Ataxia is a common neurological feature of Niemann-Pick disease type C (NPC). In this disease, unesterified cholesterol accumulates in lysosomes of the central nervous system and hepatic cells. Oxidation by reactive oxygen species produces oxysterols that can be metabolised to specific bile acids. These bile acids have been suggested as useful biomarkers to detect NPC. Concentrations of 3β,5α,6β-trihydroxycholanyl glycine (3β,5α,6β-triOH-Gly) and 3β,7β-dihydroxy-5-cholenyl glycine (3β,7β-diOH-Δ5-Gly) were measured in plasma of 184 adults with idiopathic ataxia. All patients were tested with whole genome sequencing containing hereditary ataxia panels, which include NPC1 and NPC2 mutations and other genetic causes of ataxia. Plasma 3β,5α,6β-triOH-Gly above normal (>90 nM) was found in 8 out of 184 patients. One patient was homozygous for the p.(Val1165Met) mutation in the NPC1 gene. The remaining seven included one patient with Friedreich\'s ataxia and three patients with autoimmune diseases. Oxidative stress is known to be increased in Friedreich\'s ataxia and in autoimmune diseases. Therefore, this subset of patients possibly shares a common mechanism that determines the increase of this bile acid. In a large cohort of adults with ataxia, plasma 3β,5α,6β-triOH-Gly was able to detect the one patient in the cohort with NPC1 disease, but also detected oxidation of cholesterol by ROS in other disorders. Plasma 3β,7β-diOH-Δ5-Gly is not a potential biomarker for NPC1.

Niemann-Pick disease type C (NPC) is one of the lysosomal storage disorders. It is caused by biallelic pathogenic variants in NPC1 or NPC2, which results in a defective cholesterol trafficking inside the late endosome and lysosome. There is a high clinical variability in the age of presentation and the phenotype of this disorder making the diagnosis challenging. Here, we report a patient with an infantile onset global developmental delay, microcephaly and dysmorphic features, homozygous for c.3560C>T (p.A1187V) variant in NPC1. His plasma oxysterol levels were normal on two occasions. His lyso-sphingomyelin-509 (lyso-SM 509) and urinary bile acid levels were normal. Based on the phenotype and biochemical features, the diagnosis of NPC was excluded in this patient. We emphasize the importance of functional characterization in the classification of novel variants to prevent a misdiagnosis. Matching the phenotype and biochemical evidence with the molecular genomic tests is crucial for the confirmation of genetic diagnoses.

Niemann-Pick type C (NPC, ORPHA: 646) is a neuro-visceral, psychiatric disease caused predominantly by pathogenic variants in the NPC1 gene or seldom in NPC2. The rarity of the disease, and its wide range of clinical phenotypes and ages of onset, turn the diagnosis into a significant challenge. Other than the detailed clinical history, the typical diagnostic work-up for NPC includes the quantification of pathognomonic metabolites. However, the molecular basis diagnosis is still of utmost importance to fully characterize the disorder. Here, the authors provide an overview of splicing variants in the NPC1 and NPC2 genes and propose a new workflow for NPC diagnosis. Splicing variants cover a significant part of the disease-causing variants in NPC. The authors used cDNA analysis to study the impact of such variants, including the collection of data to classify them as leaky or non-leaky pathogenic variants. However, the presence of naturally occurring spliced transcripts can misdiagnose or mask a pathogenic variant and make the analysis even more difficult. Analysis of the NPC1 cDNA in NPC patients in parallel with controls is vital to assess and detect alternatively spliced forms. Moreover, nonsense-mediated mRNA decay (NMD) analysis plays an essential role in evaluating the naturally occurring transcripts during cDNA analysis and distinguishing them from other pathogenic variants\' associated transcripts.

BACKGROUND: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results.
METHODS: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann-Pick type C. We used specific markers for each pathology.
CONCLUSIONS: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

BACKGROUND: Niemann-Pick is a rare metabolic disease distinguished by lysosomal storage defects. This disease is characterized by sphingomyelinase acid deficiency, causing its accumulation in various organs such as the kidneys, spleen, liver, brain, and nerves. Niemann-Pick disease is categorized into four groups: A, B, C, and D. Peripheral neuropathy is an extremely rare complication in patients with Niemann-Pick type C, which certainly leads to neurologic deterioration.
METHODS: We report a case of Niemann-Pick type C disease in a 3-year-old Iranian Azeri female patient who was hospitalized twice. The first time was at 1 month of age with symptoms of splenomegaly, jaundice, and elevated liver enzymes, and the second time was at around age 2 for loss of mental and physical abilities. The patient presented with failure to thrive. According to paraclinical examinations, mildly delayed myelination along with a nonspecific periventricular hypersignal intensity was seen. Interestingly, the patient\'s Niemann-Pick type C enzymatic function was evaluated twice and was negative on both occasions, while she was positive for NPC1 and NPC2 gene examinations.
CONCLUSIONS: In this study, despite the enzymatic study being negative, Niemann-Pick type C disease was finally confirmed, revealing the importance of mutations in Niemann-Pick type C pathogenesis. Besides, peripheral neuropathy was diagnosed in this patient as a very rare symptom of Niemann-Pick type C.

Niemann-Pick type C (NPC) disease is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol in the late endo-lysosomal system of cells. Progressive neurological deterioration and the onset of symptoms, such as ataxia, seizures, cognitive decline, and severe dementia, are pathognomonic features of the disease. In addition, different pathological similarities, including degeneration of hippocampal and cortical neurons, hyperphosphorylated tau, and neurofibrillary tangle formation, have been identified between NPC disease and other neurodegenerative pathologies. However, the underlying pathophysiological mechanisms are not yet well understood, and even a real cure to counteract neurodegeneration has not been identified. Therefore, the combination of current pharmacological therapies, represented by miglustat and cyclodextrin, and non-pharmacological approaches, such as physical exercise and appropriate diet, could represent a strategy to improve the quality of life of NPC patients. Based on this evidence, in our review we focused on the neurodegenerative aspects of NPC disease, summarizing the current knowledge on the molecular and biochemical mechanisms responsible for cognitive impairment, and suggesting physical exercise and nutritional treatments as additional non-pharmacologic approaches to reduce the progression and neurodegenerative course of NPC disease.

Niemann-Pick type C (NPC) disease is a rare autosomal recessive inherited childhood neurodegenerative disease characterized by the accumulation of cholesterol and glycosphingolipids, involving the autophagy-lysosome system. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that metabolizes epoxy fatty acids (EpFAs) to 12-diols, exerts beneficial effects in modulating inflammation and autophagy, critical features of the NPC disease. This study aims to evaluate the effects of UB-EV-52, an sEH inhibitor (sEHi), in an NPC mouse model (Npc) by administering it for 4 weeks (5 mg/kg/day). Behavioral and cognitive tests (open-field test (OF)), elevated plus maze (EPM), novel object recognition test (NORT) and object location test (OLT) demonstrated that the treatment produced an improvement in short- and long-term memory as well as in spatial memory. Furthermore, UB-EV-52 treatment increased body weight and lifespan by 25% and reduced gene expression of the inflammatory markers (i.e., Il-1β and Mcp1) and enhanced oxidative stress (OS) markers (iNOS and Hmox1) in the treated Npc mice group. As for autophagic markers, surprisingly, we found significantly reduced levels of LC3B-II/LC3B-I ratio and significantly reduced brain protein levels of lysosomal-associated membrane protein-1 (LAMP-1) in treated Npc mice group compared to untreated ones in hippocampal tissue. Lipid profile analysis showed a significant reduction of lipid storage in the liver and some slight changes in homogenated brain tissue in the treated NPC mice compared to the untreated groups. Therefore, our results suggest that pharmacological inhibition of sEH ameliorates most of the characteristic features of NPC mice, demonstrating that sEH can be considered a potential therapeutic target for this disease.

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

Lysosomal storage diseases are the most common cause of neurodegeneration in children. They are characterised at the cellular level by the accumulation of storage material within lysosomes. There are very limited therapeutic options, and the search for novel therapies has been hampered as few good small animal models are available. Here, we describe the use of light sheet microscopy to assess lipid storage in drug and morpholino induced zebrafish models of two diseases of cholesterol homeostasis with lysosomal dysfunction: First, Niemann-Pick type C disease (NPC), caused by mutations in the lysosomal transmembrane protein NPC1, characterised by intralysosomal accumulation of cholesterol and several other lipids. Second, Smith-Lemli-Opitz syndrome (SLOS), caused by mutations in 7-dehydrocholesterol reductase, which catalyses the last step of cholesterol biosynthesis and is characterised by intralysosomal accumulation of dietary cholesterol. This is the first description of a zebrafish SLOS model. We find that zebrafish accurately model lysosomal storage and disease-specific phenotypes in both diseases. Increased cholesterol and ganglioside GM1 were observed in sections taken from NPC model fish, and decreased cholesterol in SLOS model fish, but these are of limited value as resolution is poor, and accurate anatomical comparisons difficult. Using light sheet microscopy, we were able to observe lipid changes in much greater detail and identified an unexpected accumulation of ganglioside GM1 in SLOS model fish. Our data demonstrate, for the first time in zebrafish, the immense potential that light sheet microscopy has in aiding the resolution of studies involving lysosomal and lipid disorders.

Lysosomes are of major importance for the regulation of cellular cholesterol homeostasis. Food-derived cholesterol and cholesterol esters contained within lipoproteins are delivered to lysosomes by endocytosis. From the lysosomal lumen, cholesterol is transported to the inner surface of the lysosomal membrane through the glycocalyx; this shuttling requires Niemann-Pick C (NPC) 1 and NPC2 proteins. The lysosomal membrane proteins lysosomal-associated membrane protein (LAMP)-2 and lysosomal integral membrane protein (LIMP)-2/SCARB2 also bind cholesterol. LAMP-2 may serve as a cholesterol reservoir, whereas LIMP-2, like NPC1, is able to transport cholesterol through a transglycocalyx tunnel. Contact sites and fusion events between lysosomes and other organelles mediate the distribution of cholesterol. Lysosomal cholesterol content is sensed thereby regulating mammalian target of rapamycin complex (mTORC)-dependent signaling. This review summarizes our understanding of the major steps in cholesterol handling from the moment it enters the lysosome until it leaves this compartment.