Jessie Zhang

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.

Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking

Niemann-Pick C1 (NPC1) is a key participant in cellular cholesterol trafficking. Loss of NPC1 function leads to defective suppression of SREBP-dependent gene expression and failure to appropriately activate liver X receptor-mediated (LXR-mediated) pathways, ultimately resulting in intracellular cholesterol accumulation. To determine whether NPC1 contributes to regulation of macrophage sterol homeostasis in vivo, we examined the effect of NPC1 deletion in BM-derived cells on atherosclerotic lesion development in the Ldlr-/- mouse model of atherosclerosis. High-fat diet-fed chimeric Npc1-/- mice reconstituted with Ldlr-/-Npc1-/- macrophages exhibited accelerated atherosclerosis despite lower serum cholesterol compared with mice reconstituted with wild-type macrophages. The discordance between the low serum lipoprotein levels and the presence of aortic atherosclerosis suggested that intrinsic alterations in macrophage sterol metabolism in the chimeric Npc1-/- mice played a greater role in atherosclerotic lesion formation than did serum lipoprotein levels. Macrophages from chimeric Npc1-/- mice showed decreased synthesis of 27-hydroxycholesterol (27-HC), an endogenous LXR ligand; decreased expression of LXR-regulated cholesterol transporters; and impaired cholesterol efflux. Lower 27-HC levels were associated with elevated cholesterol oxidation products in macrophages and plasma of chimeric Npc1-/- mice and with increased oxidative stress. Our results demonstrate that NPC1 serves an atheroprotective role in mice through regulation of LXR-dependent cholesterol efflux and mitigation of cholesterol-induced oxidative stress in macrophages.

Identification of Niemann-Pick C1 disease biomarkers through sphingolipid profiling

Niemann-Pick type C (NPC)1 is a rare neurodegenerative disease for which treatment options are limited. A major barrier to development of effective treatments has been the lack of validated biomarkers to monitor disease progression or serve as outcome measures in clinical trials. Using targeted metabolomics to exploit the complex lipid storage phenotype that is the hallmark of NPC1 disease, we broadly surveyed Npc1−/− mouse tissues and identified elevated species across multiple sphingolipid classes that increased with disease progression. There was a striking accumulation of sphingoid bases, monohexosylceramides (MCs), and GM2 gangliosides in liver, and sphingoid bases and GM2 and GM3 gangliosides in brain. These lipids were modestly decreased following miglustat treatment, but markedly decreased in response to treatment with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), two drugs that have shown efficacy in NPC1 animal models. Extending these studies to human subjects led to identification of sphingolipid classes that were significantly altered in the plasma of NPC1 patients. Plasma MCs and ceramides were elevated, whereas sphingoid bases were reduced in NPC1 subjects. Intervention with miglustat in NPC1 patients was accompanied by striking alterations in plasma (reductions in GM1 and GM3 gangliosides) and cerebrospinal fluid (CSF) (increased MCs) sphingolipids. Similar alterations were observed in the CSF from the NPC1 feline model following HP-β-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.

A Murine Niemann-Pick C1 I1061T Knock-In Model Recapitulates the Pathological Features of the Most Prevalent Human Disease Allele

Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol–sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1I1061T, encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1−/− mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1−/− mouse, this Npc1tm(I1061T)Dso model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1I1061T protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.

Cytisine binds with similar affinity to nicotinic α4β2 receptors on the cell surface and in homogenates

Abstract Cytisine and nicotine bound to specific sites in homogenates prepared from HEK 293 cells which stably express human neuronal nicotinic α4 and β2 subunits. The number of sites was the same for both ligands and nicotine was a full competitive inhibitor of cytisine binding. However, when binding was done to intact cells the number of binding sites per cell for nicotine was ∼4-fold the number of sites for cytisine. Nicotine fully blocked cytisine binding, but cytisine only partially blocked nicotine binding to intact cells. When cells were permeabilized with saponin, the number of sites for nicotine was unchanged, while the number of sites for cytisine was increased, and cytisine was able to fully block nicotine binding. These data indicate that cytisine binds only to surface receptors on intact cells. The apparent affinity of cytisine for surface receptors (Kd=0.8 nM) was not significantly different from that for receptors in the cell homogenate (0.3 nM).

Cholesterol homeostatic responses provide biomarkers for monitoring treatment for the neurodegenerative disease Niemann–Pick C1 (NPC1)

Niemann-Pick C1 (NPC1) disease is a rare, neurodegenerative lysosomal cholesterol storage disorder, typified by progressive cognitive and motor function impairment. Affected individuals usually succumb to the disease in adolescence. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) has emerged as a promising intervention that reduces lipid storage and prolongs survival in NPC1 disease animal models. A barrier to the development of HP-β-CD and other treatments for NPC disease has been the lack of validated biochemical measures to evaluate efficacy. Here we explored whether cholesterol homeostatic responses resulting from HP-β-CD-mediated redistribution of sequestered lysosomal cholesterol could provide biomarkers to monitor treatment. Upon direct CNS delivery of HP-β-CD, we found increases in plasma 24(S)-HC in two independent NPC1 disease animal models, findings that were confirmed in human NPC1 subjects receiving HP-β-CD. Since circulating 24(S)-HC is almost exclusively CNS-derived, the increase in plasma 24(S)-HC provides a peripheral, non-invasive measure of the CNS effect of HP-β-CD. Our findings suggest that plasma 24(S)-HC, along with the other cholesterol-derived markers examined in this study, can serve as biomarkers that will accelerate development of therapeutics for NPC1 disease.

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.

Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

Niemann-Pick type C disease (NP-C) is a progressive lysosomal lipid storage disease caused by mutations in the NPC1 and NPC2 genes. NPC1 is essential for transporting cholesterol and other lipids out of lysosomes, but little is known about the mechanisms that control its cellular abundance and localization. Here we show that a reduction of TMEM97, a cholesterol-responsive NPC1-binding protein, increases NPC1 levels in cells through a post-transcriptional mechanism. Reducing TMEM97 through RNA-interference reduces lysosomal lipid storage and restores cholesterol trafficking to the endoplasmic reticulum in cell models of NP-C. In TMEM97 knockdown cells, NPC1 levels can be reinstated with wild type TMEM97, but not TMEM97 missing an ER-retention signal suggesting that TMEM97 contributes to controlling the availability of NPC1 to the cell. Importantly, knockdown of TMEM97 also increases levels of residual NPC1 in NPC1-mutant patient fibroblasts and reduces cholesterol storage in an NPC1-dependent manner. Our findings propose TMEM97 inhibition as a novel strategy to increase residual NPC1 levels in cells and a potential therapeutic target for NP-C.

The Activators of Cyclin-Dependent Kinase 5 p35 and p39 Are Essential for Oligodendrocyte Maturation, Process Formation, and Myelination

The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35−/− mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation. SIGNIFICANCE STATEMENT The development of oligodendrocytes and myelination is essential for normal CNS function and cyclin-dependent kinase 5 (Cdk5) activity is critical for oligodendrocyte maturation, but how Cdk5 activity is controlled is unclear. Here we show that the coactivators of Cdk5, p35 and p39, regulate distinct stages of oligodendrocyte development and myelination. Loss of p35 perturbs oligodendrocyte progenitor cell differentiation, whereas loss of p39 delays oligodendrocyte maturation. Loss of both completely inhibits oligodendrogenesis and myelination. Disruption of oligodendrocyte development was more pronounced in p35−/−;p39 shRNA cells than loss of Cdk5 alone and could not be rescued by Cdk5 overexpression, suggesting that p35 and p39 have Cdk5-independent roles during oligodendrocyte development. These studies provide novel targets for therapeutic intervention in conditions in which myelination is perturbed.