The Association Between C24:0/C16:0 Ceramide Ratio and Cardiorespiratory Fitness is Robust to Effect Modifications by Age and Sex.

Ceramides and cardiorespiratory (CR) fitness are both related to cardiovascular diseases. The associations of three blood plasma ceramides (C16:0, C22:0, and C24:0) with CR fitness in the population-based Study of Health in Pomerania (SHIP-START-1; n = 1,102; mean age 50.3 years, 51.5% women) are investigated. In addition, subgroup analysis according to age (

A Comparative Biochemical and Pathological Evaluation of Brain Samples from Knock-In Murine Models of Gaucher Disease.

Gaucher disease (GD) is a lysosomal storage disorder stemming from biallelic mutations in GBA1, characterized by glucocerebrosidase dysfunction and glucocerebroside and glucosylsphingosine accumulation. Since phenotypes of murine models of GD often differ from those in patients, the careful characterization of Gba1 mutant mice is necessary to establish their ability to model GD. We performed side-by-side comparative biochemical and pathologic analyses of four murine Gba1 models with genotypes L444P/L444P (p.L483P/p.L483P), L444P/null, D409H/D409H (p.D448H/p.D448H) and D409H/null, along with matched wildtype mice, all with the same genetic background and cage conditions. All mutant mice exhibited significantly lower glucocerebrosidase activity (p < 0.0001) and higher glucosylsphingosine levels than wildtype, with the lowest glucocerebrosidase and the highest glucosylsphingosine levels in mice carrying a null allele. Although glucocerebrosidase activity in L444P and D409H mice was similar, D409H mice showed more lipid accumulation. No Gaucher or storage-like cells were detected in any of the Gba1 mutant mice. Quantification of neuroinflammation, dopaminergic neuronal loss, alpha-synuclein levels and motor behavior revealed no significant findings, even in aged animals. Thus, while the models may have utility for testing the effect of different therapies on enzymatic activity, they did not recapitulate the pathological phenotype of patients with GD, and better models are needed.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

Prevalence of diarrheagenic Escherichia coli and impact on child health in Cap-Haitien, Haiti.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) are common pathogens infecting children during their growth and development. Determining the epidemiology and the impact of DEC on child anthropometric measures informs prioritization of prevention efforts. These relationships were evaluated in a novel setting, Cap-Haitien, Haiti. METHODS: We performed pre-specified secondary analysis of a case-control study of community-dwelling children, 6-36 months of age, enrolled 96 cases with diarrhea and 99 asymptomatic controls. Assessments were performed at enrollment and one month later at follow-up. Established endpoint PCR methodologies targeted DEC gDNA isolated from fecal swabs. The association between DEC and anthropometric z-scores at enrollment was determined using multivariate linear regression. Lastly, we assessed the association between specific biomarkers, choline and docosahexaenoic acid (DHA) and diarrheal burden. RESULTS: Enterotoxigenic Escherichia coli (ETEC) was identified in 21.9% of cases vs. 16.1% of controls with heat-stable producing ETEC significantly associated with symptomatic disease. Enteroaggregative E. coli (EAEC) was found in 30.2% of cases vs. 27.3% of controls, and typical enteropathogenic E. coli in 6.3% vs. 4.0% of cases and controls, respectively. Multivariate linear regression, controlled for case or control status, demonstrated ETEC and EAEC were significantly associated with reduced weight-age z-score (WAZ) and height-age z-score (HAZ) after adjusting for confounders. An interaction between ETEC and EAEC was observed. Choline and DHA were not associated with diarrheal burden. CONCLUSIONS: DEC are prevalent in north Haitian children. ETEC, EAEC, household environment, and diet are associated with unfavorable anthropometric measures, with possible synergistic interactions between ETEC and EAEC. Further studies with longer follow up may quantify the contribution of individual pathogens to adverse health outcomes.

Sphingomyelin 16:0 is a therapeutic target for neuronal death in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the SMPD1 gene encoding for the acid sphingomyelinase (ASM). While intravenous infusion of recombinant ASM is an effective treatment for the peripheral disease, the neurological complications of ASMD remain unaddressed. It has been shown that aberrantly high level of total brain sphingomyelin (SM) is a key pathological event leading to neurodegeneration. Using mice lacking ASM (ASMko), which mimic the disease, we here demonstrate that among the SM species, SM16:0 shows the highest accumulation and toxicity in ASMko neurons. By targeting lysosomes, SM16:0 causes permeabilization and exocytosis of these organelles and induces oxidative stress and cell death. We also show that genetic silencing of Ceramide Synthase 5, which is involved in SM16:0 synthesis and overexpressed in the ASMko brain, prevents disease phenotypes in ASMko cultured neurons and mice. The levels of SM16:0 in plasma also show a strong correlation with those in brain that is higher than in liver, even at early stages of the disease. These results identify SM16:0 both as a novel therapeutic target and potential biomarker of brain pathology in ASMD.

Glb1 knockout mouse model shares natural history with type II GM1 gangliosidosis patients.

GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of ß-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1-/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1-/- mice die six weeks earlier than male Glb1-/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1-/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1-/- mice as seen in some patients. In addition, Glb1-/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1-/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1-/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Choline, DHA, and Diarrheal Disease Associated with Growth Faltering in a Case-Control Study.

Background: Children with recurrent infectious diarrhea are susceptible to growth faltering. DHA and choline may play a role in this relationship due to their involvement in lipid metabolism, gut immunity, and inflammatory pathways. Objectives: This study aimed to characterize the contributions made by DHA and choline status and enteric damage in young children in the association between diarrheal illness and child growth. Methods: A longitudinal case-control study was conducted among children aged 6-36 mo (N = 195) in Cap-Haitien, Haiti. Mother-child dyads were recruited from community health posts and outpatient clinics. Cases were defined as children experiencing acute diarrhea within the last 3 d and matched to healthy controls. Child anthropometry, dietary intake, and blood and stool samples were collected at baseline and follow-up. Plasma DHA, choline, and betaine were determined by LC-MS/MS methods (n = 49) and intestinal fatty acid-binding protein (I-FABP) by ELISA (n = 183). Multivariate regression models were applied with mediation analyses to examine associations and adjust for confounding factors. Results: At baseline, mean plasma DHA concentrations (1.03 µg/mL; 95% CI: 0.91, 1.15) were not significantly different between cases and controls, nor was there a difference in mean plasma choline concentrations (4.5 µg/mL; 95% CI: 3.8, 5.1). Mean plasma I-FABP concentrations were significantly higher at follow-up in cases (3.34; 95% CI: 3.28, 3.40) than controls (3.20; 95% CI: 3.13, 3.27; P = 0.002). In adjusted multilinear regression models, higher plasma DHA concentrations at follow-up were associated with a negative change in weight-age z score (P = 0.016), and follow-up I-FABP was inversely associated with height-age z score (P = 0.035). No interaction or mediation effects were found. Conclusions: I-FABP concentrations were significantly higher in cases as compared with controls at follow-up, suggesting ongoing enteric damage and increased risk for malnutrition. Plasma DHA and I-FABP may have a role in childhood growth outcomes.

CRISPR-Cas9 Knock-In of T513M and G41S Mutations in the Murine ß-Galactosyl-Ceramidase Gene Re-capitulates Early-Onset and Adult-Onset Forms of Krabbe Disease.

Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme ß-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. The twitcher mouse, the most used animal model, has a nonsense mutation, which limits the study of how different mutations impact the processing and activity of GALC enzyme. To partially address this, we generated two new transgenic mouse models carrying point mutations frequently found in infantile and adult forms of KD. Using CRISPR-Cas9 gene editing, point mutations T513M (infantile) and G41S (adult) were introduced in the murine GALC gene and stable founders were generated. We show that GALC T513M/T513M mice are short lived, have the greatest decrease in GALC activity, have sharp increases of psychosine, and rapidly progress into a severe and lethal neurological phenotype. In contrast, GALC G41S/G41S mice have normal lifespan, modest decreases of GALC, and minimal psychosine accumulation, but develop adult mild inflammatory demyelination and slight declines in coordination, motor skills, and memory. These two novel transgenic lines offer the possibility to study the mechanisms by which two distinct GALC mutations affect the trafficking of mutated GALC and modify phenotypic manifestations in early- vs adult-onset KD.

AAV gene therapy for Tay-Sachs disease.

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 × 1014 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 × 1012 vg per thalamus) and i.t. infusion (3.9 × 1013 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.

A human iPSC-derived inducible neuronal model of Niemann-Pick disease, type C1.

BACKGROUND: Niemann-Pick disease, type C (NPC) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the genes NPC1 or NPC2 and characterized by impaired cholesterol homeostasis, a lipid essential for cellular function. Cellular cholesterol levels are tightly regulated, and mutations in either NPC1 or NPC2 lead to deficient transport and accumulation of unesterified cholesterol in the late endosome/lysosome compartment, and progressive neurodegeneration in affected individuals. Previous cell-based studies to understand the NPC cellular pathophysiology and screen for therapeutic agents have mainly used patient fibroblasts. However, these do not allow modeling the neurodegenerative aspect of NPC disease, highlighting the need for an in vitro system that permits understanding the cellular mechanisms underlying neuronal loss and identifying appropriate therapies. This study reports the development of a novel human iPSC-derived, inducible neuronal model of Niemann-Pick disease, type C1 (NPC1). RESULTS: We generated a null i3Neuron (inducible × integrated × isogenic) (NPC1-/- i3Neuron) iPSC-derived neuron model of NPC1. The NPC1-/- and the corresponding isogenic NPC1+/+ i3Neuron cell lines were used to efficiently generate homogenous, synchronized neurons that can be used in high-throughput screens. NPC1-/- i3Neurons recapitulate cardinal cellular NPC1 pathological features including perinuclear endolysosomal storage of unesterified cholesterol, accumulation of GM2 and GM3 gangliosides, mitochondrial dysfunction, and impaired axonal lysosomal transport. Cholesterol storage, mitochondrial dysfunction, and axonal trafficking defects can be ameliorated by treatment with 2-hydroxypropyl-ß-cyclodextrin, a drug that has shown efficacy in NPC1 preclinical models and in a phase 1/2a trial. CONCLUSION: Our data demonstrate the utility of this new cell line in high-throughput drug/chemical screens to identify potential therapeutic agents. The NPC1-/- i3Neuron line will also be a valuable tool for the NPC1 research community to explore the pathological mechanisms contributing to neuronal degeneration.

Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.

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.

Natural history of Tay-Sachs disease in sheep.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disease caused by a deficiency of the enzyme ß-N-acetylhexosaminidase A (HexA). TSD naturally occurs in Jacob sheep is the only experimental model of TSD. TSD in sheep recapitulates neurologic features similar to juvenile onset and late onset TSD patients. Due to the paucity of human literature on pathology of TSD, a better natural history in the sheep TSD brain, which is on the same order of magnitude as a child's, is necessary for evaluating therapy and characterizing the pathological events that occur. To provide clinicians and researchers with a clearer understanding of longitudinal pathology in patients, we compare spectrum of clinical signs and brain pathology in mildly symptomatic (3-months), moderately symptomatic (6-months), or severely affected TSD sheep (humane endpoint at ~9-months of age). Increased GM2 ganglioside in the CSF of TSD sheep and a TSD specific biomarker on MRS (taurine) correlate with disease severity. Microglial activation and reactive astrocytes were observed globally on histopathology in TSD sheep with a widespread reduction in oligodendrocyte density. Myelination is reduced primarily in the forebrain illustrated by loss of white matter on MRI. GM2 and GM3 ganglioside were increased and distributed differently in various tissues. The study of TSD in the sheep model provides a natural history to shed light on the pathophysiology of TSD, which is of utmost importance due to novel therapeutics being assessed in human patients.

Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann-Pick disease type C1 mice.

Niemann-Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1 m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1 m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1 m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.

Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.

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.

A phase 1/2 open label nonrandomized clinical trial of intravenous 2-hydroxypropyl-ß-cyclodextrin for acute liver disease in infants with Niemann-Pick C1.

INTRODUCTION: Niemann-Pick C (NPC) is an autosomal recessive disease due to defective NPC1 or NPC2 proteins resulting in endo-lysosomal storage of unesterified cholesterol in the central nervous system and liver. Acute liver disease in the newborn period may be self-limited or fatal. 2-hydroxypropyl-ß-cyclodextrin (2HPBCD) is a cholesterol-binding agent that reduces lysosomal cholesterol storage. We have enrolled 3 infants 0-6 months old with direct hyperbilirubinemia due to NPC1 or NPC2 liver disease in a Phase I/II open label clinical trial of intravenous 2HPBCD. METHODS: Infants received intravenous 2HPBCD twice a week for 6 weeks, followed by monthly infusion for 6-months. Primary outcome measure was reduction of plasma (3ß,5α,6ß-trihydroxy-cholan-24-oyl) glycine (TCG), a bile acid generated from cholesterol sequestered in lysosome. RESULTS: Three participants completed this protocol. A fourth patient received intravenous 2HPBCD under an emergency investigational new drug study but later expired from her underlying condition. The three protocol patients are living and have improved liver enzymes and TCG. No patient has experienced a drug-related adverse event. CONCLUSION: Intravenous 2HPBCD was tolerated in three infants with liver disease due to NPC.

Examination of a blood-brain barrier targeting ß-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase ß-galactosidase (ß-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing ß-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (ß-gal-/-). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult ß-gal-/- mice showed clearance of enzyme activity in the plasma and an increase in ß-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating ß-gal-/- mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated ß-gal-/- mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed ß-gal-/- mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in ß-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active ß-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to ß-gal-/- mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in ß-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).

4ß-Hydroxycholesterol is a prolipogenic factor that promotes SREBP1c expression and activity through the liver X receptor.

Oxysterols are oxidized derivatives of cholesterol that play regulatory roles in lipid biosynthesis and homeostasis. How oxysterol signaling coordinates different lipid classes such as sterols and triglycerides remains incompletely understood. Here, we show that 4ß-hydroxycholesterol (HC) (4ß-HC), a liver and serum abundant oxysterol of poorly defined functions, is a potent and selective inducer of the master lipogenic transcription factor, SREBP1c, but not the related steroidogenic transcription factor SREBP2. By correlating tracing of lipid synthesis with lipogenic gene expression profiling, we found that 4ß-HC acts as a putative agonist for the liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of the LXR, 4ß-HC induced expression of the lipogenic program downstream of SREBP1c and triggered de novo lipogenesis both in primary hepatocytes and in the mouse liver. In addition, 4ß-HC acted in parallel to insulin-PI3K-dependent signaling to stimulate triglyceride synthesis and lipid-droplet accumulation. Thus, 4ß-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis.

Enhanced Efficacy and Increased Long-Term Toxicity of CNS-Directed, AAV-Based Combination Therapy for Krabbe Disease.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.