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.
Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms ...that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.
Tay-Sachs disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Here, we describe an adeno-associated virus (AAV) gene therapy expanded-access trial in ...two patients with infantile TSD (IND 18225) with safety as the primary endpoint and no secondary endpoints. Patient TSD-001 was treated at 30 months with an equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB administered intrathecally (i.t.), with 75% of the total dose (1 × 10
vector genomes (vg)) in the cisterna magna and 25% at the thoracolumbar junction. Patient TSD-002 was treated at 7 months by combined bilateral thalamic (1.5 × 10
vg per thalamus) and i.t. infusion (3.9 × 10
vg). Both patients were immunosuppressed. Injection procedures were well tolerated, with no vector-related adverse events (AEs) to date. Cerebrospinal fluid (CSF) HexA activity increased from baseline and remained stable in both patients. TSD-002 showed disease stabilization by 3 months after injection with ongoing myelination, a temporary deviation from the natural history of infantile TSD, but disease progression was evident at 6 months after treatment. TSD-001 remains seizure-free at 5 years of age on the same anticonvulsant therapy as before therapy. TSD-002 developed anticonvulsant-responsive seizures at 2 years of age. This study provides early safety and proof-of-concept data in humans for treatment of patients with TSD by AAV gene therapy.
Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the ...accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing “psychosine hypothesis.” We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients.We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized L-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.
Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and ...lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.
Background
Recent studies suggest that circulating concentrations of specific ceramide species may be associated with coronary risk and mortality. We sought to determine the relations between the ...most abundant plasma ceramide species of differing acyl chain lengths and the risk of coronary heart disease (CHD) and mortality in community‐based samples.
Methods and Results
We developed a liquid chromatography/mass spectrometry assay to quantify plasma C24:0, C22:0, and C16:0 ceramides and ratios of these very–long‐chain/long‐chain ceramides in 2642 FHS (Framingham Heart Study) participants and in 3134 SHIP (Study of Health in Pomerania) participants. Over a mean follow‐up of 6 years in FHS, there were 88 CHD and 90 heart failure (HF) events and 239 deaths. Over a median follow‐up time in SHIP of 5.75 years for CHD and HF and 8.24 years for mortality, there were 209 CHD and 146 HF events and 377 deaths. In meta‐analysis of the 2 cohorts and adjusting for standard CHD risk factors, C24:0/C16:0 ceramide ratios were inversely associated with incident CHD (hazard ratio per average SD increment, 0.79; 95% confidence interval, 0.71–0.89; P<0.0001) and inversely associated with incident HF (hazard ratio, 0.78; 95% confidence interval, 0.61–1.00; P=0.046). Moreover, the C24:0/C16:0 and C22:0/C16:0 ceramide ratios were inversely associated with all‐cause mortality (C24:0/C16:0: hazard ratio, 0.60; 95% confidence interval, 0.56–0.65; P<0.0001; C22:0/C16:0: hazard ratio, 0.65; 95% confidence interval, 0.60–0.70; P<0.0001).
Conclusions
The ratio of C24:0/C16:0 ceramides in blood may be a valuable new biomarker of CHD risk, HF risk, and all‐cause mortality in the community.
Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing ...cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.
Lysosomal function is impaired in Niemann-Pick disease type C1 (NPC1), a rare and inherited neurodegenerative disorder, resulting in late endosomal/lysosomal accumulation of unesterified cholesterol. ...The precise pathogenic mechanism of NPC1 remains incompletely understood. In this study, we employed metabolomics to uncover secondary accumulated substances in NPC1. Our findings unveiled a substantial elevation in the levels of three alkyl-lysophosphatidylcholine alkyl-LPC, also known as lyso-platelet activating factor (PAF) species in NPC1 compared to controls across various tissues, including brain tissue from individuals with NPC1, liver, spleen, cerebrum, cerebellum, and brain stem from NPC1 mice, as well as in both brain and liver tissue from NPC1 cats. The three elevated alkyl-LPC species were as follows: LPC O-16:0, LPC O-18:1, and LPC O-18:0. However, the levels of PAF 16:0, PAF 18:1, and PAF 18:0 were not altered in NPC1. In the NPC1 feline model, the brain and liver alkyl-LPC levels were reduced following 2-hydroxypropyl-β-cyclodextrin (HPβCD) treatment, suggesting that alkyl-LPCs are secondary storage metabolites in NPC1 disease. Unexpectedly, cerebrospinal fluid (CSF) levels of LPC O-16:0 and LPC O-18:1 were decreased in individuals with NPC1 compared to age-appropriate comparison samples, and their levels were increased in 80% of participants 2 years after intrathecal HPβCD treatment. The fold increases in CSF LPC O-16:0 and LPC O-18:1 levels were more pronounced in responders compared to nonresponders. This study identified alkyl-LPC species as secondary storage metabolites in NPC1 and indicates that LPC O-16:0 and LPC O-18:1, in particular, could serve as potential biomarkers for tracking treatment response in NPC1 patients.
PDF
