The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that becomes activated at the lysosome in response to nutrient cues. Here we identify cholesterol, an essential building block for cellular growth, as a nutrient input that drives mTORC1 recruitment and activation at the lysosomal surface. The lysosomal transmembrane protein, SLC38A9, is required for mTORC1 activation by cholesterol through conserved cholesterol-responsive motifs. Moreover, SLC38A9 enables mTORC1 activation by cholesterol independently from its arginine sensing function. Conversely, the Niemann-Pick C1 (NPC1) protein, which regulates cholesterol export from the lysosome, binds to SLC38A9 and inhibits mTORC1 signaling through its sterol transport function. Thus, lysosomal cholesterol drives mTORC1 activation and growth signaling through the SLC38A9-NPC1 complex.

Background Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterized by progressive neurodegeneration. In preclinical testing 2-hydroxypropyl-β-cyclodextrins (HPβCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological signs, and increased lifespan in murine and feline models of NPC1. Methods Safety and clinical efficacy of intrathecal HPβCD were evaluated in an open-label, dose- escalation phase 1/2a study. Intrathecal doses ranging from 50–1200 mg were evaluated in 14 neurologically affected NPC1 participants treated monthly for 12 to 18 months. Three additional participants were treated every two weeks for 18 months. Serum and CSF 24(S)- hydroxycholesterol, which served as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NSS) were used to compare disease progression in HPβCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. Findings No drug-related serious adverse events were observed. Mid- to high-frequency hearing loss, an expected adverse event, was documented. When managed with hearing aids, this did not have an appreciable impact on daily communication. Biomarker studies were consistent with improved neuronal cholesterol homeostasis and decreased neuronal pathology. The NSS score for the 14 participants treated monthly increased at a rate of 122 ± 0 34 points/year compared to 2 92 ± 0 27 points/year (p=0 0002) for the comparison group. Decreased progression was observed for NSS domains of ambulation (p=0 0622), cognition (p=0 0040) and speech (p=0 0423). Interpretation This phase 1/2a study of intrathecal HPβCD for the treatment of NPC1 demonstrated an acceptable safety profile and slowing of disease progression.

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes via the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signaling are unknown. We show that Oxysterol Binding Protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA/B, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, mTORC1 recruitment by the Rag GTPases is inhibited due to impaired cholesterol transport to lysosomes. Conversely, OSBPmediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signaling and restores autophagic function in cellular models of NPC. Thus, ER-lysosome contacts are signaling hubs that enable cholesterol sensing by mTORC1, and targeting their sterol-transfer activity could be beneficial in NPC.The exchange of contents and signals between organelles is key to the execution of cellular programs for growth and homeostasis, and failure of this communication can drive disease. A form of organelle communication involves exchange of cholesterol and other lipids by Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Supplementary key words cholesterol • diagnostic tools • liquid chromatography/tandem mass spectrometry • Niemann-Pick disease • oxysterols • neurodegenerationNiemann-Pick type C (NPC) disease is a rare, primarily pediatric disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system. Approximately 95% of NPC cases are caused by mutations of the NPC1 gene, whereas the remaining 5% are caused by mutations in the NPC2 gene ( 1, 2 ). Affected individuals typically present in early childhood with ataxia and progressive impairment of motor and intellectual function and usually die in adolescence. There are currently no FDA-approved therapies for this progressively fatal neurodegenerative disorder. However, a recent controlled study and a series of case reports suggests effi cacy for miglustat ( 3 ), an inhibitor of glycosphingolipid biosynthesis that is now licensed for use as a disease modifying therapy in multiple countries, including the European Union, Russia, Brazil, Australia, Canada, and Taiwan.Abstract Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifi cally cholestane-3 ␤ ,5 ␣ ,6 ␤ -triol (3 ␤ ,5 ␣ ,6 ␤ -triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specifi c LC-MS/ MS method for quantifying 3 ␤ ,5 ␣ ,6 ␤ -triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3 ␤ ,5 ␣ ,6 ␤ -triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantifi cation of 3 ␤ ,5 ␣ ,6 ␤ -triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specifi city between control and NPC1 subjects, and offers for the fi rst time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1

Globoid cell leukodystrophy (GLD, Krabbe disease) is a lysosomal storage disease (LSD) caused by a deficiency in galactocerebrosidase (GALC) activity. In the absence of GALC activity, the cytotoxic lipid, galactosylsphingosine (psychosine), accumulates in the CNS and peripheral nervous system. Oligodendrocytes and Schwann cells are particularly sensitive to psychosine, thus leading to a demyelinating phenotype. Although hematopoietic stem-cell transplantation provides modest benefit in both presymptomatic children and the murine model (Twitcher), there is no cure for GLD. In addition, GLD has been relatively refractory to virtually every experimental therapy attempted. Here, Twitcher mice were simultaneously treated with CNS-directed gene therapy, substrate reduction therapy, and bone marrow transplantation to target the primary pathogenic mechanism (GALC deficiency) and two secondary consequences of GALC deficiency (psychosine accumulation and neuroinflammation). Simultaneously treating multiple pathogenic targets resulted in an unprecedented increase in life span with improved motor function, persistent GALC expression, nearly normal psychosine levels, and decreased neuroinflammation. Treating the primary pathogenic mechanism and secondary targets will likely improve therapeutic efficacy for other LSDs with complex pathological and clinical presentations.