Alterations in Proteostasis Mechanisms in Niemann-Pick Type C Disease.

Niemann-Pick Type C (NPC) represents an autosomal recessive disorder with an incidence rate of 1 in 150,000 live births, classified within lysosomal storage diseases (LSDs). The abnormal accumulation of unesterified cholesterol characterizes the pathophysiology of NPC. This phenomenon is not unique to NPC, as analogous accumulations have also been observed in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. Interestingly, disturbances in the folding of the mutant protein NPC1 I1061T are accompanied by the aggregation of proteins such as hyperphosphorylated tau, α-synuclein, TDP-43, and ß-amyloid peptide. These accumulations suggest potential disruptions in proteostasis, a regulatory process encompassing four principal mechanisms: synthesis, folding, maintenance of folding, and protein degradation. The dysregulation of these processes leads to excessive accumulation of abnormal proteins that impair cell function and trigger cytotoxicity. This comprehensive review delineates reported alterations across proteostasis mechanisms in NPC, encompassing changes in processes from synthesis to degradation. Additionally, it discusses therapeutic interventions targeting pharmacological facets of proteostasis in NPC. Noteworthy among these interventions is valproic acid, a histone deacetylase inhibitor (HDACi) that modulates acetylation during NPC1 synthesis. In addition, various therapeutic options addressing protein folding modulation, such as abiraterone acetate, DHBP, calnexin, and arimoclomol, are examined. Additionally, treatments impeding NPC1 degradation, exemplified by bortezomib and MG132, are explored as potential strategies. This review consolidates current knowledge on proteostasis dysregulation in NPC and underscores the therapeutic landscape targeting diverse facets of this intricate process.

Long-term efficacy of intrathecal cyclodextrin in patients with Niemann-Pick disease type C.

BACKGROUND AND OBJECTIVES: Niemann-Pick type C (NPC) is a rare lysosomal storage disease characterized by hepatosplenomegaly and progressive neurological deterioration due to abnormal intracellular cholesterol transport. Cyclic oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (HPBCD) is an effective treatment for NPC; however, few reports have shown its long-term efficacy and safety. To demonstrate long-term efficacy and safety of intrathecal HPBCD (IT-HPBCD) treatment for NPC, we herein reports five patients with NPC treated using IT-HPBCD for 4-11 years. CASES AND RESULTS: Patients' ages at the onset ranged from 1.5 to 20 years. Notably, all patients showed rapid disease progression despite treatment with miglustat before IT-HPBCD treatment. Similarly, some patients showed transient improvement; however, all patients' conditions stabilized after long-term IT-HPBCD therapy. Mild-to-moderate hearing loss was observed in three patients. Furthermore, long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, patients still experience some disease progression. CONCLUSIONS: Long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, the treatment outcome is dependent on the neurological status at the time of diagnosis, and disease progression is not completely inhibited. Awareness of the disease and newborn screening is needed for earlier disease detection. In addition, further optimization of the treatment protocol and additional treatments are needed to improve patient outcomes.

Endo-lysosomal dysfunction and neuronal-glial crosstalk in Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a rare progressive lysosomal lipid storage disorder that manifests with a heterogeneous spectrum of clinical syndromes, including visceral, neurological and psychiatric symptoms. This monogenetic autosomal recessive disease is largely caused by mutations in the NPC1 gene, which controls intracellular lipid homeostasis. Vesicle-mediated endo-lysosomal lipid trafficking and non-vesicular lipid exchange via inter-organelle membrane contact sites are both regulated by the NPC1 protein. Loss of NPC1 function therefore triggers intracellular accumulation of diverse lipid species, including cholesterol, glycosphingolipids, sphingomyelin and sphingosine. The NPC1-mediated dysfunction of lipid transport has severe consequences for all brain cells, leading to neurodegeneration. Besides the cell-autonomous contribution of neuronal NPC1, aberrant NPC1 signalling in other brain cells is critical for the pathology. We discuss here the importance of endo-lysosomal dysfunction and a tight crosstalk between neurons, oligodendrocytes, astrocytes and microglia in NPC pathology. We strongly believe that a cell-specific rescue may not be sufficient to counteract the severity of the NPC pathology, but targeting common mechanisms, such as endo-lysosomal and lipid trafficking dysfunction, may ameliorate NPC pathology. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Mitochondrial dysfunction in NPC1-deficiency is not rescued by drugs targeting the glucosylceramidase GBA2 and the cholesterol-binding proteins TSPO and StARD1.

Niemann-Pick type C disease (NPCD) is a rare neurodegenerative disorder most commonly caused by mutations in the lysosomal protein Niemann-Pick C1 (NPC1), which is implicated in cholesterol export. Mitochondrial insufficiency forms a significant feature of the pathology of this disease, yet studies attempting to address this are rare. The working hypothesis is that mitochondria become overloaded with cholesterol which renders them dysfunctional. We examined two potential protein targets-translocator protein (TSPO) and steroidogenic acute regulatory protein D1 (StARD1)-which are implicated in cholesterol transport to mitochondria, in addition to glucocerbrosidase 2 (GBA2), the target of miglustat, which is currently the only approved treatment for NPCD. However, inhibiting these proteins did not correct the mitochondrial defect in NPC1-deficient cells.

Differential Interferon Signaling Regulation and Oxidative Stress Responses in the Cerebral Cortex and Cerebellum Could Account for the Spatiotemporal Pattern of Neurodegeneration in Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative condition caused by genetic mutations of the NPC1 or NPC2 genes that encode the NPC1 and NPC2 proteins, respectively, which are believed to be responsible for cholesterol efflux from late-endosomes/lysosomes. The pathogenic mechanisms that lead to neurodegeneration in NPC are not well understood. There are, however, well-defined spatiotemporal patterns of neurodegeneration that may provide insight into the pathogenic process. For example, the cerebellum is severely affected from early disease stages, compared with cerebral regions, which remain relatively spared until later stages. Using a genome-wide transcriptome analysis, we have recently identified an aberrant pattern of interferon activation in the cerebella of pre-symptomatic Npc1-/- mice. Here, we carried out a comparative transcriptomic analysis of cerebral cortices and cerebella of pre-symptomatic Npc1-/- mice and age-matched controls to identify differences that may help explain the pathological progression within the NPC brain. We report lower cerebral expression of genes within interferon signaling pathways, and significant differences in the regulation of oxidative stress, compared with the cerebellum. Our findings suggest that a delayed onset of interferon signaling, possibly linked to lower oxidative stress, may account for the slower onset of cerebral cortical pathology in the disease.

Impact of miR-29c-3p in the Nucleus Accumbens on Methamphetamine-Induced Behavioral Sensitization and Neuroplasticity-Related Proteins.

Methamphetamine (METH) abuse inflicts both physical and psychological harm. While our previous research has established the regulatory role of miR-29c-3p in behavior sensitization, the underlying mechanisms and target genes remain incompletely understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique in conjunction with Ingenuity pathway analysis (IPA) to probe the putative molecular mechanisms of METH sensitization through miR-29c-3p inhibition. Through a microinjection of AAV-anti-miR-29c-3p into the nucleus accumbens (NAc) of mice, we observed the attenuation of METH-induced locomotor effects. Subsequent iTRAQ analysis identified 70 differentially expressed proteins (DEPs), with 22 up-regulated potential target proteins identified through miR-29c-3p target gene prediction and IPA analysis. Our focus extended to the number of neuronal branches, the excitatory synapse count, and locomotion-related pathways. Notably, GPR37, NPC1, and IREB2 emerged as potential target molecules for miR-29c-3p regulation, suggesting their involvement in the modulation of METH sensitization. Quantitative PCR confirmed the METH-induced aberrant expression of Gpr37, Npc1, and Ireb2 in the NAc of mice. Specifically, the over-expression of miR-29c-3p led to a significant reduction in the mRNA level of Gpr37, while the inhibition of miR-29c-3p resulted in a significant increase in the mRNA level of Gpr37, consistent with the regulatory principle of miRNAs modulating target gene expression. This suggests that miR-29c-3p potentially influences METH sensitization through its regulation of neuroplasticity. Our research indicates that miR-29c-3p plays a crucial role in regulating METH-induced sensitization, and it identified the potential molecular of miR-29c-3p in regulating METH-induced sensitization.

Trial of N-Acetyl-l-Leucine in Niemann-Pick Disease Type C.

BACKGROUND: Niemann-Pick disease type C is a rare lysosomal storage disorder. We evaluated the safety and efficacy of N-acetyl-l-leucine (NALL), an agent that potentially ameliorates lysosomal and metabolic dysfunction, for the treatment of Niemann-Pick disease type C. METHODS: In this double-blind, placebo-controlled, crossover trial, we randomly assigned patients 4 years of age or older with genetically confirmed Niemann-Pick disease type C in a 1:1 ratio to receive NALL for 12 weeks, followed by placebo for 12 weeks, or to receive placebo for 12 weeks, followed by NALL for 12 weeks. NALL or matching placebo was administered orally two to three times per day, with patients 4 to 12 years of age receiving weight-based doses (2 to 4 g per day) and those 13 years of age or older receiving a dose of 4 g per day. The primary end point was the total score on the Scale for the Assessment and Rating of Ataxia (SARA; range, 0 to 40, with lower scores indicating better neurologic status). Secondary end points included scores on the Clinical Global Impression of Improvement, the Spinocerebellar Ataxia Functional Index, and the Modified Disability Rating Scale. Crossover data from the two 12-week periods in each group were included in the comparisons of NALL with placebo. RESULTS: A total of 60 patients 5 to 67 years of age were enrolled. The mean baseline SARA total scores used in the primary analysis were 15.88 before receipt of the first dose of NALL (60 patients) and 15.68 before receipt of the first dose of placebo (59 patients; 1 patient never received placebo). The mean (±SD) change from baseline in the SARA total score was -1.97±2.43 points after 12 weeks of receiving NALL and -0.60±2.39 points after 12 weeks of receiving placebo (least-squares mean difference, -1.28 points; 95% confidence interval, -1.91 to -0.65; P<0.001). The results for the secondary end points were generally supportive of the findings in the primary analysis, but these were not adjusted for multiple comparisons. The incidence of adverse events was similar with NALL and placebo, and no treatment-related serious adverse events occurred. CONCLUSIONS: Among patients with Niemann-Pick disease type C, treatment with NALL for 12 weeks led to better neurologic status than placebo. A longer period is needed to determine the long-term effects of this agent in patients with Niemann-Pick disease type C. (Funded by IntraBio; ClinicalTrials.gov number, NCT05163288; EudraCT number, 2021-005356-10.).

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders.

Lysosomal storage disorders (LSDs), which are characterized by genetic and metabolic lysosomal dysfunctions, constitute over 60 degenerative diseases with considerable health and economic burdens. However, the mechanisms driving the progressive death of functional cells due to lysosomal defects remain incompletely understood, and broad-spectrum therapeutics against LSDs are lacking. Here, we found that various gene abnormalities that cause LSDs, including Hexb, Gla, Npc1, Ctsd and Gba, all shared mutual properties to robustly autoactivate neuron-intrinsic cGAS-STING signalling, driving neuronal death and disease progression. This signalling was triggered by excessive cytoplasmic congregation of the dsDNA and DNA sensor cGAS in neurons. Genetic ablation of cGAS or STING, digestion of neuronal cytosolic dsDNA by DNase, and repair of neuronal lysosomal dysfunction alleviated symptoms of Sandhoff disease, Fabry disease and Niemann-Pick disease, with substantially reduced neuronal loss. We therefore identify a ubiquitous mechanism mediating the pathogenesis of a variety of LSDs, unveil an inherent connection between lysosomal defects and innate immunity, and suggest a uniform strategy for curing LSDs.

Novel compound heterozygous mutations of the NPC1 gene associated with Niemann-pick disease type C: a case report and review of the literature.

BACKGROUND: Niemann-Pick Disease type C is a fatal autosomal recessive lipid storage disorder caused by NPC1 or NPC2 gene mutations and characterized by progressive, disabling neurological deterioration and hepatosplenomegaly. Herein, we identified a novel compound heterozygous mutations of the NPC1 gene in a Chinese pedigree. CASE PRESENTATION: This paper describes an 11-year-old boy with aggravated walking instability and slurring of speech who presented as Niemann-Pick Disease type C. He had the maternally inherited c.3452 C > T (p. Ala1151Val) mutation and the paternally inherited c.3557G > A (p. Arg1186His) mutation using next-generation sequencing. The c.3452 C > T (p. Ala1151Val) mutation has not previously been reported. CONCLUSIONS: This study predicted that the c.3452 C > T (p. Ala1151Val) mutation is pathogenic. This data enriches the NPC1 gene variation spectrum and provides a basis for familial genetic counseling and prenatal diagnosis.

Npc1 gene mutation abnormally activates the classical Wnt signalling pathway in mouse kidneys and promotes renal fibrosis.

Niemann-Pick disease type C1 (NPC1) is a lysosomal lipid storage disease caused by NPC1 gene mutation. Our previous study found that, compared with wild-type (Npc1+/+ ) mice, the renal volume and weight of Npc1 gene mutant (Npc1-/- ) mice were significantly reduced. We speculate that Npc1 gene mutations may affect the basic structure of the kidneys of Npc1-/- mice, and thus affect their function. Therefore, we randomly selected postnatal Day 28 (P28) and P56 Npc1+/+ and Npc1-/- mice, and observed the renal structure and pathological changes by haematoxylin-eosin staining. The level of renal fibrosis was detected by immunofluorescence histochemical techniques, and western blotting was used to detect the expression levels of apoptosis-related proteins and canonical Wnt signalling pathway related proteins. The results showed that compared with Npc1+/+ mice, the kidneys of P28 and P56 Npc1-/- mice underwent apoptosis and fibrosis; furthermore, there were obvious vacuoles in the cytoplasm of renal tubular epithelial cells of P56 Npc1-/- mice, the cell bodies were loose and foam-like, and the canonical Wnt signalling pathway was abnormally activated. These results showed that Npc1 gene mutation can cause pathological changes in the kidneys of mice. As age increased, vacuoles developed in the cytoplasm of renal tubular epithelial cells, and apoptosis of renal cells, abnormal activation of the Wnt signalling pathway, and promotion of renal fibrosis increased.

Comparative Hippocampal Proteome and Phosphoproteome in a Niemann-Pick, Type C1 Mouse Model Reveal Insights into Disease Mechanisms.

Niemann-Pick disease, type C (NPC) is a neurodegenerative, lysosomal storage disorder in individuals carrying two mutated copies of either the NPC1 or NPC2 gene. Consequently, impaired cholesterol recycling and an array of downstream events occur. Interestingly, in NPC, the hippocampus displays lysosomal lipid storage but does not succumb to progressive neurodegeneration as significantly as other brain regions. Since defining the neurodegeneration mechanisms in this disease is still an active area of research, we use mass spectrometry to analyze the overall proteome and phosphorylation pattern changes in the hippocampal region of a murine model of NPC. Using 3 week old mice representing an early disease time point, we observed changes in the expression of 47 proteins, many of which are consistent with the previous literature. New to this study, changes in members of the SNARE complex, including STX7, VTI1B, and VAMP7, were identified. Furthermore, we identified that phosphorylation of T286 on CaMKIIα and S1303 on NR2B increased in mutant animals, even at the late stage of the disease. These phosphosites are crucial to learning and memory and can trigger neuronal death by altering protein-protein interactions.

Development and validation of a new genotype-phenotype correlation for Niemann-Pick disease type C1.

Hundreds of NPC1 variants cause highly heterogeneous phenotypes. This study aims to explore the genotype-phenotype correlation of NPC1, especially for missense variants. In a well-characterized cohort, phenotypes are graded into three clinical forms: mild, intermediate, and severe. Missense residue structural location was stratified into three categories: surface, partially, and fully buried. The association of phenotypes with the topography of the amino acid substitution in the protein structure was investigated in our cohort and validated in two reported cohorts. One hundred six unrelated NPC1 patients were enrolled. A significant correlation of genotype-phenotype was found in 81 classified individuals with two or one (the second was null variant) missense variant (p < 0.001): of 25 patients with at least one missense variant of surface (group A), 19 (76%) mild, six (24%) intermediate, and none severe; of 31 cases with at least one missense variant of partially buried without surface variants (group B), 11 (35%) mild, 16 (52%) intermediate, and four (13%) severe; of the remaining 25 patients with two or one buried missense variants (group C), eight (32%) mild, nine (36%) intermediate, and eight (32%) severe. Additionally, 7-ketocholesterol, the biomarker, was lower in group A than in group B (p = 0.024) and group C (p = 0.029). A model was proposed that accurately predicted phenotypes of 72 of 90 (80%), 73 of85 (86%), and 64 of 69 (93%) patients in our cohort, Italian, and UK cohort, respectively. This study proposed a novel genotype-phenotype correlation in NPC1, linking the underlying molecular pathophysiology with clinical phenotype and aiding genetic counseling and evaluation in clinical practice.

Characterization of central manifestations in patients with Niemann-Pick disease type C.

PURPOSE: Niemann-Pick disease type C (NPC) is a rare lysosomal storage disease characterized by progressive neurodegeneration and neuropsychiatric symptoms. This study investigated pathophysiological mechanisms underlying motor deficits, particularly speech production, and cognitive impairment. METHODS: We prospectively phenotyped 8 adults with NPC and age-sex-matched healthy controls using a comprehensive assessment battery, encompassing clinical presentation, plasma biomarkers, hand-motor skills, speech production, cognitive tasks, and (micro-)structural and functional central nervous system properties through magnetic resonance imaging. RESULTS: Patients with NPC demonstrated deficits in fine-motor skills, speech production timing and coordination, and cognitive performance. Magnetic resonance imaging revealed reduced cortical thickness and volume in cerebellar subdivisions (lobule VI and crus I), cortical (frontal, temporal, and cingulate gyri) and subcortical (thalamus and basal ganglia) regions, and increased choroid plexus volumes in NPC. White matter fractional anisotropy was reduced in specific pathways (intracerebellar input and Purkinje tracts), whereas diffusion tensor imaging graph theory analysis identified altered structural connectivity. Patients with NPC exhibited altered activity in sensorimotor and cognitive processing hubs during resting-state and speech production. Canonical component analysis highlighted the role of cerebellar-cerebral circuitry in NPC and its integration with behavioral performance and disease severity. CONCLUSION: This deep phenotyping approach offers a comprehensive systems neuroscience understanding of NPC motor and cognitive impairments, identifying potential central nervous system biomarkers.

Cholesterol redistribution triggered by CYP46A1 gene therapy improves major hallmarks of Niemann-Pick type C disease but is not sufficient to halt neurodegeneration.

Cholesterol 24-hydroxylase (CYP46A1) is an exclusively neuronal cytochrome P450 enzyme responsible for converting cholesterol into 24S-hydroxycholesterol, which serves as the primary pathway for eliminating cholesterol in the brain. We and others have shown that increased activity of CYP46A1 leads to reduced levels of cholesterol and has a positive effect on cognition. Therefore, we hypothesized that CYP46A1 could be a potential therapeutic target in Niemann-Pick type C (NPC) disease, a rare and fatal neurodegenerative disorder, characterized by cholesterol accumulation in endolysosomal compartments. Herein, we show that CYP46A1 ectopic expression, in cellular models of NPC and in Npc1tm(I1061T) mice by adeno-associated virus-mediated gene therapy improved NPC disease phenotype. Amelioration in functional, biochemical, molecular and neuropathological hallmarks of NPC disease were characterized. In vivo, CYP46A1 expression partially prevented weight loss and hepatomegaly, corrected the expression levels of genes involved in cholesterol homeostasis, and promoted a redistribution of brain cholesterol accumulated in late endosomes/lysosomes. Moreover, concomitant with the amelioration of cholesterol metabolism dysregulation, CYP46A1 attenuated microgliosis and lysosomal dysfunction in mouse cerebellum, favoring a pro-resolving phenotype. In vivo CYP46A1 ectopic expression improves important features of NPC disease and may represent a valid therapeutic approach to be used concomitantly with other drugs. However, promoting cholesterol redistribution does not appear to be enough to prevent Purkinje neuronal death in the cerebellum. This indicates that cholesterol buildup in neurons might not be the main cause of neurodegeneration in this human lipidosis.

Global Proteomics for Identifying the Alteration Pathway of Niemann-Pick Disease Type C Using Hepatic Cell Models.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder with progressive neurodegeneration. Although the causative genes were previously identified, NPC has unclear pathophysiological aspects, and patients with NPC present various symptoms and onset ages. However, various novel biomarkers and metabolic alterations have been investigated; at present, few comprehensive proteomic alterations have been reported in relation to NPC. In this study, we aimed to elucidate proteomic alterations in NPC and perform a global proteomics analysis for NPC model cells. First, we developed two NPC cell models by knocking out NPC1 using CRISPR/Cas9 (KO1 and KO2). Second, we performed a label-free (LF) global proteomics analysis. Using the LF approach, more than 300 proteins, defined as differentially expressed proteins (DEPs), changed in the KO1 and/or KO2 cells, while the two models shared 35 DEPs. As a bioinformatics analysis, the construction of a protein-protein interaction (PPI) network and an enrichment analysis showed that common characteristic pathways such as ferroptosis and mitophagy were identified in the two model cells. There are few reports of the involvement of NPC in ferroptosis, and this study presents ferroptosis as an altered pathway in NPC. On the other hand, many other pathways and DEPs were previously suggested to be associated with NPC, supporting the link between the proteome analyzed here and NPC. Therapeutic research based on these results is expected in the future.

Trehalose enhances mitochondria deficits in human NPC1 mutant fibroblasts but disrupts mouse Purkinje cell dendritic growth ex vivo.

Lysosomes play important roles in catabolism, nutrient sensing, metabolic signaling, and homeostasis. NPC1 deficiency disrupts lysosomal function by inducing cholesterol accumulation that leads to early neurodegeneration in Niemann-Pick type C (NPC) disease. Mitochondria pathology and deficits in NPC1 deficient cells are associated with impaired lysosomal proteolysis and metabolic signaling. It is thought that activation of the transcription factor TFEB, an inducer of lysosome biogenesis, restores lysosomal-autophagy activity in lysosomal storage disorders. Here, we investigated the effect of trehalose, a TFEB activator, in the mitochondria pathology of NPC1 mutant fibroblasts in vitro and in mouse developmental Purkinje cells ex vivo. We found that in NPC1 mutant fibroblasts, serum starvation or/and trehalose treatment, both activators of TFEB, reversed mitochondria fragmentation to a more tubular mitochondrion. Trehalose treatment also decreased the accumulation of Filipin+ cholesterol in NPC1 mutant fibroblasts. However, trehalose treatment in cerebellar organotypic slices (COSCs) from wild-type and Npc1nmf164 mice caused mitochondria fragmentation and lack of dendritic growth and degeneration in developmental Purkinje cells. Our data suggest, that although trehalose successfully restores mitochondria length and decreases cholesterol accumulation in NPC1 mutant fibroblasts, in COSCs, Purkinje cells mitochondria and dendritic growth are negatively affected possibly through the overactivation of the TFEB-lysosomal-autophagy pathway.

Challenges in the Definitive Diagnosis of Niemann-Pick Type C-Leaky Variants and Alternative Transcripts.

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.