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.

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-ß-cyclodextrin (HP-ß-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-ß-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-ß-CD-mediated redistribution of sequestered lysosomal cholesterol could provide biomarkers to monitor treatment. Upon direct CNS delivery of HP-ß-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-ß-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-ß-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.

Development and validation of sensitive LC-MS/MS assays for quantification of HP-ß-CD in human plasma and CSF.

2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-ß-CD required for clinical trials has been challenging owing to the dispersity of the HP-ß-CD. To support a phase 1 clinical trial for ICV delivery of HP-ß-CD in NPC1 patients, novel methods for quantification of HP-ß-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-ß-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-ß-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-ß-CD in NPC1 patients.

Relative acidic compartment volume as a lysosomal storage disorder-associated biomarker.

Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency­approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.

Human and mouse neuroinflammation markers in Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 (NPC1) is an autosomal recessive lipid storage disorder in which a pathological cascade, including neuroinflammation occurs. While data demonstrating neuroinflammation is prevalent in mouse models, data from NPC1 patients is lacking. The current study focuses on identifying potential markers of neuroinflammation in NPC1 from both the Npc1 mouse model and NPC1 patients. We identified in the mouse model significant changes in expression of genes associated with inflammation and compared these results to the pattern of expression in human cortex and cerebellar tissue. From gene expression array analysis, complement 3 (C3) was increased in mouse and human post-mortem NPC1 brain tissues. We also characterized protein levels of inflammatory markers in cerebrospinal fluid (CSF) from NPC1 patients and controls. We found increased levels of interleukin 3, chemokine (C-X-C motif) ligand 5, interleukin 16 and chemokine ligand 3 (CCL3), and decreased levels of interleukin 4, 10, 13 and 12p40 in CSF from NPC1 patients. CSF markers were evaluated with respect to phenotypic severity. Miglustat treatment in NPC1 patients slightly decreased IL-3, IL-10 and IL-13 CSF levels; however, further studies are needed to establish a strong effect of miglustat on inflammation markers. The identification of inflammatory markers with altered levels in the cerebrospinal fluid of NPC1 patients may provide a means to follow secondary events in NPC1 disease during therapeutic trials.

Altered distribution and function of natural killer cells in murine and human Niemann-Pick disease type C1.

Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesized that NK-cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model, we found the frequency of NK cells was altered and phenocopied S1P5-deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicity due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK-cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.

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-ß-cyclodextrin (HP-ß-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-ß-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.

A somatic cell defect is associated with the onset of neurological symptoms in a lysosomal storage disease.

Mutations in individuals with the lysosomal storage disorder Niemann-Pick disease, type C1 (NPC1) are heterogeneous, not localized to specific protein domains, and not correlated to time of onset or disease severity. We demonstrate direct correlation of the time of neurological symptom onset with the severity of lysosomal defects in NPC1 patient-derived fibroblasts. This is a novel assay for NPC1 individuals that may be predictive of NPC1 disease progression and broadly applicable to other lysosomal disorders.

Efficacy of N-acetylcysteine in phenotypic suppression of mouse models of Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 (NPC1), which arises from a mutation in the NPC1 gene, is characterized by abnormal cellular storage and transport of cholesterol and other lipids that leads to hepatic disease and progressive neurological impairment. Oxidative stress has been hypothesized to contribute to the NPC1 disease pathological cascade. To determine whether treatments reducing oxidative stress could alleviate NPC1 disease phenotypes, the in vivo effects of the antioxidant N-acetylcysteine (NAC) on two mouse models for NPC1 disease were studied. NAC was able to partially suppress phenotypes in both antisense-induced (NPC1ASO) and germline (Npc1-/-) knockout genetic mouse models, confirming the presence of an oxidative stress-related mechanism in progression of NPC1 phenotypes and suggesting NAC as a potential molecule for treatment. Gene expression analyses of NAC-treated NPC1ASO mice suggested NAC affects pathways distinct from those initially altered by Npc1 knockdown, data consistent with NAC achieving partial disease phenotype suppression. In a therapeutic trial of short-term NAC administration to NPC1 patients, no significant effects on oxidative stress in these patients were identified other than moderate improvement of the fraction of reduced CoQ10, suggesting limited efficacy of NAC monotherapy. However, the mouse model data suggest that the distinct antioxidant effects of NAC could provide potential treatment of NPC1 disease, possibly in concert with other therapeutic molecules at earlier stages of disease progression. These data also validated the NPC1ASO mouse as an efficient model for candidate NPC1 drug screening, and demonstrated similarities in hepatic phenotypes and genome-wide transcript expression patterns between the NPC1ASO and Npc1-/- models.

Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight.

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Apolipoprotein E genotype and neurological disease onset in Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 (NPC1) is a lipid storage disorder that results in progressive neurological impairment. The NPC1 phenotype is extremely variable and at the individual level is likely influenced by other genetic traits. In addition to residual function of NPC1 protein, we hypothesize that modifier genes, as frequently observed with other autosomal recessive diseases, influence the NPC phenotype. The NPC1 phenotype includes progressive dementia, and the NPC pathology has some overlap with the pathology of Alzheimer disease (AD). Thus, we examined apolipoprotein E (ApoE) and microtubule-associated protein tau (MAPT) polymorphisms in a cohort of 15 NPC1 patients with well characterized longitudinal disease progression. Although we did not find any correlations between disease severity and tau polymorphisms, we found significant associations between ApoE polymorphisms and phenotypic severity. Specifically, ApoE4 and ApoE2 alleles were associated, respectively, with increased and decreased disease severity in this cohort of NPC1 patients. These data support the hypothesis that ApoE may play a role in modulating NPC1 neuropathology.

Invariant natural killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C.

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development.

Growth charts for individuals with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a rare multiple congenital anomaly neurodevelopmental syndrome of impaired cholesterol synthesis. Growth restriction and developmental delay are very common clinical manifestations of SLOS. The degree, etiology, and consequences of growth restriction in SLOS remain an area of limited knowledge to the scientific community. There have been no studies describing the growth parameters and providing reference growth charts for individuals with SLOS. Our longitudinal data from 78 patients between the ages of 0.1 and 16 years with SLOS show a growth restriction of about two standard deviations below the Centers for Disease Control (CDC) norms for age. This study represents comprehensive anthropometric data from the largest cohort available, and proposes growth charts for widespread use in the management and study of individuals with SLOS.

Miglustat treatment may reduce cerebrospinal fluid levels of the axonal degeneration marker tau in niemann-pick type C.

INTRODUCTION: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder that leads to progressive neurodegeneration. The glucosylceramide synthase blocker miglustat is being used to treat NPC, but monitoring of disease progression and treatment response is difficult. NPC patients have elevated cerebrospinal fluid (CSF) levels of total-tau (T-tau) indicating axonal degeneration, and increased CSF amyloid ß (Aß) indicating abnormal brain amyloid metabolism, but it is unknown if start of miglustat treatment affects these biomarker levels. METHODS: Biomarkers were measured in serial CSF samples from NPC patients who started miglustat between samplings (N=5), were untreated at both samplings (N=5) or received treatment during the whole study (N=6) (median time between samplings 309 days [range 175-644]). CSF was analyzed for Aß(38), Aß(40), Aß(42), α-cleaved soluble APP, ß-cleaved soluble APP, T-tau and phospho-tau. RESULTS: T-tau levels decreased in patients who started miglustat treatment (median 955 [range 338-1,271]ng/L at baseline vs. 382 [187-736]ng/L at follow-up, p=0.043). Untreated patients and continuously treated patients had stable levels (p>0.05). No changes were seen in the other biomarkers. CONCLUSION: Reduced CSF T-tau suggests that miglustat treatment might affect axonal degeneration in NPC. However, the results must be interpreted with caution and verified in future studies, since this pilot study was small, treatment was not randomized, and patients starting treatment had higher baseline CSF T-tau than untreated patients.

Defining natural history: assessment of the ability of college students to aid in characterizing clinical progression of Niemann-Pick disease, type C.

Niemann-Pick Disease, type C (NPC) is a fatal, neurodegenerative, lysosomal storage disorder. It is a rare disease with broad phenotypic spectrum and variable age of onset. These issues make it difficult to develop a universally accepted clinical outcome measure to assess urgently needed therapies. To this end, clinical investigators have defined emerging, disease severity scales. The average time from initial symptom to diagnosis is approximately 4 years. Further, some patients may not travel to specialized clinical centers even after diagnosis. We were therefore interested in investigating whether appropriately trained, community-based assessment of patient records could assist in defining disease progression using clinical severity scores. In this study we evolved a secure, step wise process to show that pre-existing medical records may be correctly assessed by non-clinical practitioners trained to quantify disease progression. Sixty-four undergraduate students at the University of Notre Dame were expertly trained in clinical disease assessment and recognition of major and minor symptoms of NPC. Seven clinical records, randomly selected from a total of thirty seven used to establish a leading clinical severity scale, were correctly assessed to show expected characteristics of linear disease progression. Student assessment of two new records donated by NPC families to our study also revealed linear progression of disease, but both showed accelerated disease progression, relative to the current severity scale, especially at the later stages. Together, these data suggest that college students may be trained in assessment of patient records, and thus provide insight into the natural history of a disease.

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-3ß,5α,6ß-triol (3ß,5α,6ß-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 3ß,5α,6ß-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3ß,5α,6ß-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 3ß,5α,6ß-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.

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1(-/-) mouse model and found several cholesterol oxidation products that were elevated in Npc1(-/-) mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Oxidative stress in Niemann-Pick disease, type C.

Niemann-Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder due to impaired intracellular cholesterol and lipid transport. Increased oxidative stress has been reported in human NPC1 mutant fibroblasts and in tissues from Npc1 mutant mice. However, oxidative stress in NPC patients has not been established. In this study, we demonstrated increased oxidative stress in NPC patients. Evaluation of serum from 37 NPC patients, compared to control values, showed significant decreases (p<.01) in both the fraction of reduced coenzyme Q10 (CoQ10) and trolox equivalent antioxidant capacity (TEAC). Both findings are consistent with increased oxidative stress in NPC. Supplementation with CoQ10 was not effective in correcting the decreased fraction of reduced CoQ10. Increased oxidative stress may be a contributing factor to the pathology of NPC, and demonstration of increased oxidative stress in NPC patients provides both a rationale and the biomarkers necessary to test the efficacy of antioxidant therapy in NPC.

Linear clinical progression, independent of age of onset, in Niemann-Pick disease, type C.

Niemann-Pick disease, type C is a neurodegenerative, lysosomal storage disorder with a broad clinical spectrum and a variable age of onset. The absence of a universally accepted clinical outcome measure is an impediment to the design of a therapeutic trial for NPC. Thus, we developed a clinical severity scale to characterize and quantify disease progression. Clinical signs and symptoms in nine major (ambulation, cognition, eye movement, fine motor, hearing, memory, seizures, speech, and swallowing) and eight minor (auditory brainstem response, behavior, gelastic cataplexy, hyperreflexia, incontinence, narcolepsy, psychiatric, and respiratory problems) domains were scored. Data were collected from 18 current NPC patients and were extracted from records of 19 patients. Both patient cohorts showed a linear increase in severity scores over time. Cross-sectional evaluation of current patients showed a linear increase in the severity score. Longitudinal chart review of historical data demonstrated that although age of onset varied significantly, the rate of progression appeared linear, independent of age of onset, and similar in all patients. Combining the data from both cohorts, disease progression could be modeled by the following equation: s(t0+x) = s(t0) + 1.87x; where s(t0) is the initial score and s(t0+x) is the predicted future score after x years. Our observation that disease progression is similar across patients and independent of age of onset is consistent with a biphasic pathological model for NPC. This scale may prove useful in the characterization of potential biomarkers, and as an outcome measure to monitor disease progression in NPC patients.