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.

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.

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.

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).

Application of a glycinated bile acid biomarker for diagnosis and assessment of response to treatment in Niemann-pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3ß,5α,6ß-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3ß,5α,6ß-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPßCD treatment.

Application of N-palmitoyl-O-phosphocholineserine for diagnosis and assessment of response to treatment in Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder caused by mutations in either the NPC1 or the NPC2 gene. A new class of lipids, N-acyl-O-phosphocholineserines were recently identified as NPC biomarkers. The most abundant species in this class of lipid, N-palmitoyl-O-phosphocholineserine (PPCS), was evaluated for diagnosis of NPC disease and treatment efficacy assessment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) in NPC. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated to measure PPCS in human plasma and cerebrospinal fluid (CSF). A cutoff of 248 ng/mL in plasma provided a sensitivity of 100.0% and specificity of 96.6% in identifying NPC1 patients from control and NPC1 carrier subjects. PPCS was significantly elevated in CSF from NPC1 patients, and CSF PPCS levels were significantly correlated with NPC neurological disease severity scores. Plasma and CSF PPCS did not change significantly in response to intrathetical (IT) HPßCD treatment. In an intravenous (IV) HPßCD trial, plasma PPCS in all patients was significantly reduced. These results demonstrate that plasma PPCS was able to diagnose NPC1 patients with high sensitivity and specificity, and to evaluate the peripheral treatment efficacy of IV HPßCD treatment.

A novel gene editing system to treat both Tay-Sachs and Sandhoff diseases.

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme ß-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex ß subunit, causes Tay-Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay-Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n = 10, p < 0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p < 0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p < 0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results demonstrate the potential of developing a treatment of in vivo genome editing for Tay-Sachs and Sandhoff patients.

N-acyl-O-phosphocholineserines: structures of a novel class of lipids that are biomarkers for Niemann-Pick C1 disease.

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H]+ ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

2-Hydroxypropyl-ß-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B.

Deficiencies in the lysosomal hydrolase ß-galactosidase (ß-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse ß-gal encoding gene, Glb1, was targeted to generate both models of ß-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of ß-gal (ß-gal-/-). Glb1W274L mice showed a significant reduction in ß-gal enzyme activity (8.4-13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, ß-gal-/- mice were devoid of ß-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). ß-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of ß-galactosidase deficiency with residual enzyme activity has been developed.

Diagnosis of niemann-pick C1 by measurement of bile acid biomarkers in archived newborn dried blood spots.

BACKGROUND: Niemann-Pick disease type C1 (NPC1) is a rare, neurodegenerative cholesterol storage disorder. Diagnostic delay of >5 years is common due to the rarity of the disease and non-specific early symptoms. To improve diagnosis and facilitate early intervention, we previously developed a newborn screening assay based on newly identified plasma bile acid biomarkers. Because the newborn screen had been validated using dried blood spots (DBS) from already diagnosed NPC1 patients, an unanswered question was whether the screen would be able to detect individuals with NPC1 at birth. METHODS: To address this critical question, we obtained the newborn DBS for already diagnosed NPC1 subjects (n = 15) and carriers (n = 3) residing in California, New York, and Michigan states that archive residual DBS in biorepositories. For each of the DBS, we obtained two neighbor controls - DBS from patients born on the same day and in the same hospital as the NPC1 patients and carriers. 3ß,5α,6ß-trihydroxycholanic acid (bile acid A) and trihydroxycholanic acid glycine conjugate (bile acid B) were measured in the DBS using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. RESULTS: Bile acid B, the more specific biomarker for which the fully validated DBS assay was developed, was detected in 8/15 NPC1 patients, and elevated above the cut-off in 2/15 patients (the two samples with the shortest storage time). Bile acid B was detected in 2/2, 6/10, and 0/7 NPC1 samples that have been stored for <10.5 years, 13-20 years, and > 20 years, respectively, indicating that the glycine conjugate is detectable in DBS but may have reduced long-term stability compared with bile acid A, the precursor trihydroxycholanic acid, which was elevated in 15/15 NPC1 subjects, but not in carriers and controls. CONCLUSIONS: These results demonstrate that newborn screening for NPC1 disease is feasible using bile acid biomarkers.

Oxysterol Signatures Distinguish Age-Related Macular Degeneration from Physiologic Aging.

Macrophage aging is pathogenic in numerous diseases, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. Although prior studies have explored the functional consequences of macrophage aging, less is known about its cellular basis or what defines the transition from physiologic aging to disease. Here, we show that despite their frequent self-renewal, macrophages from old mice exhibited numerous signs of aging, such as impaired oxidative respiration. Transcriptomic profiling of aged murine macrophages revealed dysregulation of diverse cellular pathways, especially in cholesterol homeostasis, that manifested in altered oxysterol signatures. Although the levels of numerous oxysterols in human peripheral blood mononuclear cells and plasma exhibited age-associated changes, plasma 24-hydroxycholesterol levels were specifically associated with AMD. These novel findings demonstrate that oxysterol levels can discriminate disease from physiologic aging. Furthermore, modulation of cholesterol homeostasis may be a novel strategy for treating age-associated diseases in which macrophage aging is pathogenic.

Ceramide Remodeling and Risk of Cardiovascular Events and Mortality.

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.

Macrophage microRNA-150 promotes pathological angiogenesis as seen in age-related macular degeneration.

Macrophage aging is pathogenic in diseases of the elderly, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. However, the role of microRNAs, which modulate immune processes, in regulating macrophage dysfunction and thereby promoting age-associated diseases is underexplored. Here, we report that microRNA-150 (miR-150) coordinates transcriptomic changes in aged murine macrophages, especially those associated with aberrant lipid trafficking and metabolism in AMD pathogenesis. Molecular profiling confirmed that aged murine macrophages exhibit dysregulated ceramide and phospholipid profiles compared with young macrophages. Of translational relevance, upregulation of miR-150 in human peripheral blood mononuclear cells was also significantly associated with increased odds of AMD, even after controlling for age. Mechanistically, miR-150 directly targets stearoyl-CoA desaturase-2, which coordinates macrophage-mediated inflammation and pathologic angiogenesis, as seen in AMD, in a VEGF-independent manner. Together, our results implicate miR-150 as pathogenic in AMD and provide potentially novel molecular insights into diseases of aging.

A HILIC-MS/MS method for simultaneous quantification of the lysosomal disease markers galactosylsphingosine and glucosylsphingosine in mouse serum.

Deficiencies of galactosylceramidase and glucocerebrosidase result in the accumulation of galactosylsphingosine (GalSph) and glucosylsphingosine (GluSph) in Krabbe and Gaucher diseases, respectively. GalSph and GluSph are useful biomarkers for both diagnosis and monitoring of treatment effects. We have developed and validated a sensitive, accurate, high-throughput assay for simultaneous determination of the concentration of GalSph and GluSph in mouse serum. GalSph and GluSph and their deuterated internal standards were extracted by protein precipitation in quantitative recoveries, baseline separated by hydrophilic interaction chromatography and detected by positive-ion electrospray mass spectrometry in multiple reaction monitoring mode. Total run time was 7 min. The lower limit of quantification was 0.2 ng/mL for both GalSph and GluSph. Sample stability, assay precision and accuracy, and method robustness were demonstrated. This method has been successfully applied to measurement of these lipid biomarkers in a natural history study in twitcher (Krabbe) mice.

Eggs early in complementary feeding increase choline pathway biomarkers and DHA: a randomized controlled trial in Ecuador.

Background: Choline status has been associated with stunting among young children. Findings from this study showed that an egg intervention improved linear growth by a length-for-age z score of 0.63.Objective: We aimed to test the efficacy of eggs introduced early in complementary feeding on plasma concentrations of biomarkers in choline pathways, vitamins B-12 and A, and essential fatty acids.Design: A randomized controlled trial, the Lulun ("egg" in Kichwa) Project, was conducted in a rural indigenous population of Ecuador. Infants aged 6-9 mo were randomly assigned to treatment (1 egg/d for 6 mo; n = 80) and control (no intervention; n = 83) groups. Socioeconomic data, anthropometric measures, and blood samples were collected at baseline and endline. Household visits were made weekly for morbidity surveillance. We tested vitamin B-12 plasma concentrations by using chemiluminescent competitive immunoassay and plasma concentrations of choline, betaine, dimethylglycine, retinol, essential fatty acids, methionine, dimethylamine (DMA), trimethylamine, and trimethylamine-N-oxide (TMAO) with the use of liquid chromatography-tandem mass spectrometry.Results: Socioeconomic factors and biomarker concentrations were comparable at baseline. Of infants, 11.4% were vitamin B-12 deficient and 31.7% marginally deficient at baseline. In adjusted generalized linear regression modeling, the egg intervention increased plasma concentrations compared with control by the following effect sizes: choline, 0.35 (95% CI: 0.12, 0.57); betaine, 0.29 (95% CI: 0.01, 0.58); methionine, 0.31 (95% CI: 0.03, 0.60); docosahexaenoic acid, 0.43 (95% CI: 0.13, 0.73); DMA, 0.37 (95% CI: 0.37, 0.69); and TMAO, 0.33 (95% CI: 0.08, 0.58). No significant group differences were found for vitamin B-12, retinol, linoleic acid (LA), α-linolenic acid (ALA), or ratios of betaine to choline and LA to ALA.Conclusion: The findings supported our hypothesis that early introduction of eggs significantly improved choline and other markers in its methyl group metabolism pathway. This trial was registered at clinicaltrials.gov as NCT02446873.

Intrathecal 2-hydroxypropyl-ß-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1-2 trial.

BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterised by progressive neurodegeneration. In preclinical testing, 2-hydroxypropyl-ß-cyclodextrins (HPßCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological manifestations, and increased lifespan in mouse and cat models of NPC1. The aim of this study was to assess the safety and efficacy of lumbar intrathecal HPßCD. METHODS: In this open-label, dose-escalation phase 1-2a study, we gave monthly intrathecal HPßCD to participants with NPC1 with neurological manifestation at the National Institutes of Health (NIH), Bethesda, MD, USA. To explore the potential effect of 2-week dosing, three additional participants were enrolled in a parallel study at Rush University Medical Center (RUMC), Chicago, IL, USA. Participants from the NIH were non-randomly, sequentially assigned in cohorts of three to receive monthly initial intrathecal HPßCD at doses of 50, 200, 300, or 400 mg per month. A fifth cohort of two participants received initial doses of 900 mg. Participants from RUMC initially received 200 or 400 mg every 2 weeks. The dose was escalated based on tolerance or safety data from higher dose cohorts. Serum and CSF 24(S)-hydroxycholesterol (24[S]-HC), which serves as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NNSS) were used to compare disease progression in HPßCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. FINDINGS: Between Sept 21, 2013, and Jan 19, 2015, 32 participants with NPC1 were assessed for eligibility at the National Institutes of Health. 18 patients were excluded due to inclusion criteria not met (six patients), declined to participate (three patients), pursued independent expanded access and obtained the drug outside of the study (three patients), enrolled in the RUMC cohort (one patient), or too late for the trial enrolment (five patients). 14 patients were enrolled and sequentially assigned to receive intrathecal HPßCD at a starting dose of 50 mg per month (three patients), 200 mg per month (three patients), 300 mg per month (three patients), 400 mg per month (three patients), or 900 mg per month (two patients). During the first year, two patients had treatment interrupted for one dose, based on grade 1 ototoxicity. All 14 patients were assessed at 12 months. Between 12 and 18 months, one participant had treatment interrupted at 17 months due to hepatocellular carcinoma, one patient had dose interruption for 2 doses based on caregiver hardship and one patient had treatment interrupted for 1 dose for mastoiditis. 11 patients were assessed at 18 months. Between Dec 11, 2013, and June 25, 2014, three participants were assessed for eligibility and enrolled at RUMC, and were assigned to receive intrathecal HPßCD at a starting dose of 200 mg every 2 weeks (two patients), or 400 mg every two weeks (one patient). There were no dropouts in this group and all 3 patients were assessed at 18 months. Biomarker studies were consistent with improved neuronal cholesterol homoeostasis and decreased neuronal pathology. Post-drug plasma 24(S)-HC area under the curve (AUC8-72) values, an indicator of neuronal cholesterol homoeostasis, were significantly higher than post-saline plasma 24(S)-HC AUC8-72 after doses of 900 mg (p=0·0063) and 1200 mg (p=0·0037). CSF 24(S)-HC concentrations in three participants given either 600 or 900 mg of HPßCD were increased about two fold (p=0·0032) after drug administration. No drug-related serious adverse events were observed. Mid-frequency to high-frequency hearing loss, an expected adverse event, was documented in all participants. When managed with hearing aids, this did not have an appreciable effect on daily communication. The NNSS for the 14 participants treated monthly increased at a rate of 1·22, SEM 0·34 points per year compared with 2·92, SEM 0·27 points per year (p=0·0002) for the 21 patient comparison group. Decreased progression was observed for NNSS domains of ambulation (p=0·0622), cognition (p=0·0040) and speech (p=0·0423). INTERPRETATION: Patients with NPC1 treated with intrathecal HPßCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPßCD. FUNDING: National Institutes of Health, Dana's Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samantha's Search for the Cure Foundation, National Niemann-Pick Disease Foundation, Support of Accelerated Research for NPC Disease, Vtesse, Janssen Research and Development, a Johnson & Johnson company, and Johnson & Johnson.

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation.

Fatty acid synthesis configures the plasma membrane for inflammation in diabetes.

Dietary fat promotes pathological insulin resistance through chronic inflammation. The inactivation of inflammatory proteins produced by macrophages improves diet-induced diabetes, but how nutrient-dense diets induce diabetes is unknown. Membrane lipids affect the innate immune response, which requires domains that influence high-fat-diet-induced chronic inflammation and alter cell function based on phospholipid composition. Endogenous fatty acid synthesis, mediated by fatty acid synthase (FAS), affects membrane composition. Here we show that macrophage FAS is indispensable for diet-induced inflammation. Deleting Fasn in macrophages prevents diet-induced insulin resistance, recruitment of macrophages to adipose tissue and chronic inflammation in mice. We found that FAS deficiency alters membrane order and composition, impairing the retention of plasma membrane cholesterol and disrupting Rho GTPase trafficking-a process required for cell adhesion, migration and activation. Expression of a constitutively active Rho GTPase, however, restored inflammatory signalling. Exogenous palmitate was partitioned to different pools from endogenous lipids and did not rescue inflammatory signalling. However, exogenous cholesterol, as well as other planar sterols, did rescue signalling, with cholesterol restoring FAS-induced perturbations in membrane order. Our results show that the production of endogenous fat in macrophages is necessary for the development of exogenous-fat-induced insulin resistance through the creation of a receptive environment at the plasma membrane for the assembly of cholesterol-dependent signalling networks.