) and rescue HSP neurons in SPG4 (Rehbach (2019) Sci. Rep. 9:9615). However, there is no effective cure for SPG11, partially due to the lack of human neuronal models to study this disease. It has been shown thatSummary

[0008] Provided herein is a method for treating or ameliorating a motor neuron disease or condition associated with a SPG11 pathogenic variant by administering to a subject in need thereof a Liver X Receptor (LXR) agonist thereby treating or ameliorating the subject's disease or condition associated with the SPG11 pathogenic variant.Brief Description of the Drawings

[0009] FIG. 1, Quantification of the amount of filipin staining colocalized with LAMP1 in SPG11 knockdown groups treated with GW3965, RGX104, or DMSO (vehicle control). The proportion of cholesterol in lysosome was significantly increased in SPG11 neurons, which was mitigated by the treatment with either LXR agonist. Data were presented as Mean + SD. *p < 0.05, **p < 0.01 versus DMSO group by Dunnett's test after ANOVA. n.s.= not significant.

[0010] FIG. 2, Neurite outgrowth quantification revealed a significant elongation of Tau+axons in SPG11 knockdown neurons after the treatment of GW3965, RGX104, or DMSO(Vehicle control). Data were presented as Mean ± SD. Specificp values, *p < 0.05, **p < 0.01 versus DMSO group by Dunnett's test after ANOVA. n.s.= not significant.

[0011] FIG. 3, Quantification of axonal swelling density in long-term cultures revealed a significant reduction of increased axonal swellings in SPG11 knockdown groups (SPG11- A and SPG11-B) by the treatment of GW3965 and RGX104. Both drugs did not show significant effects on axonal swellings density in control Luc neurons. Data were presented as Mean ± SD, *p < 0.05, **p < 0.01 versus SPG11 DMSO group by Dunnett's test after ANOVA.

[0012] FIG. 4, Relative Caspase 3 / 7 activity was significantly increased in two independent SPG11 knockdown groups (SPG11-A and SPG11-B) compared to Luc control neurons. Data were presented as Mean + SD, ***p < 0.01 versus luciferase group by Dunnett's test.

[0013] FIG. 5, Relative Caspase 3 / 7 activity in both SPG11 knockdown treated with different drugs for 1 week. After treatment with GW3965 or RGX104, the caspase activity was significantly decreased. Data were presented as Mean ± SD, *p < 0.05, **p < 0.01, versus DMSO group by two-sided t-test.

[0014] FIG. 6, Quantification of the relative intensity of filipin staining in plasma membrane (PM) of cortical neurons after treatment of GW3965, RGX104, and DMSO (vehicle). Data were presented as Mean ± SD. **p < 0.01 versus SPG11 DMSO group by Dunnett's test after ANOVA.

[0015] FIG. 7, Quantification of axonal swelling density in long-term cultures revealed a significant reduction of increased axonal swellings in SPGll-mutated neurons after the treatment of GW3965 and RGX104. Both drugs did not show significant effects on axonal swellings density in control Luc neurons. Data were presented as Mean ± SD. **p < 0.01 versus SPG11 DMSO group by Dunnett's test after ANOVA.

[0016] FIG. 8, After treatment with LXR agonists and vehicle, the apoptosis levels in SPG11 p.Gln40X neurons were examined by analyzing Caspase 3 / 7 activities. Data were presented as Mean ± SD. **p < 0.01 versus SPG11 DMSO group by Dunnett's test after ANOVA.

[0017] FIG. 9, Effects of non-lipogenic ABCA1 inducers on the apoptosis levels in SPG11 neurons. Cortical neurons were differentiated from SPG11 and normal control human pluripotent stem cells. To examine the effects, of ABCA1 inducers, HSP neurons were treated with different doses of ABCAl-inducing compound, CL3-3, at 0.1 pM, 1 pM, and 3 pM) for one week. The levels of Caspase 3 / 7 activities in these neurons were then examined using the Caspase 3 / 7 Gio® assay. Data were presented as Mean ± SD,< 0.01 versus Control DMSO group, and ##P < 0.01 versus SPG11 DMSO group byDunnett's test after ANOVA.Detailed Description

[0018] Spastic paraplegia type 11 (SPG11) is a common autosomal recessive form of hereditary spastic paraplegia characterized by the degeneration of cortical motor neuron axons, leading to muscle spasticity and weakness. Using hESC- based SPG11 models created by SFGll-knockdown and diseasemutation knock-in, disease-relevant axonal defects were examined. In particular, the mutant hESC cells were differentiated into cortical projection neurons (PNs), which exhibited reduced axonal outgrowth, impaired axonal transport, axonal swellings, accumulation of cholesterol in lysosomes and reduced levels of cholesterol in plasma membranes, and elevated apoptosis. Using the SPGll-deficient neuron models, it was determined whether axonal defects could be rescued by targeting cholesterol trafficking. Interestingly, treatment of SPGll-knockdown neurons and SPG11mutation knock-in neurons (p.Gln40-STOP) with liver X receptor (LXR) agonists, GW3965 and RGX104, significantly mitigated the increased lysosomal cholesterol, indicating that LXR agonists can rescue aberrant lysosomal cholesterol accumulation in SPGll-deficient neurons. Notably, the axonal deficits of both SPGll-knockdown and disease mutation knock- in cortical neurons, including reduced axonal outgrowth, axonal swelling, and impaired axonal transport, were significantly mitigated by treatment with GW3965 and RGX104.

[0019] The accumulated cholesterol in lysosomes and the reduced plasma membrane cholesterol in SPGll-deficient neurons indicated that the efflux of cholesterol from lysosomes and the trafficking of cholesterol from the lysosome to the plasma membrane are impaired in these neurons. These cholesterol trafficking processes are regulated by ATP- binding cassette (ABC) transporters including ABCA1 and ABCA2 . Accordingly, compounds that induce ABCA1 expression and agonist LXRp were tested using the SPGll-deficient neuron models. After treatment with the ABCA1 inducers, apoptosis in SPGll-deficient neurons was significantly mitigated. Accordingly, these data demonstrate impaired cholesterol trafficking underlying axonal degeneration of SPG11 human neurons, and highlight the therapeutic use of LXR agonists and / or ABCA1 inducers to restore cholesterol homeostasis and treat diseases associated with SPG11 pathogenic variants, e.g.fhereditary spastic paraplegias or amyotrophic lateral sclerosis .

[0020] Accordingly, provided herein are methods for treating or ameliorating a motor neuron disease or condition associated with, caused by, or otherwise attributed to a SPG11 pathogenic variant by administering to a subject in need thereof a LXR agonist thereby treating or ameliorating the subject's disease or condition associated with the SPG11pathogenic variant. "Treating" or "treatment" as used herein covers the treatment of a motor neuron disease or condition described herein, in a subject, preferably a human, and includes inhibiting a motor neuron disease or condition, i.e., arresting its development; relieving a motor neuron disease or condition, i.e., causing regression of the motor neuron disease or condition; slowing progression of the motor neuron disease or condition; and / or inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment results in a measurable (i.e., at a statistically significant level) decrease or reduction in one or more signs or symptoms of a motor neuron disease or condition.

[0021] "Subject" or "subject in need thereof" refers to a warm-blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential to be afflicted with one or motor neuron diseases or conditions described herein. Subjects benefiting from any one of the above referenced treatments include, but are not limited to, subjects at risk of having, having or predisposed to have a motor neuron disease or condition associated with the SPG11 pathogenic variant.

[0022] As used herein, a "SPG11 pathogenic variant" refers to a complete or partial deletion or mutation in the SPG11 locus resulting in SPG11 deficiency, e.g., the absence of expression or absence of a functional gene product (e.g., SPG11 mRNA or SPG11 protein). SPG11 pathogenic variants are known in the art and include, but are not limited to, c.H 8C>T (p.Gln4O-STOP), C.3291+1G>T, c.4307_4308delAA (p.Glnl436Argfs), c.200 203delCTTT (p.Ser67fs), c.2987_2989delGTT (p.Cys996del), c.2163dupT (p.Ile722Tyrfs), c .5410___5411delTG (p .Cysl804Profs), c.l203del (p.Asp402fs), C.2316+ 1G-A, c.3121C>T (p.Argl041Ter), p.AlalO6Leufs*15,p.Thrl92AsnfsTerll, p .Met245Valfs*2, p.Met245ValfsTer2, p.Ser216Glufs*22, p.Trp362Ter, p.Lys405Thr, p.Ser412*, p.Ser412Ter, p.Glu426Argfs*3, p.Ile450AsnfsTer26, p.Gln478Ter, p.Gln498*, p.Asn579_Glu874delinsLys, p.Glu582*, p.Pro585ThrfsTerll, p.Arg651*, p.Arg651Ter, p.Gln716Ter, p.Ile722Tyrfs*10, p.Glu774Leufs*21,Val773Argfs*5, p.Gln811*, p.Gln811Ter, p.Trp899Ter, p.Gln914Ter, p.Leu920Aspf s*3, p.Leu850Trpfs*13, p.Cys996del, p .Tyrl006Ter, p.GlulO26Argfs*4, p.Vall098MetfsTer20, p.Lysll90*, p.Glnl436Argfs*7, p.Glnl466*, p .Glul495Ter, p.Leul605Profs*10, p.Glyl636Aspfs*12, p .Hisl717thrfs*3, p.Alal726Glnfs*112, p.Glnl785Ter, p .Glul798Lys, p.Glnl875*, p.Thrl956Serfs*18, p.Cysl996Leufs*4, and p.Leul997MetfsTer60 with the transcript provided under GENBANK Accession No. NM_025137.3 used as the reference sequence. See, e.g.zKang et al. (2023) Front. Neurol. 14:1198728; and Leiden Open Variation Database.

[0023] A motor neuron disease or condition "attributed to a SPG11 pathogenic variant, " "caused by a SPG11 pathogenic variant," or "associated with a SPG11 pathogenic variant" refers to a motor neuron disease, disorder, or condition in which a SPG11 pathogenic variant (e.g., SPG11 deficiency) has been observed to occur or in which a SPG11 pathogenic variant is known or thought to be associated with or contribute to motor neuron disease etiology, progression, or symptoms, or in which a SPG11 pathogenic variant is known or thought to occur as the motor neuron disease progresses. SPG11 pathogenic variants are associated with a spectrum of clinical manifestations resulting from first and / or second motor neuron degeneration. Motor neuron diseases associated with a SPG11 pathogenic variant include, but are not limited to, a pure or complex hereditary spastic paraplegia (Stevanin et al. (2008) Brain 131:772-84), amyotrophic lateralsclerosis (Orlacchio et al. (2010) Brain 133:591-8; Daoud et al. (2012) Neurobiol. Aging 33:839.e5-9; Manole et al. (2016) J. Neurol. 263:2278-86) and Charcot-Marie-Tooth neuropathy(Montecchiani et al. (2016) Brain 139:73-85; Sanghani et al. (2020) Peripheral Neuropath. 94(15_suppl.)). Individuals with multiple sclerosis have also been found to have SPG11 pathogenic variants (Romagnolo et al. (2014) Eur. J. Neurol. 21:el4-5; Mukai et al. (2018) Intern Med. 57:3183-6).

[0024] In some embodiments, the motor neuron disease or condition is a hereditary spastic paraplegia. Hereditary spastic paraplegia is characterized by progressive spasticity and weakness of the lower limbs freguently associated with mild intellectual disability with learning difficulties in childhood and / or progressive cognitive decline, peripheral neuropathy, pseudobulbar involvement, and increased reflexes in the upper limbs. The diagnosis of hereditary spastic paraplegia is established in a proband with characteristic clinical and MRI findings and biallelic pathogenic variants in SPG11 identified on molecular genetic testing.

[0025] In some embodiments, the motor neuron disease or condition is amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS), formerly known as Lou Gehrig's disease, is a neurological disorder that affects motor neurons, the nerve cells in the brain and spinal cord that control voluntary muscle movement and breathing. As motor neurons degenerate and die, they stop sending messages to the muscles, which causes the muscles to weaken, start to twitch (fasciculations), and waste away (atrophy). Early symptoms include muscle twitches, muscle cramps, tight and stiff muscles (spasticity), muscle weakness, slurred and nasal speech, and difficulty chewing and swallowing. As the disease progresses, muscle weakness and atrophy spread throughout the body.

[0026] In some embodiments, the motor neuron disease or condition is the Charcot-Marie-Tooth neuropathy. Charcot- Marie-Tooth disease results in smaller, weaker muscles and may cause a loss of sensation and muscle contractions, and difficulty walking. Foot deformities such as hammertoes and high arches also are common. Symptoms usually begin in the feet and legs, but they may eventually affect the hands and arms.

[0027] In some embodiments, the motor neuron disease or condition is multiple sclerosis (MS). MS damages the protective cover around nerves called myelin in your central nervous system. It can cause muscle weakness, vision changes, numbness and memory issues. In addition, common symptoms include fatigue, dizziness, loss of balance, mood changes, and muscle stiffness.

[0028] A "liver X receptor agonist" or "LXR agonist" refers to an agent capable of activating, enhancing, increasing, or otherwise stimulating one or more functions of the target LXR. In some embodiments, the LXR agonist increases the activity of the target LXR by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, as compared to the target LXR in the absence of the agonist. In some embodiments, the LXR is LXRa. In some embodiments, the target is LXRp. In some embodiments, the LXR agonist selectively agonizes LXRa. In some embodiments, the LXR agonist is a nonselective LXR agonist that agonizes LXRa and LXRp. In some embodiments, the LXR agonist selectively agonizes LXR[}. In some embodiments, the agonist exhibits at least 2.5-fold selectively for LXRp over LXRa.

[0029] Examples of suitable LXR agonists include, but are not limited to, T0901317 (CAS No. 293754-55-9), GW3965 (3- [3-[[[2-Chloro-3-(trifluoromethyl)phenyl]methyl](2,2- diphenylethyl)amino]propoxy]benzeneacetic acid; CAS No.405911-17-30, RGX-104 (2-[3-[(3R)-3-[[2-chloro-3-(trifluoromethyl)phenyl]methyl- (2,2- diphenylethyl)amino]butoxy]phenyl]acetic acid; CAS No. 610318-54-2), GW6340 (CAS No. 405910-78-3), Acetyl podocarpic acid anhydride (CAS No. 344327-48-6), LXR-623 (CAS No.875787-07-8), AZ876 (CAS No. 898800-26-5), IMB-808 (CAS No.870768-70-0), DMHCA (CAS No. 79066-03-8), BMS-779788 (CAS No.918348-67-1), XL041 (CAS No. 1256918-39-4), GSK3987 (CAS No.264206-85-1), or a pharmaceutically acceptable salt thereof.In some embodiments, the LXR agonist is RGX-104.

[0030] In some embodiments, the LXR agonist is a compound that increases or induces ABCA1 expression. Examples of such compounds are described in, e.g., US 2024 / 0199572 Al, incorporated herein by reference in its entirety. In some embodiments, the LXR agonist is a compound of Formula (I), or a pharmaceutically acceptable salt or prodrug thereof:Formula (I) wherein,X1, X2, X3, X4, X5, X6 X7, X8, X9, X10, and Xu are each independently selected from the group of carbon (C), nitrogen (N), sulfur (S), oxygen (O), NH, or CYi; each occurrence of Yi is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl,hydroxyalkyl, hydroxy, cyano, halo, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, a 5- or 6-membered monocyclic heteroaryl, and a 5- or 6-membered fused-ring;Zi, Z2, Z3, Z4, Zg, and Zg are each independently selected from the group of hydrogen, halo, 0, S, N, NH, C(=0), CH2, hydroxy, or CY2;Z5 is S;Zi is N or C, and each occurrence of Y2 is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, and halo, with the proviso that either Zi is not hydrogen, or Zg is alkyl when Z7 is N. Accordingly, in some embodiments, the compound is not F420, wherein Zi is hydrogen, and Zg is hydrogen when Z7 is N,

[0031] In some embodiments, the LXR agonist is a compound of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof:Formula (II) wherein,X1 and X2 are each independently selected from the group of C, N, S, O, NH, or CYi;each occurrence of Yi is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, and halo;Zi, Z2, Z3, Z4, Ze, and Z$ are each independently selected from the group of hydrogen, halo, 0, S, N, NH, C(=0), CH2, hydroxy, or CY2;Z5 is S;Z? is N or C; and each occurrence of Y2 is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, and halo; mi and m2 are independently 0, 1, 2, 3, or 4; m and n2 are independently 0, 1, 2, 3, or 4; andRi, R2, R3, and R4 are each independently selected from the group of hydrogen, halo, hydroxy, nitro, C1-C6 alkyl, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, C1-C6 fluoroalkyl, cyano, -O(C1-C6 alkyl), and -0 (C1-C6fluoroalkyl), with the proviso that either Zi is not hydrogen, or Z@ is alkyl when Z7is N.

[0032] In some embodiments, the LXR agonist is a compound of Formula (III), or a pharmaceutically acceptable salt or prodrug thereof:Formula (III) wherein,X is S, 0, or NH;Zi, Z2, Z3, Z5, and Ze are each independently selected from the group of hydrogen, halo, 0, S, N, NH, CH2, hydroxy, orCYi;Z4 is S; each occurrence of Yi is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, and halo; mi and m2 are independently 0, 1, 2, 3, or 4; m and n2 are independently 0, 1, 2, 3, or 4; andRi, R2, R3, and Ro are each independently selected from the group of hydrogen, halo, hydroxy, nitro, C1-C6 alkyl, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, C1-C6 fluoroalkyl, cyano, -O(C1-C6 alkyl), and -0 (C1-C6fluoroalkyl, with the proviso that either Zi is not hydrogen, or Ze is alkyl.

[0033] In some embodiments, the LXR agonist is a compound of Formula (IV), or a pharmaceutically acceptable salt or prodrug thereof:Formula (IV) wherein,X is S, 0, or NH;Zi, Z2, Z3, Z5, Z6,Z7and Zg are each independently selected from the group of hydrogen, halo, 0, S, N, NH, CH2, hydroxy, keto (C=0) or CYi;Z4is S; each occurrence of Yi is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, and halo; mi and m2 are independently 0, 1, 2, 3, or 4; ni is independently 0, 1, 2, 3, or 4;01 and 02 are independently 0, 1, 2, 3, or 4; andRi, R2, R3, R4and R5 are each independently selected from the group of hydrogen, halo, hydroxy, nitro, C1-C6 alkyl, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, C1-C6 fluoroalkyl, cyano, “O(C1-C6 alkyl), and -0 (C1-C6 fluoroalkyl), with the proviso that either Z1 is not hydrogen, or Z6 is alkyl.

[0034] In some embodiments, the LXR agonist is a compound of Formula (V), or a pharmaceutically acceptable salt or prodrug thereof:Formula (V) wherein,Z1 and Z2 are each independently selected from the group of N, S, 0, NH, CH, or NYi, each occurrence of Yi is independently selected from the group consisting of alkyl, haloalkyl, and hydroxyalkyl; mi and m2 are independently 0, 1, 2, 3, or 4; nx and n2 are independently 0, 1, 2, 3, or 4; andRi, R2, R3 and R4 are each independently selected from the group of hydrogen, halo, hydroxy, nitro, C1-C6alkyl, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, C1-C6 fluoroalkyl, cyano, -OfC1-C6 alkyl), and - O(C1-C6 fluoroalkyl).

[0035] In some embodiments, the LXR agonist is a compound of Formula (VI), or a pharmaceutically acceptable salt or prodrug thereof:Formula (VI) wherein,Zi, Z2, Z3, and Z4 are each independently selected from the group of N, S, 0, NH, CH, or NYi; each occurrence of Yi is independently selected from the group consisting of alkyl, haloalkyl, and hydroxyalkyl; mi and m2 are independently 0, 1, 2, 3, or 4; ni and n2 are independently 0, 1, 2, 3, or 4; andRi, R2, R3 and R4 are each independently selected from the group of hydrogen, halo, hydroxy, nitro, C1-C6alkyl, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, C1-C6 fluoroalkyl, cyano, -O(C1-C6 alkyl), or - O(C1-C6fluoroalkyl) .

[0036] In some embodiments, the LXR agonist is a compound of Formula (VII), or a pharmaceutically acceptable salt or prodrug thereof:Formula (VII) wherein,Z1, Z2, Z3, Z4and Z5 are each independently selected from the group of N, S, 0, C(=0), NH, CH, or NYi; each occurrence of Yi is independently selected from the group consisting of alkyl, haloalkyl, and hydroxyalkyl; mi and m2 are independently 0, 1, 2, 3, or 4; ni and n2 are independently 0, 1, 2, 3, or 4; andRi, R2, R3 and R4 are each independently selected from the group of hydrogen, halo, hydroxy, nitro, C1-C6alkyl, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, C1-C6 fluoroalkyl, cyano, -O(C1-C6 alkyl), or - O(C1-C6fluoroalkyl) .

[0037] In some embodiments, the LXR agonist is a compound of Formula (VIII), or a pharmaceutically acceptable salt or prodrug thereof:Formula (VIII) wherein,Ring B is selected from

[0038] In some embodiments, the LXR agonist is a compound of Formula (IX), or a pharmaceutically acceptable salt or prodrug thereof:wherein,Ring B is selected fromRi is hydrogen, halo, alkyl, alkoxy, COaMe, CChEt, cyano, or OCF3; andR2 and R3 are each independently selected from the group of alkoxy, alkyl, halo, or cyano.

[0039] In some embodiments, the LXR agonist is a compound of Formula (X), or a pharmaceutically acceptable salt or prodrug thereof :Formula (X) wherein,Ring B is selected fromR1 and R2 are each independently selected from the group of hydrogen, halo, alkyl, alkoxy, C02Me, COsEt, cyano, or OCF3; andR3 and R4 are each independently selected from the group of alkoxy, alkyl, halo, or cyano.

[0040] In some embodiments, the LXR agonist is a compound ofFormula (XI), or a pharmaceutically acceptable salt orRi and R2 are each independently selected from the group of hydrogen, halo, alkyl, alkoxy, COzMe, COzEt, cyano, or OCF3; andR3 and Rs are each independently selected from the group of alkoxy, alkyl, halo, or cyano.

[0041] The term "halo" or "halogen" refers to a fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I) group. In certain aspects, "halo" or "halogen" refers to a Cl or F group. "Cyano" refers to a -CN group, "hydroxy" refers to a -OH group, and "nitro" refers to a “NO2 group. "0" as a substituent refers to oxo (=0).

[0042] The term "alkyl" as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms unless otherwise specified. Representative examples ofalkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tertbutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3- methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and n-decyl. When an "alkyl" group is a linking group between two other moieties, then it may also be a straight or branched chain; examples include, but are not limited to -CH2-, "CH2CH2-, -CH2CH2CHC(CH3)-, and -CH2CH (CH2CH3)CH2-.

[0043] In some embodiments, the groups disclosed herein are delineated by a specified number of carbon atoms, e.g., Ci- C6- The numerical range "1 to 6" or "1-6" refers to each integer in the given range, e.g., "1 to 6 carbon atoms" means that a group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 6 carbon atoms. By way of example, "C1-C4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from among methyl, ethyl, propyl, iso-propyl, n- butyl, iso-butyl, sec-butyl, and t-butyl. Thus, C1-C4 alkyl includes C1-C2 alkyl and C1-C3 alkyl.

[0044] "Alkoxy" means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0045] The term "haloalkyl" as used herein, means at least one halogen, as defined herein, is appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl. In certain aspects, each "haloalkyl" is a fluoroalkyl, for example, a polyfluoroalkyl such as a substantially perfluorinated alkyl.

[0046] As used herein, the term "hydroxyalkyl" means an alkyl group, as defined herein, with one or more (e.g., 1, 2 or 3) hydroxy substituents.

[0047] The term "ketone" as used herein refers to the structure -C(=0)-Ra, wherein Ramay be alkyl (e.g., -C(=0)- CH3) or aryl.

[0048] The term "ester" as used herein refers to the structure -C (=0)-O-Rc, where Rcmay be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl.

[0049] "Carboxamide" refers to a group having the structure —C (=0)-N (RaRe), where Rd and Reindependently represent hydrogen or alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl groups.

[0050] The term "sulfide" refers to a group having the structure -S-Rf, where Rf may be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl. Examples of sulfide groups include a methylthio group, an ethylthio group, a t-butylthio group, and a tert-butylthio group.

[0051] "Sulfinyl" or "sulfoxide" refers to the —S(=O)—Rggroup, wherein for "sulfinyl" Rgis hydrogen, and for "sulfoxide" Rgmay be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl.

[0052] "Sulfone" or "sulfonyl" refers to a -S (=0)2-Rh group, wherein Rh is selected from alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl.

[0053] "Sulfonamide" refers to a group having the structure -S (=0)2-N (RiRj), where Ri and Rj independently represent hydrogen or alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl groups.

[0054] As used herein, the term "ring" refers to any covalently closed structure. Rings include, for example, carbocycles (e.g., aryls and cycloalkyls), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics(e.g., aryls and heteroaryls), and non-aromatics (e.g., cycloalkyls and non-aromatic heterocycles). Rings can be monocyclic or fused-ring, i.e., polycyclic. Rings can be optionally substituted.

[0055] The term "membered ring" can embrace any cyclic structure. The term "membered" is meant to denote the number of skeletal atoms that constitute the ring. Thus, for example, cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, and thiophene are 5- membered rings.

[0056] "Ring system substituent" means a substituent attached to an aromatic or non-aromatic ring system, which, for example, replaces an available hydrogen on the ring system. Ring system substituents may be the same or different, each being independently selected from the group of C (=NH)(NH2), -NEC (=NH)(NH2), alkyl, alkenyl, alkynyl, alkoxy, acyl, alkylcarbonylamino, carboxy, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, cyano, nitro, alkylthio, halo, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, aminocarbonyl, alkyl aminocarbonyl, dialkylaminocarbonyl, alkyl sulfonyl, cycloalkylsulfonyl, alkylsulfonylamino, alkylaminosulfonyl, haloalkylamino, oxo, hydroxy, hydroxyalkyl, hydroxyalkyloxy, hydroxyalkyloxyalkyl, alkoxyalkyloxyalkyl, aryl, heteroaryl, cycloalkyl, cycloalkylamino, cycloalkyloxy, heteroaralkyloxy, aminoalkyl, aminoalkyloxy, alkoxyalkyl, alkoxyalkylcarbonyl, alkoxyalkyloxy, haloalkoxyalkyl, aryloxyalkyl, heteroaryloxyalkyl, heterocycloalkyl, heterocycloalkyloxyalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy, or heterocycloalkyloxy. "Ring system substituent" may also mean a single moiety that simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) on a ring system.Examples of such moieties are methylenedioxy, ethylenedioxy, -C(CH3)2“ and the like.

[0057] The term "aryl," as used herein, means a phenyl (i.e., monocyclic aryl), or a bicyclic ring system containing at least one phenyl ring or an aromatic bicyclic ring containing only carbon atoms in the aromatic bicyclic ring system. The bicyclic aryl can be azulenyl, naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocyclyl. The bicyclic aryl is attached to the parent molecular moiety through any carbon atom contained within the phenyl portion of the bicyclic system, or any carbon atom with the napthyl or azulenyl ring. The fused monocyclic cycloalkyl or monocyclic heterocyclyl portions of the bicyclic aryl are optionally substituted with one or two oxo and / or thia groups. Representative examples of the bicyclic aryls include, but are not limited to, azulenyl, naphthyl, dihydroinden-l-yl, dihydroinden-2-yl, dihydroinden-3-yl, dihydroinden-4-yl, 2,3-dihydroindol-4-yl,2.3-dihydroindol-5-yl, 2, 3~dihydroindol-6-yl, 2,3- dihydroindol-7-yl, inden-l-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen~l~yl , 5,6,7,8- tetrahydronaphthalen-l-yl, 5,6,7, 8-tetrahydronaphthalen-2- yl, 2,3-dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl,2.3-dihydrobenzofuran-6-yl, 2,3-dihydrobenzofuran-7-yl, benzo [d][1,3]dioxol-4-yl, benzo[d][1,3]dioxol-5-yl, 2H- chromen-2-on-5-yl, 2H-chromen-2-on-6-yl, 2H-chromen-2-on-7- yl, 2H-chromen-2-on-8-yl, isoindoline-1,3-dion-4-yl, isoindoline-1 ,3-dion-5-yl, inden-l-on-4-yl, inden-l-on-5-yl, inden-l-on-6-yl, inden-l-on-7-yl, 2,3- dihydrobenzo [b][1,4]dioxan-5-yl, 2,3- dihydrobenzo [b][1,4]dioxan-6-yl, 2H- benzo[b] [1,4]oxazin3(4H)-on-5-yl, 2H-benzo [b][1,4]oxazin3(4H)-on-6-yl, 2H- benzo[b][1,4]oxazin3 (4H)-on-7-yl, 2H- benzo[b][1,4]oxazin3 (4H)-on-8-yl, benzo[d]oxazin-2(3H)-on-5- yl, benzo[d]oxazin-2 (3H)-on-6-yl, benzo[d]oxazin-2(3H)-on-7- yl, benzo[d]oxazin-2(3H)-on-8-yl, quinazolin-4(3H)-on-5-yl, quinazolin-4 (3H)-on-6-yl, quinazolin-4 (3H)-on-7-yl, quinazolin-4 (3H)-on-8-yl, quinoxalin-2 (1H)-on-5-yl, quinoxalin-2 (1H)-on-6-yl, quinoxalin-2 (1H)-on-7-yl, quinoxalin-2 (1H)-on-8-yl, benzo [d]thiazol-2(3H)-on-4-yl, benzo [d]thiazol-2(3H)-on-5-yl, benzo [d]thiazol-2(3H)-on-6- yl, and, benzo[d]thiazol-2(3H)-on-7-yl. In certain aspects, the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, or a 5- or 6-membered monocyclic heterocyclyl , wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted. In certain embodiments of the disclosure, the aryl group is phenyl or naphthyl. In certain other embodiments, the aryl group is phenyl.

[0058] The term "cycloalkyl" as used herein, means a monocyclic or a bicyclic cycloalkyl ring system. Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In certain aspects, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings. Bridged monocyclic rings contain a monocyclic cycloalkyl ring where two nonadjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form -(CH2)W“, where wis 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo [3.1.1]heptane, bicyclo [2.2.1]heptane, bicyclo [2.2.2]octane, bicyclo [3.2.2]nonane, bicyclo [3.3.1]nonane, and bicyclo[4.2.1]nonane. Fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. The bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In certain aspects, the fused bicyclic cycloalkyl is a 5- or 6-membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5- or 6- membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted.

[0059] The term "heteroaryl,” as used herein, means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring. The monocyclic heteroaryl can be a 5- or 6-membered ring. The 5-membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom. The 6-membered ring consists of three double bonds and one, two, three or four nitrogen atoms. The 5- or 6-membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl. Representative examples of monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclicheteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. The fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted. When the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl, or heterocyclyl ring, then the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon or nitrogen atom contained within the monocyclic heteroaryl portion of the bicyclic ring system. When the bicyclic heteroaryl is a monocyclic heteroaryl fused to a phenyl ring, then the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon atom or nitrogen atom within the bicyclic ring system. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl, cinnolinyl, 5,6- dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-l-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl , quinolinyl, purinyl, 5,6,7,8- tetrahydroquinolin-2-yl, 5, 6,7,8-tetrahydroquinolin-3-yl,5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8- tetrahydroisoquinolin-l-yl, thienopyridinyl, 4,5,6,7- tetrahydrobenzo [c][1,2,5]oxadiazolyl, and 6,7- dihydrobenzo [c][1,2,5]oxadiazol-4(5H)-onyl. In certain aspects, the fused bicyclic heteroaryl is a 5- or 6-membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted.

[0060] The term "heterocyclyl" as used herein, means a monocyclic heterocycle or a bicyclic heterocycle. The monocyclic heterocycle is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of 0, N, and S where the ring is saturated or unsaturated, but not aromatic. The 5-membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The 6-membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of 0, N' and S. The monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl , isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl , oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl , thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl,1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl. The bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system. Representative examples of bicyclicheterocyclyls include, but are not limited to, 2,3- dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin- 1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2- yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro- iH-indolyl, and octahydrobenzofuranyl. Heterocyclyl groups are optionally substituted. In certain embodiments, the bicyclic heterocyclyl is a 5- or 6-membered monocyclic heterocyclyl ring fused to phenyl ring, a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted.

[0061] Terms used herein may be preceded and / or followed by a single dash, "-", or a double dash,to indicate the bond order of the bond between the named substituent and its parent moiety; a single dash indicates a single bond and a double dash indicates a double bond. In the absence of a single or double dash it is understood that a single bond is formed between the substituent and its parent moiety; further, substituents are intended to be read "left to right" unless a dash indicates otherwise. For example, C1-C6 alkoxycarbonyloxy and -OC (=0)-C1-C6 alkyl indicate the same functionality; similarly, arylalkyl and alkylaryl indicate the same functionality.

[0062] In all structural formulas used herein, it is understood that all carbon valences not shown here are satisfied by the groups illustrated and by hydrogen atoms.

[0063] The term "saturated" as used herein means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like.

[0064] The term '''unsaturated" as used herein means the referenced chemical structure contains at least one multiple carbon-carbon bond but is not aromatic. For example, an unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.

[0065] The compounds herein include the compounds themselves as well as tautomers, salts, solvates, amides, and / or esters thereof.

[0066] Exemplary compounds of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), or Formula (VIII), Formula (IX), Formula (X), or Formula (XI), include, but are not limited to:pharmaceutically acceptable salt thereof.

[0068] "Pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the present compounds. Salts of the present compounds can be prepared during the final isolation and purification of the compounds or separately by reactingthe compound with an acid having a suitable cation. The pharmaceutically acceptable salts of the present compounds can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric. Nonlimiting examples of salts of compounds of the disclosure include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p- toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds herein is intended to include the present compounds as well as pharmaceutically acceptable salts thereof.

[0069] For use in the methods herein, the LXR agonist may be provided in a pharmaceutical composition comprising the LXR agonist, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, excipients, or carriers. The pharmaceutical composition may be used, for example, for treating motor neuron diseases such as amyotrophic lateral sclerosis and hereditary spastic paraplegias .

[0070] In some embodiments, a pharmaceutical composition is provided that includes a LXR agonist together with one or more pharmaceutically acceptable excipients or vehicles, and optionally other therapeutic and / or prophylactic ingredients. Such excipients include liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like.

[0071] The term "pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient or carrier with which a LXR agonist of the disclosure is administered. The terms "effective amount" or "pharmaceutically effective amount" refer to a nontoxic but sufficient amount of the agent to provide the desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0072] "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, latest edition. For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agentscan be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used.

[0073] Suitable excipients for non-liquid formulations are also known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington's Pharmaceutical Sciences, latest edition.

[0074] Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, can be present in such vehicles. A biological buffer can be any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate-buffered saline, Trisbuffered saline, Hank's-buffered saline, and the like.

[0075] Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semisolid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, can include other pharmaceutical agents, adjuvants, diluents, buffers, and the like.

[0076] In general, the compositions herein will be administered in a therapeutically effective amount by any of the accepted modes of administration. Suitable dosage ranges depend upon numerous factors such as the severity of thedisease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication toward which the administration is directed, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compositions of the disclosure for a given disease.

[0077] Thus, the compositions herein may be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The preferred manner of administration is intravenous or oral using a convenient daily dosage regimen which can be adjusted according to the degree of affliction.

[0078] For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, latest edition.

[0079] In another embodiment is the use of permeation enhancer excipients including polymers such as: polycations (chitosan and its quaternary ammonium derivatives, poly-L- arginine, aminated gelatin); polyanions (N-carboxymethyl chitosan, poly-acrylic acid); and, thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).

[0080] For oral administration, the composition will generally take the form of a tablet, capsule, a softgel capsule or can be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. Typically, the compositions of the disclosure can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0081] When liquid suspensions are used, the active agent can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like and with emulsifying and suspending agents. If desired, flavoring, coloring and / or sweetening agents can be added as well. Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.

[0082] Parenteral formulations may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.

[0083] Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Administration via certain parenteral routes can involve introducing the formulations of the disclosure into the body of a patient through a needle or a catheter, propelled by a sterile syringe or some other mechanical device such as a continuous infusion system. A formulation provided by the disclosure can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration.

[0084] Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.

[0085] Preparations according to the disclosure for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol,polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They can be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

[0086] Sterile injectable solutions are prepared by incorporating one or more of the LXR agonists herein in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-f iltered solution thereof. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

[0087] Alternatively, the pharmaceutical compositions herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable nonirritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols .

[0088] The pharmaceutical compositions herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents.

[0089] Preferred formulations for topical drug delivery are ointments and creams. Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent, are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing .

[0090] Formulations for buccal administration include tablets, lozenges, gels and the like. Alternatively, buccal administration can be effected using a transmucosal delivery system as known to those skilled in the art. The compounds of the disclosure can also be delivered through the skin or muscosal tissue using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the agent is typically contained within a laminated structure that serves as a drug delivery device to be affixed to the body surface. In such a structure, the drug composition is typically contained in a layer, or "reservoir, " underlying an upper backing layer. The laminated device can contain a single reservoir, or it can contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, can be either a polymeric matrix as described above, or it can be a liquid or gel reservoir, or can take some other form. The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing layer should be substantially impermeable to the active agent and any other materials that are present.

[0091] The LXR agonists herein may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The agonist willgenerally have a small particle size for example of the order of 5 microns or less. Such a particle size can be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol can conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively, the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition can be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder can be administered by means of an inhaler.

[0092] A pharmaceutically or therapeutically effective amount of the composition will be delivered to the subject. The precise effective amount will vary from subject to subject and will depend upon the species, age, the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration. Thus, the effective amount for a given situation can be determined by routine experimentation. For purposes of the disclosure, generally a therapeutic amount will be in the range of about 0.01 mg / kg to about 250 mg / kg body weight, more preferably about 0.1 mg / kg to about 10 mg / kg, in at least one dose. In larger mammals the indicated daily dosage can be from about 1 mg to 300 mg, one or more times per day, more preferably in the range of about 10 mgto 200 mg. The subject can be administered as many doses as is required to reduce and / or alleviate the signs, symptoms, or causes of the disorder in question, or bring about any other desired alteration of a biological system. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.

[0093] The LXR agonist may be administered alone or in combination with other therapeutics (e.g., antispasmodic medications, medications for treating cognitive symptoms, and / or disease-modifying therapies) in the treatment of the motor neuron disease or disorder. Such therapeutics include, e.g., riluzole, edaravone, sodium phenylbutyrate / taurursodiol, tofersen, baclofen, tizandine, botulinum, gabapentin, nortriptyline, donepezil, beta interferon, glatiramer acetate, ofatumumab, alemtzuumab, natalizumab, rituximab, ocrelizumab, ublituximab, cladribine, dimethyl fumarate, diroximel fumarate, fingolimod, siponimod, ponesimod, oxanimod, and teriflunomide ,

[0094] The foregoing may be better understood by reference to the following methods, results and discussion which are presented for purposes of illustration and are not intended to limit the scope of the invention.Example 1: Materials and Methods

[0095] Lentivirus Production and Transfection of hESCs, To knockdown spatacsin, shRNA sequences that target SPG11 and luciferase (as controls) were cloned into pLKO.l. shRNAs that target two different regions of the SPG11 gene (SPG11 shRNA- A and SPG11 shRNA-B) were used to minimize off-target effects. SPG11 shRNA-A targets SPG11 sequences 5'- GAGGAACCCATAGAGCTTAAATG-3' (SEQ ID NO:1) and SPG11 shRNA-Btargets SPG11 sequences 5'-GCTCATTGACGATCAAGATATCA-3' (SEQ ID NO:2). Lentiviruses were packaged using a calcium phosphate transfection method as described previously (Denton et al. (2014) Stem Cells 32:414-423). Briefly, pLKO.l shRNA plasmid, psPAX2 packaging plasmid and pMD2.G envelope plasmid were mixed and then incubated in calcium phosphate solution with 2x HEPES-buffered saline for 1 minute. The mixture was transferred into dishes for co-culturing the cocktail with HEK293T cells. After 2 to 3 days, the viral particles from the cell culture medium were collected through 20,000 rpm ultracentrifugation (SW28 rotor, Beckman) at 16°C for 2 hours and the pellet was resuspended in hESC medium. For stable transfection of H9 ESCs (WA09, WiCell Research Institute), cells were dissociated to small clusters and then incubated with viruses at 37°C for 30 minutes. With the Blasticidin selection, the hESCs with stable transfection survive while other cells including feeder cells gradually die. The Blasticidin-resistant cells were then dissociated to select clonal SPG11 knockdown hESC cell lines. With knockdown efficacy confirmed by qPCR, both SPG11 knockdown and Luciferase knockdown lines (as controls) were generated for differentiation .

[0096] Neural Differentiation of Pluripotent Stem Cells. Human ESCs were maintained in ESC medium and differentiated into cortical PNs as described previously (Boisvert et al. (2013) J. Vis. Exp. (74):50321; Li et al. (2009) Development 136:4055-4063). Stem cells were cultured on a feeder layer in the hESC medium and 10 pg / mL bFGF. After being dissociated and detached from the feeder, the stem cell aggregates / clusters were cultured in suspension with hESC medium to initiate the differentiation (day 0 of the neural differentiation process). These hESC aggregates were then cultured in Neural Induction Medium (NIM) and attached toculture dishes as previously described (Boisvert et al. (2013) J. Vis. Exp. (74):50321). At approximately day 15 of differentiation, the neuroepithelial clusters were isolated and cultured in suspension culture. The neurospheres were cultured in suspension for 3-4 weeks and were then plated onto polyornithine-coated coverslips for terminal differentiation. At about 2 days after plating (total 6 weeks), the cortical PNs were fixed to examine early axonal outgrowth and extension. With further culture, the long-term cultured cortical PNs (over 10 weeks) were used to analyze axonal degeneration. Cells from three independent coverslips in each group were measured and compared.

[0097] Immunostaining and Image Acquisition. Immunostaining analyses were performed as previously described (Denton et al. (2014) Stem Cells 32:414-423). Coverslips were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (PFA). Primary antibodies used in this study included anti-Ctip2 (Abeam, Rat IgG), anti-Tau (Sigma, rabbit IgG), anti-LAMPl (Abeam, Rabbit IgG). After incubation with primary antibody at 4°C overnight, cells were subsequently washed and incubated with corresponding fluorescence- conjugated secondary antibodies. With cells stained with Hoechst (Sigma) as indicator, coverslips were mounted on glass slides. Triplicate coverslips were used for each treatment or cell type and at least three blindly-selected images per coverslip were acquired and analyzed (Chen et al. (2022) Brain 145(11):4016-4031).

[0098] Colocalization Analysis of Cholesterol and Lysosome Staining . Similar to immunostaining, coverslips were fixed in 4% PFA for 20 minutes at room temperature and subsequently treated with 0.05 mg / mL filipin in PBS with 10% FBS for 2 hours at room temperature. Cells were incubated in primary antibody, anti-LAMP-1, with 5% BSA in PBS overnight at 4 °C.Then cells were processed for secondary antibody incubation in 5% BSA for 45 minutes at room temperature and mounted on microscope slides. Triplicate coverslips were used to acquire images by using 60x objective of the fluorescence microscope. The colocalization images were analyzed with Imaged software. The LAMPl-positive lysosomal area (first) and the soma of the cell (second area) were selected and added to ROI manager. Total fluorescent intensity of the first and the second selection area under the blue channel (cholesterol stained) were quantified, which represent the relative cholesterol level in lysosome and soma separately. The ratio between two values is the proportion of cholesterol accumulation in lysosome (Boutry et al. (2019) Commun. Biol. 2:380). The proportion of cholesterol accumulated in lysosome was calculated by % = (filipin intensity in selected area of LAMP1+) / (filipin intensity in whole cell) x 100 %.

[0099] Plasma Membrane Labeling and Analysis of PM Cholesterol Levels. Plasma was stained with Deep Red CellMask™ plasma membrane stain (Invitrogen, 1:1000) for 10 minutes at 37°C. Cells were subsequently washed, fixed and stained with Filipin III using cell-based cholesterol detection kit following the manufacture's protocol (Cayman Chemical) . Images were then taken using a Keyence microscope. To analyze membrane cholesterol levels, plasma membrane was selected in ImageJ and then Filipin levels were measured by overlapping with plasma membrane area.

[0100] Neurite Outgrowth Analysis. After dissociating and plating the neurospheres on coverslips for 2 days, cells were fixed and subjected to neurite outgrowth analysis (Denton et al. (2016) Methods Mol. Biol. 1353:309-321; Zhu et al. (2014) Hum. Mol. Genet. 23:5638-5648). The longest process, which also had the greatest Tau intensity, was measured using the "Simple Neurite Tracer" plugin in ImageJ software. Inparticular, the neurons were stained not only with anti-Tau but also anti-Ctip2 antibody as a cortical marker. Triplicate coverslips were used for imaging and analyzing.

[0101] Axonal Swellings Analysis . To analyze axonal swellings, cortical neuron cultures at approximately 3 months were stained with the axonal marker, Tau. Axonal swellings are defined as a diameter more than 2 times of the contiguous axons (Cheng et al. (2013) J. Pain 14:941-947; Lauria et al. (2003) Neurology 61:631-636). The swelling density was calculated by swelling number over axonal length in the selected area by using Imaged software as described previously (Mou et al. (2020) Acta Neuropathol. Commun. 8:214).

[0102] Gene Expression Analysis. Total RNA was isolated and 1 pg of RNA was taken for reverse transcription using iScriptcDNA Synthesis Kit (BioRad). For quantitative PCR (qPCR), reactions were performed in a 20 pL mixture containing cDNA, primers, and SYBR® Green Master Mix in the QuantStudio 6 Flex Real-Time PCR System per the manufacturer' s instructions (Applied Biosystems). Gene expression levels were compared with the housekeeping gene GAPDH, and calculated using the comparative CT method. Primers used for qPCR include SPG11, 5'-CTCCTAGTGTCCTGCCATCTGA-3' (SEQ ID NO:3), 5'-GGGCTAGAGAAATGTGGGAGATG-3' (SEQ ID NO:4); GAPDH, 5'-ATGACATCAAGAAGGTGGTG-3' (SEQ ID NO:5), 5'- CATACCAGGAAATGAGCTTG-3' (SEQ ID NO:6).

[0103] Caspase 3 / 1 Activity Assay. Caspase 3 and 7 activities are highly related to apoptosis levels in cells and were examined using Caspase-Gio® 3 / 7 Assay kit (Promega) following the manufacturer's instructions. Briefly, neurons were dissociated with Accutase®, a mild enzyme that shows increased survival for neural cells (Wachs et al. (2003) Lab. Invest. 83:949-962), into single cell suspension. Cells werethen seeded onto 96-well plates with the density of 5000 cells / well in 50 pL, lysed and incubated with illuminance substrate by adding 50 pL of caspase-3 / 7 reagents following the manufacture's protocol (Promega). After incubation for 45 minutes at room temperature, luminescence from each well was measured using a Gen5 microplate reader (BioTek).

[0104] Live Cell Imaging with MitoTracker® Dye. To examine mitochondrial transport, neurons (about 10-week) were stained with 50 nM MitoTracker® Red for 4 minutes and then the coverslips were washed with image medium. The coverslips were picked up and placed upside down in 35 mm glass bottom dishes with pre-added 200 pL image medium. Live-cell imaging was performed with an Olympus 1X83 microscope equipped with an incubation chamber. The cells were kept at 37°C with 5% CO2 while imaging. Images were taken every 5 seconds for 5 minutes. The mitochondrial motile percentage was analyzed by ImageJ software with MultiKymograph plugin and Bio-formats Package, as described previously (Mou et al. (2020) J. Vis. Exp. (156):10.3791 / 60548).

[0105] Generation of SPG11 Mutation Knock-in hESC Lines Using CRISPR-Cas9-Mediated Gene Editing. To further examine the role of SPG11 disease mutation, isogenic H9 hESC lines with the disease-specific SPG11 mutation were established (Chen et al. (2022) Brain 145(11):4016-4031). Briefly, using CRISPR-Cas9-mediated gene editing, a C.118O T (p. Gln40X) homozygous mutation was knocked-into H9 hESC lines by the Cell and Genome Engineering Core at the University of Connecticut Health Center. H9 hESCs serve as isogenic controls. Both H9 control and H9-Gln40X were then differentiated into cortical PNs as described in the above section for the examination of the effects caused by this disease-specific mutation.

[0106] Statistical Analysis. The Shapiro-Wilk test, skewness and kurtosis were used to assess whether the data were normally or approximately normally distributed (Kim (2013) Restor. Dent. Ended. 38:52-54). A two-tailed Student t-test was used to compare mean values between two groups, after confirming that the data were normally or approximately normally distributed. When comparing mean values among three or more groups, a one-way ANOVA was conducted to assess group- wise differences for normally or approximately normally distributed data; alternatively, a Kruskal-Wallis H test (also known as one-way ANOVA on ranks) was used for data that were not normally or approximately normally distributed. Post hoc Dunnett tests were performed to further compare the differences between the treatment groups to the control group. The statistical significance level was set at p < 0.05 and SAS 9.4 (SAS Institute Inc, Cary, NC) was used for statistical analysis.Example 2: Generation and Differentiation of SPG11 Knockdown hESC Lines

[0107] To examine the effects of loss of SPG11 function on human neurons, SPG11 knockdown hESC lines were established by combining lentiviruses and RNA interference (RNAi). pLKO.l vectors were adopted to express small hairpin RNA (shRNA) sequences that target SPG11 (SPG11 shRNA) and luciferase (Luc shRNA, as controls). Two different SPG11 RNAi sequences, SPG11 RNAi-A (SPG11-A) and SPG11 RNAi-B (SPG11-B) were used to minimize off-target effects. The constructed vectors were first transfected into HEK293T cells (transient transfection) to check the knockdown efficacy. After confirming the knockdown efficiency of SPG11 expression by both shRNAs, the lentiviruses containing SPG11 and Luciferase shRNA were produced and collected from HEK293T cells, and subsequentlyused to infect hESCs to generate RNAi hESC lines. After drug selection and passages, clonal hESC lines with uniform GFP expression indicative of shRNA expression were generated. The hESCs were expanded by those GFP positive clusters and differentiated into neurons. It was confirmed that hESCs were differentiated into cortical projection neurons by checking the expression of Ctip2, a marker for subcerebral cortical PNs (Arlotta et al. (2005) Nennon 45:207-221; Li et al. (2009) Development 136:4055-4063). During the differentiation process, knockdown efficacy in every stage was monitored by quantifying the mRNA expression levels. The data showed that both SPGll-shRNA knockdown groups maintained a significant reduction in SPG11 mRNA transcripts through the differentiation process, revealing the successful establishment of an hESC-based model of SPG11.Example 3: Characterization of SPG11 Axonal Defects in hESC- Derived Neurons

[0108] To observe the effects of SPG11 on axonal development, hESCs were differentiated into cortical projection neurons using established methods (Boisvert et al. (2013) J. Vis. Exp. (74):50321; Li et al. (2009) Development 136:4055-4063). Axons are generally derived by the longest neurite, which is involved in the formation of the complex neuronal architecture during development (Tosney & Landmesser (1985) Dev. Biol. 109:193-214). Axonal development was examined and axonal length of Ctip2+cortical PNs was measured. The data showed that the average length of Tau+axons was significantly decreased in SPG11 knockdown neurons (e.g., an average length of approximately 75-80 pm) compared to that in Ctip2+cortical PNs derived from Luc control hESCs (e.g., an average length of approximately 105 pm)(p < 0.01 versus luciferase group). Similar reductions of axonal length were observed inknockdown neurons in both shRNA-A and shRNA-B groups, suggesting that neurite outgrowth in neurons is sensitive to spatacsin levels.

[0109] Next, the density of axonal swellings, a marker for degenerated axons, was compared. Accumulation of axonal swellings is a characteristic pathological change observed in HSP, which is formed by the accumulation of transported cargos caused by impaired transport (Carpenter (1968) Neurology 18:841-851). To determine whether the knockdown of SPG11 in neurons leads to axonal phenotypes, Tau immunostaining was performed to visualize axonal swellings. The number of axonal swellings was guantified and divided by axonal length according to established methods (Denton et al. (2014) Stem Cells 32:414-423; Mou et al. (2020) Acta Neuropathol. Commun. 8:214). Under control conditions, neurons mostly exhibited long smooth neurites. In SPG11- knockdown neural cultures, accumulated axonal swellings were observed. The quantification results revealed a significant increase in the number of axonal swellings in neurons from both SPG11-A and SPG11-B groups (approximately a 3-fold change), as compared to control neurons (p < 0.01 versus luciferase group). These data indicate that loss of spatacsin function leads to impaired axonal transport and accumulated axonal swellings in human neurons.Example 4: Loss of Spatacsin Results in Abnormal Cholesterol Distribution in Neurons.

[0110] Given that spatacsin is important for maintaining normal lysosomal tubule structure and cholesterol trafficking (Boutry et al. (2019) Commun. Biol. 2:380), it was posited that loss of spatacsin in human neurons results in cholesterol accumulation in lysosomes, disrupting cholesterol distribution to plasma membrane. To test this, thecholesterol distribution in neurons was analyzed using Filipin III (Vienken et al. (2017) Sai. Rep. 7:43575), a dye that stains for free cholesterol in Luc- and SPGll-shRNA hESC- derived neurons. Clear membrane Filipin staining was observed in control neurons, which was disrupted in SPGll-deficient neurons. To confirm the aberrant cholesterol in the plasma membrane of SPGll-knocodwn neurons, Deep Red CellMask™ was used to label plasma membrane. This analysis showed a significant reduction of cholesterol in the plasma membrane of SPG11 shRNA-A and shRNA-B neurons. These data indicate that the loss of spatacsin leads to an alteration of cholesterol distribution in SPG11 neurons.

[0111] To further examine the localization of cholesterol, cholesterol was labelled with the main SPGll-targeted organelle, lysosomes, for cholesterol accumulation quantification (Varga et al. (2015) PLoS Genet. 11:61005454). After the double staining of filipin and LAMP1, the colocalization analysis was performed to quantify the cholesterol in lysosome. The proportion of cholesterol accumulated in lysosome was compared between SPG11 knockdown and control neurons. The data showed that the proportion of cholesterol in lysosome was significantly increased in SPG11 neurons compared to control neurons (approximately 12-13% vs. approximately 8%; p < 0.01 versus DMSO group by Dunnett's test after ANOVA). Together with the filipin analysis, these data indicate that spatacsin deficiency impairs cholesterol homeostasis and leads to the accumulation of cholesterol in lysosomes and reduction of cholesterol in plasma membranes.Example 5: RGX104 Reduces Lysosomal Cholesterol Accumulation in SPG11 Cortical Neurons

[0112] Given that the liver X receptors (LXRs) can regulate cholesterol homeostasis, which has been shown to enhance theefflux of free form cholesterol from the late endosomes / lysosomes (Rigamonti et al. (2005) Circ. Res. 97:682-689), it is possible that LXR agonists may rescue axonal defects through regulating cholesterol trafficking. Accordingly, the effects of LXR agonists on cholesterol trafficking in SPG11 neurons was analyzed. Given that SPG11 is critical for lysosomal function and cholesterol can accumulate in lysosome in SPG11 knockdown neurons, the effect of LXR agonists on cholesterol distribution in lysosome was examined after applying 1 pM GW3965 or RGX104, every 2 days for 1 week. Interestingly, the increased proportion of cholesterol in the lysosome was significantly mitigated by the application of LXR agonists in SPGll-kncokdown neurons. Both GW3965 and RGX104 significantly reduced the accumulated cholesterol in neurons derived from both SPGll-shRNA-A and SPGll-shRNA-B groups (FIG. 1). Furthermore, in control luciferase-knockdown neurons, treatment of GW 3965 and RGX104 did not significantly alter the lysosomal cholesterol contents in these normal neurons. Together, these data confirm that LXR activators can restore cholesterol homeostasis .Example 6: RGX104 Rescues Axonal Outgrowth and Transport Defects in SPG11 Knockdown Neurons

[0113] Given that LXR agonist can restore normal cholesterol trafficking and distribution, it was determined whether LXR agonists have any protective effects against axonal defects of SPG11 knockdown neurons. It was demonstrated herein that the absence of spatacsin impaired the neurite outgrowth. To evaluate the effects of synthetic LXRs agonists in SPG11 neurons, the SPG11 neurons were treated with cholesterol- targeting drugs (GW3965 or RGX104 at 1 pM final concentration) (Collins et al. (2002) J. Med. Chem. 45:1963-1966; Tavazoieet al. (2018) Cell 172:825-840 e818) and vehicle control (DMSO), and were cultured for 2 days. By measuring the average length of Tau+axons, both GW3965 and RGX104 significantly increased the neurite length of SPG11 neurons to a level similar to control neurons (FIG. 2). In addition, there was no significant difference in neurite length between normal neurons treated with and without those drugs (FIG. 2), indicating that these drugs did not exert a distinct effect on healthy neurite outgrowth during neuronal development. Taken together, these data revealed that LXR agonists can ameliorate the neurite outgrowth abnormalities observed in hESC-derived SPG11 neurons.

[0114] Considering that aberrant transport along axons is a characteristic change in SPG11, the effects of LXR agonists on axonal transport were investigated. Increased axonal swellings in SPG11 knockdown neurons indicates that loss of spatacsin impairs axonal transport. To confirm this, axonal transport of mitochondria was examined to monitor fast axonal transport in live neuron cultures (Hollenbeck & Saxton (2005) J. Cell Sci. 118:5411-5419; Mandal & Drerup (2019) Front. Cell Neurosci. 13:373). Cortical PNs derived from Luc-, SPGll-shRNA-A, and SPGll-shRNA-B hESCs were stained with MitoTracker® dye followed by washing and live cell imaging. The kymographs of mitochondria were analyzed, which can track the trajectories of each mitochondrion within the selected axons segment. The result showed that there was a significant reduction in the percentage of motile mitochondria in SPG11 neurons; over 30% of the mitochondria were motile in control (luciferase knockdown) neurons, whereas in SPG11 neurons, only around 20% of the mitochondria were motile. Thus, these data indicated that loss of spatacsin reduced axonal transport of mitochondria and decreased the proportion of motile mitochondria. After treatment with 1 pM GW3965 orRGX104 for 1 week, the mitochondrial motility of SPG11 neurons was measured. There were significant increases in the percentage of motile mitochondria in both GW3965- and RGX104- treated groups compared with vehicle-treated SPGll-shRNA-A groups. A similar increase of motile mitochondria by GW3965 and RGX104 was observed in SPGll-shRNA-B groups. Taken together, both GW3965 and RGX104 can significantly raise the percentage of motile mitochondria, revealing that the disruption of axonal transport can be rescued by LXR agonists.Example 7: RGX104 Mitigates Axonal Swellings and Apoptosis in SPG11 Long-Term Cultures

[0115] The protective effects of LXR agonists on axonal swellings levels was also examined in long-term cultures. SPG11 knockdown and control neurons were treated with 1 pM RGX104 and GW3965 for 1 week. Interestingly, both RGX104 and GW3965 treatment significantly reduced the number of swellings in SPG11 knockdown neurons (FIG. 3). The increased axonal swellings in both shRNA-A and shRNA-B groups can be effectively inhibited by the treatment of RGX104 or GW3965. These data demonstrate that relieving the disruption of axonal transport by the treatment of LXR agonists can mitigate accumulated axonal swellings in SPG11 neurons.

[0116] To study the protection effect and toxic potency of those drugs in SPG11 neurons, apoptosis levels were determined. Given that both caspases 3 and 7 are known as executioner caspases and are activated universally during apoptosis process (Walsh et al. (2008) Proc. Natl. Acad. Sci. USA 105:12815-12819), caspase 3 / 7 activities were compared between control and SPG11 neurons. The results showed that caspase 3 / 7 activity in SPG11 neurons was significantly higher than control neurons (FIG. 4). To evaluate the effects of GW3965 and RGX104, the apoptosis level of neurons wasdetermined to check the mortality rate with treatments. Apoptosis levels in SPG11 neurons were significantly reduced after GW3965 treatment (FIG. 5). Similarly, Caspase 3 / 7 levels were much lower in RGX104-treated group compared to that in the Vehicle (DMSO)-treated group, confirming the ability of LXR agonists to attenuate caspase 3 / 7 activity in SPG11 neurons. Taken together, these data revealed that both LXR agonists, GW3965 and RGX104, are able to mitigate axonal defects and partially rescue the mortality rate of SPG11 cortical neurons.Example 8: SPG11 Disease-Causing Mutation Results in Aberrant Cholesterol Accumulation and Degeneration in Human Cortical Neurons

[0117] SPG11 is a common dominant recessive form of HSP, and can be caused by compound or homozygous mutations of the SPG11 gene (Southgate et al. (2010) Neurogenetics 11:379-389; Stevanin et al. (2008) Brain 131:772-784). To confirm that SPG11 disease-specific mutation can directly impair cholesterol trafficking in SPG11 patients, the diseasecausing mutation (c. 118C>T, p.Gln40X) was knocked into H9 hESCs using CRSPR-Cas9-mediated gene editing. After drug selection, gene sequence analysis confirmed the successful generation of H9 SPG11 Gln40X homozygous lines. These H9 SPG11 Gln40X lines, as well as normal H9 (isogenic control), were efficiently differentiated into CTIP2+ cortical PNs. The expression of SPG11 mRNA was significantly reduced upon differentiation at both neural progenitors and neuron stages, confirming that loss of Spatacsin function is implicated in SPG11. Notably, in SPG11 Gln40X neurons, the localization of cholesterol in lysosomes, as indicated by the co-localization of Filipin (stained for total Cholesterol) and LAMP1 (marker for lysosomes), was significantly increased. These dataconfirm the involvement of aberrant cholesterol accumulation in lysosome in SPG11, and indicated that impaired cholesterol homeostasis underlies the degeneration of these SPG11 neurons.

[0118] To further demonstrate this, LXR agonists were applied to SPG11 Gln40X neurons, and it was examined whether their degeneration could be rescued by restoring cholesterol homeostasis. SPG11 Gln40X neurons were treated with GW3965 (1 pM), RGX104 (1 pM), or DMSO (Vehicle control). At 1-week after treatment, the localization of cholesterol in lysosome was compared between different groups. The data revealed that the percentage of Filipin staining co-localized with LAMP1 was significantly reduced after GW3965 or RGX treatment. To further determine the effects of LXR agonists on cholesterol distribution, cholesterol levels in the plasma membrane were examined in H9 and SPG11 Gln40X neurons by performing Deep Red CellMask™ plasma membrane and Filipin staining. Filipin intensity overlapped with Deep Red CellMask™ plasma membrane stain as determined using Image J. Interestingly, the plasma membrane cholesterol levels were significantly reduced in SPG11 Gln40X neurons, and this reduction was mitigated in SPG11 neurons after GW3965 and RGX104 treatment (FIG. 6). These data indicated that GW3965 and RGX104 treatment mitigated the aberrant cholesterol accumulation in lysosome and restored cholesterol homeostatic in SPGll-mutated neurons.

[0119] To identify the role of LXR agonists on axonal degeneration, the accumulation of axonal swelling in H9 and SPG11 40X cortical neurons was compared after treatment with GW3965, RGX104, and DMSO (Vehicle control). There was a significant increase in the axonal swelling density in cortical neurons derived from SPG11 40X iPSCs compared to normal H9 cortical neurons (FIG. 7). After treatment withGW3965 and RGX104, the accumulated axonal swellings were significantly reduced (FIG. 7), confirming that LXR agonist can ameliorate axonal degeneration in long-term neurons. Furthermore, the apoptosis of these neurons, as indicated by Caspase 3 / 7 levels, was also significantly inhibited by GW3965 or RGX treatment (FIG. 8). Taken together, the data reveal that aberrant cholesterol accumulation underlies the degeneration of SPG11 neurons; and LXR agonists can rescue the degeneration of SPG11 neurons through regulating cholesterol homeostasis.Example 9: Non-Lipogenic ABCA1 Inducer

[0120] As demonstrated herein, the LXR agonist, RGX-104, exhibits protective effects by mitigating axonal degeneration via regulating cholesterol trafficking in SPGll-mutated neurons. To determine whether non-lipogenic genes may play a role, an additional screening assay was conducted, and it was observed that non-lipogenic ABCA1 inducers can also affect HSP neurons.

[0121] A non-lipogenic ABCA1 inducers, i.e., CL3-3, was tested using SPGll-mutated neurons. Cortical neurons were generated from SPG11 disease-mutated human pluripotent stem cells as described herein. These neurons were then treated with different doses of the compound and Vehicle (i.e., DMSO) for one week. Normal neurons derived from normal human pluripotent stem cells were used as controls. To examine the effects of the compound on neurodegeneration, disease-related apoptosis was compared by measuring Caspase 3 / 7 activities using the Caspase-Gio® 3 / 7 assay (Promega). The results of this analysis indicated a significant increase in Caspase 3 / 7 activity in SPG11 mutated neurons compared to normal control neurons, revealing the disease-related increase of apoptosis in SPG11-mutated neurons. Interestingly, after treatment withABCA1 inducer, the increased apoptosis in SPG11 neurons was significantly mitigated by CL3-3 (1 pM and 3 pM), indicating the use of this compound, or similar compounds having the structure of any one of Formulae (I)-(XI), in reducing the disease-related degeneration.

Claims

What is claimed is:

1. A method of treating or ameliorating a motor neuron disease or condition associated with a spastic paraplegia type 11 (SPG11) pathogenic variant comprising administering to a subject in need thereof a Liver X Receptor (LXR) agonist thereby treating or ameliorating the subject's disease or condition associated with the SPG11 pathogenic variant.

2. The method of claim 1, wherein the motor neuron disease or condition associated with a SPG11 pathogenic variant is a hereditary spastic paraplegia or amyotrophic lateral sclerosis .

3. The method of claim 1, wherein the LXR agonist is RGX-104 (2-[3-[(3R)-3-[[2-chloro-3-(trifluoromethyl)phenyl]methyl-(2,2-diphenylethyl)amino] butoxy]phenyl] acetic acid).

4. The method of claim 1, wherein the LXR agonist is a compound having the structure of Formula (I), or a pharmaceutically acceptable salt or prodrug thereof:wherein,X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, and Xu are each independently selected from the group of carbon (C), nitrogen (N), sulfur (S), oxygen (0), NH, or CYi; each occurrence of Yi is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, halo, alkoxy, ketone, ester, carboxamide, sulfide, sulfoxide, sulfone, sulfonamide, a 5- or 6-membered monocyclic heteroaryl, and a 5- or 6-membered fused-ring;Z1, Z2, Z3, Z4, Z6, and Zs are each independently selected from the group of hydrogen, halo, 0, S, N, NH, C(=0), CH2, hydroxy, or CY2;Z5 is S;Z7is N or C, and each occurrence of Y2is independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, hydroxyalkyl, hydroxy, cyano, and halo, with the proviso that either Z1 is not hydrogen, or Z8 is alkyl when Z7 is N.

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