Xuntian Jiang

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

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, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-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 specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-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 quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Alterations in lipid homeostasis of mouse dorsal root ganglia induced by apolipoprotein E deficiency: a shotgun lipidomics study

One of the fundamental goals of lipidomics research is to identify the linkage of an individual gene with a given lipidome, thereby revealing the role of that gene in lipid metabolism, transport, and homeostasis. In this study, we have identified four apolipoprotein E (apoE)‐induced alterations in the lipidome of mouse dorsal root ganglia (DRG) through utilizing the technology of shotgun lipidomics. First, apoE mediates sulfatide mass content in mouse DRG, which is comparable to its role in the CNS. Second, apoE contributes to galactosylceramide and ceramide homeostasis in mouse DRG. Third, apoE significantly modulates cholesterol levels in mouse DRG. The latter two functions of apoE are distinct from those in the CNS. Finally, mice null for apoE have dramatically less triacylglycerol mass content in DRG which are opposite to the effects observed in the peripheral organs and vascular system. Collectively, this study identifies the specific alterations in the DRG lipidome induced by apoE knockout and suggests the potential roles of apoE in lipid transport and homeostasis in a tissue specific manner, thereby providing insights into the biochemical mechanisms underlying the functions of apoE in the PNS.

Shotgun lipidomics reveals the temporally dependent, highly diversified cardiolipin profile in the mammalian brain: temporally coordinated postnatal diversification of cardiolipin molecular species with neuronal remodeling.

Large-scale neuronal remodeling through apoptosis occurs shortly after birth in all known mammalian species. Apoptosis, in large part, depends upon critical interactions between mitochondrial membranes and cytochrome c. Herein, we examined the hypothesis that the large-scale reorganization of neuronal circuitry after birth is accompanied by profound alterations in cardiolipin (CL) content and molecular species distribution. During embryonic development, over 100 CL molecular species were identified and quantitated in murine neuronal tissues. The embryonic CL profile was notable for the presence of abundant amounts of relatively short aliphatic chains (e.g., palmitoleic and oleic acids). In sharp contrast, after birth, the CL profile contained a remarkably complex repertoire of CL molecular species, in which the signaling fatty acids (i.e., arachidonic and docosahexaenoic acids) were markedly increased. These results identify the rapid remodeling of CL in the perinatal period with resultant alterations in the physical properties of the mitochondrial membrane. The complex distribution of aliphatic chains in the neuronal CL pool is separate and distinct from that in other organs (e.g., heart, liver, etc.), where CL molecular species contain predominantly only one major type of aliphatic chain (e.g., linoleic acid). Analyses of mRNA levels by real-time quantitative polymerase chain reactions suggested that the alterations in CL content were due to the combined effects of both attenuation of de novo CL biosynthesis and decreased remodeling of CL. Collectively, these results provide a new perspective on the complexity of CL in neuronal signaling, mitochondrial bioenergetics, and apoptosis.

Lysosomal cholesterol activates mTORC1 via an SLC38A9-Niemann-Pick C1 signaling complex

A cholesterol-mTORC1 axis may play a role in metabolic homeostasis in normal and disease states. Lysosomal cholesterol activates mTORC1 The mTORC1 kinase is a master nutrient sensor that governs cellular metabolism. When dysregulated, this kinase drives several human diseases, including cancer and diabetes. Recent work has delineated a pathway through which amino acids regulate mTORC1. In contrast, little is known about how sterols may affect mTORC1 signaling. Castellano et al. provide detailed mechanistic evidence for how cholesterol, derived from the processing of low-density lipoprotein in the lysosomal lumen, drives mTORC1 signaling. They identify the key players that couple lysosomal cholesterol levels to mTORC1 activation. Unexpectedly, the putative amino acid transporter SLC38A9, which is implicated in mTORC1 regulation by arginine, is essential for mTORC1 activation by cholesterol. Furthermore, the authors uncover a physical and functional interaction between SLC38A9 and the major lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1) protein. The SLC38A9-NPC1 complex is key to the ability of mTORC1 to respond to variations in dietary lipid supply. Science, this issue p. 1306 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.

Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach

Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO3]−) in the negative-ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/μl concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies.

Niemann-Pick disease type C clinical database: cognitive and coordination deficits are early disease indicators

BackgroundThe neurodegenerative lysosomal storage disorder Niemann-Pick disease type C (NP-C) is characterized by a broad clinical variability involving neurological, psychiatric and systemic signs. Diverse patterns of disease manifestation and progression considerably delay its diagnosis. Here we introduce the NP-C clinical database (NPC-cdb) to systematically obtain, store and analyze diagnostic and clinical findings in patients with NP-C. We apply NPC-cdb to study NP-C temporal expression in a large German-Swiss patient cohort.MethodsCurrent and past medical history was systematically acquired from 42 patients using tailored questionnaires. Manifestation of 72 distinct neuropsychiatric signs was modeled over the course of disease. The sequence of disease progression was re-constructed by a novel clinical outcome scale (NPC-cdb score).ResultsThe efficiency of current clinical diagnostic standards negatively correlates with duration of disease (p<3.9x10-4), suggesting insufficient sensitivity in patients early in the disease process. Neurological signs considered as typical for NP-C were frequent (e.g., cognitive impairment 86%, ataxia 79%, vertical supranuclear gaze palsy 76%) and their presence co-occurred with accelerated diagnosis. However, less specific neuropsychiatric signs were reported to arise considerably more early in the disease process (e.g., clumsiness -4.9±1.1 y before diagnosis). Most patients showed a steady disease progression that correlated with age at neurological onset. However, a distinct subcohort (n=6) with initially steadily progressing disease later showed a 2.9-fold accelerated progression that was associated with the onset of seizures (p<7x10-4), suggesting seizures as predictive for a poor prognosis.ConclusionsConsidering early, but less specific neuropsychiatric signs may accelerate the path to diagnosing NP-C in a patient.

Mechanism-Based Combination Treatment Dramatically Increases Therapeutic Efficacy in Murine Globoid Cell Leukodystrophy

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.

Development of a bile acid–based newborn screen for Niemann-Pick disease type C

A newborn screen for Niemann-Pick disease type C was developed on the basis of discovery of a bile acid marker. Expanding the newborn screen Niemann-Pick disease type C (NPC) is a fatal neurologic disorder caused by the deficiency of an enzyme involved in cholesterol storage. Although this disease was untreatable in the past, new therapeutics are now in clinical trials, but they are most likely to be effective if treatment is started as early as possible, before neurodegeneration has occurred. Jiang et al. identified three bile acids that are greatly increased in the blood of patients with NPC compared to healthy controls. The authors also demonstrated that one of these bile acids can be reliably measured in dried blood spots using mass spectrometry, suggesting that this bile acid test should be evaluated for potential addition to neonatal screening programs. Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, it is imperative to improve diagnostics and facilitate early intervention. We used metabolomic profiling to identify potential markers and discovered three unknown bile acids that were increased in plasma from NPC but not control subjects. The bile acids most elevated in the NPC subjects were identified as 3β,5α,6β-trihydroxycholanic acid and its glycine conjugate, which were shown to be metabolites of cholestane-3β,5α,6β-triol, an oxysterol elevated in NPC. A high-throughput mass spectrometry–based method was developed and validated to measure the glycine-conjugated bile acid in dried blood spots. Analysis of dried blood spots from 4992 controls, 134 NPC carriers, and 44 NPC subjects provided 100% sensitivity and specificity in the study samples. Quantification of the bile acid in dried blood spots, therefore, provides the basis for a newborn screen for NPC that is ready for piloting in newborn screening programs.

Intrathecal 2-hydroxypropyl-β-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-β-cyclodextrins (HPβCD) 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 HPβCD. METHODS In this open-label, dose-escalation phase 1-2a study, we gave monthly intrathecal HPβCD 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 HPβCD 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 HPβCD-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 HPβCD 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 HPβCD 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 HPβCD 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 HPβCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPβCD. FUNDING National Institutes of Health, Danas Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samanthas 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.

Endosomes and lysosomes play distinct roles in sulfatide-induced neuroblastoma apoptosis: potential mechanisms contributing to abnormal sulfatide metabolism in related neuronal diseases.

Alterations in sulfatide metabolism, trafficking and homoeostasis are present at the earliest clinically recognizable stages of Alzheimers disease and are associated with metachromatic leukodystrophy. However, the role of sulfatide in these disease states remains unknown. In the present study, we investigated the sequelae of NB (neuroblastoma) cells upon sulfatide supplementation and the biochemical mechanisms contributing to the sulfatide-induced changes. By using shotgun lipidomics, we showed dramatic accumulations of sulfatide, ceramide and sphingosine in NB cells in a time- and dose-dependent manner. Further studies utilizing subcellular fractionation and shotgun lipidomics analyses demonstrated that most of the increased ceramide content was generated in the endosomal compartment, whereas sulfatides predominantly accumulated in lysosomes. In addition, we determined that the sulfatide-mediated increase in endosomal ceramide content mainly resulted from beta-galactosidase activity, which directly hydrolyses sulfatide to ceramide without a prior desulfation step. Substantial cell apoptosis occurred in parallel with the accumulation of sulfatides and ceramides, as revealed by mitochondrial membrane depolarization, by phosphatidylserine translocation and by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. These findings were also demonstrated with primary neuron cultures. Collectively, our results demonstrate that abnormal sulfatide metabolism can induce cell apoptosis due to endosome-mediated ceramide generation and the accumulation of cytotoxic levels of sulfatides in lysosomes.