Niemann-Pick Type Research Articles

Investigating p.Ala1035Val in NPC1: New Cellular Models for Niemann–Pick Type C Disease

Niemann–Pick type C (NPC) is a lysosomal storage disorder (LSD) caused by pathogenic variants in either the NPC1 or NPC2 genes, which encode proteins involved in the lysosomal export of unesterified cholesterol. In patients of Western European descent, the p.Ile1061Thr variant in NPC1 is especially prevalent. However, mounting evidence has positioned p.Ala1035Val as the most common variant in Portugal and the second most prevalent variant worldwide. By analyzing 10 Portuguese NPC patients homozygous for p.Ala1035Val, we found an SNP in cis on position 858 (p.Ile858Val), which we hypothesize could have a disease-modifying effect. To address this query, we created variant-specific in vitro models of NPC by stably transducing NPC1−/− ARPE-19 cells with constructs encoding different fluorescently-tagged variants of NPC1, which we used, alongside patient-derived skin fibroblasts, to investigate lysosomal positioning and the trafficking routes elicited by p.Ile1061Thr and p.Ala1035Val (with and without the p.Ile858Val SNP in cis). Our results corroborate the previously described decrease in p.Ile1061Thr-NPC1 trafficking to the lysosome and suggest a similar, if not worse, scenario for the p.Ala1035Val variant, especially when in cis with p.Ile858Val. This is the first reported functional study addressing the impact of the p.Ala1035Val variant at the cellular level, paving the way for novel therapeutic options.

How to diagnose acid sphingomyelinase deficiency (ASMD) and Niemann-Pick disease type C from bone marrow and peripheral blood smears.

How to diagnose acid sphingomyelinase deficiency (ASMD) and Niemann-Pick disease type C from bone marrow and peripheral blood smears.

Plasma phosphorylated-tau217 is increased in Niemann-Pick disease type C.

Niemann-Pick disease type C and Alzheimer's disease are distinct neurodegenerative disorders that share the presence of neurofibrillary tangle pathology. In this multicentre study, we measured plasma phosphorylated-tau217 in controls (n = 60), Niemann-Pick disease type C (n = 71) and Alzheimer's disease (n = 30 positive for amyloid and negative for tau in CSF [A+T-] and n = 30 positive for both [A+T+]). Annual Severity Increment Score and Lysotracker measurements were evaluated in the Niemann-Pick disease type C group to estimate the rate of progression and lysosomal enlargement, respectively. In the cross-sectional analysis, plasma phosphorylated-tau217 was increased in Niemann-Pick disease type C compared with controls (2.52 ± 1.93 versus 1.02 ± 0.34 pg/mL, respectively, P < 0.001) and inversely correlated with age at disease onset (R = -0.54, P < 0.001). In the longitudinal analysis, plasma phosphorylated-tau217 was associated with disease progression determined by Annual Severity Increment Score (R = 0.48, P < 0.001) and lysosomal enlargement (R = 0.26, P = 0.004). We found no differences between A+T- Alzheimer's disease and Niemann-Pick disease type C (2.67 ± 1.18 versus 2.52 ± 1. 93 pg/mL, P = 0.31); however, A+T+ Alzheimer's disease had significantly higher levels than Niemann-Pick disease type C (3.26 ± 1.36 versus 2.52 ± 1.93 pg/mL, P = 0.001). Our findings suggest that plasma p-tau217 can increase in brain disorders with isolated tau pathology. Plasma p-tau217 associations with disease progression and severity make it a potential marker in Niemann-Pick disease type C.

Structural and biochemical analysis of ligand binding in yeast Niemann-Pick type C1-related protein.

In eukaryotes, integration of sterols into the vacuolar/lysosomal membrane is critically dependent on the Niemann-Pick type C (NPC) system. The system consists of an integral membrane protein, called NCR1 in yeast, and NPC2, a luminal soluble protein that transfers sterols to the N-terminal domain (NTD) of NCR1 before membrane integration. Both proteins have been implicated in sterol homeostasis of yeast and humans. Here, we investigate sterol and lipid binding of the NCR1/NPC2 transport system and determine crystal structures of the sterol binding NTD. The NTD binds both ergosterol and cholesterol, with nearly identical conformations of the binding pocket. Apart from sterols, the NTD can also bind fluorescent analogs of phosphatidylinositol, phosphatidylcholine, and phosphatidylserine, as well as sphingosine and ceramide. We confirm the multi-lipid scope of the NCR1/NPC2 system using photo-crosslinkable and clickable lipid analogs, namely, pac-cholesterol, pac-sphingosine, and pac-ceramide. Finally, we reconstitute the transfer of pac-sphingosine from NPC2 to the NTD in vitro. Collectively, our results support that the yeast NPC system can work as versatile machinery for vacuolar homeostasis of structurally diverse lipids, besides ergosterol.

Enhanced mGluR5 intracellular activity causes psychiatric alterations in Niemann Pick type C disease

Niemann-Pick disease Type C (NPC) is caused by mutations in the cholesterol transport protein NPC1 leading to the endolysosomal accumulation of the lipid and to psychiatric alterations. Using an NPC mouse model (Npc1nmf164) we show aberrant mGluR5 lysosomal accumulation and reduction at plasma membrane in NPC1 deficient neurons. This phenotype was induced in wild-type (wt) neurons by genetic and pharmacological NPC1 silencing. Extraction of cholesterol normalized mGluR5 distribution in NPC1-deficient neurons. Intracellular accumulation of mGluR5 was functionally active leading to enhanced mGluR-dependent long-term depression (mGluR-LTD) in Npc1nmf164 hippocampal slices. mGluR-LTD was lower or higher in Npc1nmf164 slices compared with wt when stimulated with non-membrane-permeable or membrane-permeable mGluR5 agonists, respectively. Oral treatment with the mGluR5 antagonist 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP) reduced mGluR-LTD and ameliorated psychiatric anomalies in the Npc1nmf164 mice. Increased neuronal mGluR5 levels were found in an NPC patient. These results implicate mGluR5 alterations in NPC psychiatric condition and provide a new therapeutic strategy that might help patients suffering from this devastating disease.

Rosa canina L. Methanol Extract and Its Component Rutin Reduce Cholesterol More Efficiently than Miglustat in Niemann–Pick C Fibroblasts

Niemann–Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder where 95% of the cases are caused by mutations in the Niemann–Pick C1 (NPC1) gene. Loss of function in NPC1 mutants trigger the accumulation of cholesterol in late endo-lysosomes and lysosomal dysfunction. The current study examined the potential of polyphenol-rich methanol extracts from Rosa canina L. (RCME) and two of its components, rutin and quercitrin, to enhance protein trafficking of NPC1 and restore cholesterol levels in fibroblasts derived from NPC patients, in comparison with miglustat, a drug approved in Europe for NPC treatment. Interestingly, RCME improved the trafficking of the compound heterozygous mutant NPC1I1061T/P887L, homozygous mutant NPC1R1266Q, and heterozygous mutant NPC1N1156S between the endoplasmic reticulum and the Golgi and significantly reduced the levels of cellular cholesterol in the cell lines examined. Miglustat did not affect the trafficking of the three NPC1 mutants individually nor in combination with RCME. Markedly, rutin and quercitrin exerted their effects on cholesterol, but not in the trafficking pathway of NPC1, indicating that other components in RCME are implicated in regulating the trafficking of NPC1 mutants. By virtue of its dual function in targeting the trafficking of mutants of NPC1 as well as the cholesterol contents, RCME is more beneficial than available drugs that target substrate reduction and should be therefore considered in further studies for its feasibility as a therapeutic agent for NPC patients.

Hospital dental care for patients with Niemann-Pick syndrome type B: a case report

The aim of this case report is to show the hospital dental approach to a patient with Niemann-Pick disease (NPD) type B. After her caregivers reported poor oral health, a 10-year-old girl was referred to a hospital. She was diagnosed with NPD type B 4 years ago, with involvement of the brain, nerves, liver, spleen, bone marrow, and lungs. A dental physical examination revealed chronic carious lesions in ten teeth, as well as four residual roots and poor oral hygiene. We chose a surgical block treatment based on the altered complementary exams, dental demands, and the systemic compromise caused by NPD type B. All decayed elements were restored with glass ionomer cement, and residual roots were extracted. The patient had no clinical signs of infection or hemorrhage after 20 days of post-surgical follow-up. Although rare, the presence of patients with NPD type B in the dental routine can occur, necessitating the development of an individualized and multidisciplinary treatment plan.

Oncological Aspects of Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are caused by the deficient activity of a lysosomal hydrolase or the lack of a functional membrane protein, transporter, activator, or other protein. Lysosomal enzymes break down macromolecular compounds, which contribute to metabolic homeostasis. Stored, undegraded materials have multiple effects on cells that lead to the activation of autophagy and apoptosis, including the toxic effects of lyso-lipids, the disruption of intracellular Ca2+ ion homeostasis, the secondary storage of macromolecular compounds, the activation of signal transduction, apoptosis, inflammatory processes, deficiencies of intermediate compounds, and many other pathways. Clinical observations have shown that carriers of potentially pathogenic variants in LSD-associated genes and patients affected with some LSDs are at a higher risk of cancer, although the results of studies on the frequency of oncological diseases in LSD patients are controversial. Cancer is found in individuals affected with Gaucher disease, Fabry disease, Niemann-Pick type A and B diseases, alfa-mannosidosis, and sialidosis. Increased cancer prevalence has also been reported in carriers of a potentially pathogenic variant of an LSD gene, namely CLN3, SGSH, GUSB, NEU1, and, to a lesser extent, in other genes. In this review, LSDs in which oncological events can be observed are described.

Decision tree insights analytics (DTIA) tool: an analytic framework to identify insights from large data records across fields of science

Abstract Supervised machine learning (SML) techniques have been developed since the 1960s. Most of their applications were oriented towards developing models capable of predicting numerical values or categorical output based on a set of input variables (input features). Recently, SML models’ interpretability and explainability were extensively studied to have confidence in the models’ decisions. In this work, we propose a new deployment method named Decision Tree Insights Analytics (DTIA) that shifts the purpose of using decision tree classification from having a model capable of differentiating the different categorical outputs based on the input features to systematically finding the associations between inputs and outputs. DTIA can reveal interesting areas in the feature space, leading to the development of research questions and the discovery of new associations that might have been overlooked earlier. We applied the method to three case studies: (1) nuclear reactor accident propagation, (2) single-cell RNA sequencing of Niemann-Pick disease type C1 in mice, and (3) bulk RNA sequencing for breast cancer staging in humans. The developed method provided insights into the first two. On the other hand, it showed some of the method’s limitations in the third case study. Finally, we presented how the DTIA’s insights are more agreeable with the abstract information gain calculations and provide more in-depth information that can help derive more profound physical meaning compared to the random forest’s feature importance attribute and K-means clustering for feature ranking.

NPC1 links cholesterol trafficking to microglial morphology via the gastrosome.

Microglia play important roles in brain development and homeostasis by removing dying neurons through efferocytosis. Morphological changes in microglia are hallmarks of many neurodegenerative conditions, such as Niemann-Pick disease type C. Here, NPC1 loss causes microglia to shift from a branched to an ameboid form, though the cellular basis and functional impact of this change remain unclear. Using zebrafish, we show that NPC1 deficiency causes an efferocytosis-dependent expansion of the microglial gastrosome, a collection point for engulfed material. In vivo and in vitro experiments on microglia and mammalian macrophages demonstrate that NPC1 localizes to the gastrosome, and its absence leads to cholesterol accumulation in this compartment. NPC1 loss and neuronal cell death synergistically affect gastrosome size and cell shape, increasing the sensitivity of NPC1-deficient cells to neuronal cell death. Finally, we demonstrate conservation of cholesterol accumulation and gastrosome expansion in NPC patient-derived fibroblasts, offering an interesting target for further disease investigation.

Molecular determinants of phospholipid treatment to reduce intracellular cholesterol accumulation in NPC1 deficiency

Niemann-Pick type C (NPC) disease, caused by mutations in the NPC1 or NPC2 genes, leads to abnormal intracellular cholesterol accumulation in late endosomes/lysosomes (LE/LY). Exogenous enrichment with lysobisphosphatidic acid (LBPA), also known as bis-monoacylglycerol phosphate or BMP, either directly or via the LBPA precursor phosphatidylglycerol (PG), has been investigated as a therapeutic intervention to reduce cholesterol accumulation in NPC disease. Here we report the effects of stereoisomer configuration and acyl chain composition of LBPA on cholesterol clearance in NPC1-deficient cells. We find that S,R, S,S, and S,R LBPA stereoisomers behaved similarly, with all 3 compounds leading to comparable reductions in filipin staining in two NPC1-deficient human fibroblast cell lines. Examination of several LBPA molecular species containing one or two mono- or polyunsaturated acyl chains showed that all LBPA species containing one 18:1 chain significantly reduced cholesterol accumulation, whereas the shorter chain species di-14:0 LBPA had little effect on cholesterol clearance in NPC1 deficient cells. Since cholesterol accumulation in NPC1 deficient cells can also be cleared by PG incubation, we used non-hydrolyzable PG analogues to determine whether conversion to LBPA is required for sterol clearance, or whether PG itself is effective. The results showed that non-hydrolyzable PG species were not appreciably converted to LBPA and showed virtually no cholesterol clearance efficacy in NPC1 deficient cells, supporting the notion that LBPA is the active agent promoting LE/LY cholesterol clearance. Overall these studies are helping to define the molecular requirements for potential therapeutic use of LBPA as an option for addressing NPC disease.

Graphene Microelectrode Arrays, 4D Structured Illumination Microscopy, and a Machine Learning Spike Sorting Algorithm Permit the Analysis of Ultrastructural Neuronal Changes During Neuronal Signaling in a Model of Niemann-Pick Disease Type C.

Simultaneously recording network activity and ultrastructural changes of the synapse is essential for advancing understanding of the basis of neuronal functions. However, the rapid millisecond-scale fluctuations in neuronal activity and the subtle sub-diffraction resolution changes of synaptic morphology pose significant challenges to this endeavor. Here, specially designed graphene microelectrode arrays (G-MEAs) are used, which are compatible with high spatial resolution imaging across various scales as well as permit high temporal resolution electrophysiological recordings to address these challenges. Furthermore, alongside G-MEAs, an easy-to-implement machine learning algorithm is developed to efficiently process the large datasets collected from MEA recordings. It is demonstrated that the combined use of G-MEAs, machine learning (ML) spike analysis, and 4D structured illumination microscopy (SIM) enables monitoring the impact of disease progression on hippocampal neurons which are treated with an intracellular cholesterol transport inhibitor mimicking Niemann-Pick disease type C (NPC), and show that synaptic boutons, compared to untreated controls, significantly increase in size, leading to a loss in neuronal signaling capacity.

Metronome-Guided Balance Exercises to Aid in Dynamic Balance and Postural Control in a Novel Genetic Presentation of Niemann–Pick Disease Type C: A Case Report

Abstract Niemann–Pick disease type C is a rare progressive lysosomal lipid storage disorder. The disorder is characterized by a range of neurological consequences and complications at different points of its progression including spasticity, incoordination, ataxia, supranuclear gaze palsy, asthenia, and balance impairment, with a higher risk of falls. Among various approaches to managing such a debilitating condition, the role of physical therapy in managing the symptoms of such patients is often limited or underreported in the literature. A combination of spasticity and ataxia leads to impaired timing of agonist–antagonist control. Therefore, balance loss and falls are the most prevalent symptoms. We advocated a 12-week metronome-guided balance training program. Niemann–Pick type C disease is a rare genetic phenomenon that often presents with multi-system changes at structural and functional levels for the patient. Metronome-guided balance training is beneficial for improving dynamic balance variables, including movement velocity, center of gravity sway velocity, and step width.

Global and Targeted Metabolomics for Revealing Metabolomic Alteration in Niemann-Pick Disease Type C Model Cells.

Niemann-Pick disease type C (NPC) is an inherited disorder characterized by a functional deficiency of cholesterol transport proteins. However, the molecular mechanisms and pathophysiology of the disease remain unknown. In this study, we identified several metabolite characteristics of NPC that may fluctuate in a cellular model of the disease, using both global and targeted metabolomic analyses by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Three cell lines, HepG2 cells (wild-type[WT]) and two NPC model HepG2 cell lines in which NPC1 was genetically ablated (knockout [KO]1 and KO2), were used for metabolomic analysis. Data were subjected to enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The enrichment analysis of global metabolomics revealed that 8 pathways in KO1 and 16 pathways in KO2 cells were notably altered. In targeted metabolomics for 15 metabolites, 4 metabolites in KO1 and 10 metabolites in KO2 exhibited statistically significant quantitative changes in KO1 or KO2 relative to WT. Most of the altered metabolites were related to creatinine synthesis and cysteine metabolism pathways. In the future, our objective will be to elucidate the relationship between these metabolic alterations and pathophysiology.

Assessment of the reliability, responsiveness, and meaningfulness of the scale for the assessment and rating of ataxia (SARA) for lysosomal storage disorders.

To evaluate the reliability, responsiveness, and validity of theScale for the Assessment and Rating of Ataxia (SARA) in patients with lysosomal storage disorders (LSDs) who present with neurological symptoms, and quantify the threshold for a clinically meaningful change. We analyzed data from three clinical trial cohorts (IB1001-201, IB1001-202, and IB1001-301) of patients with Niemann-Pick disease type C (NPC) and GM2 Gangliosidoses (Tay-Sachs and Sandhoff disease) comprising 122 patients and 703 visits. Reproducibility was described as re-test reliability between repeat baseline visits or baseline and post-treatment washout visits. Responsiveness was determined in relation to the Investigator's, Caregiver's, and Patient's Clinical Global Impression of Improvement (CGI-I). The CGI-I data was also used to quantify a threshold for a clinically meaningful improvement on the SARA scale. Using a qualitative methods approach, patient/caregiver interviews from the IB1001-301 trial were further used to assess a threshold of meaningful change as well as the breadth of neurological signs and symptoms captured and evaluated by the SARA scale. The Inter-Class Correlation (ICC) was 0.95 or greater for all three trials, indicating a high internal consistency/reliability. The mean change in SARA between repeat baseline and post-treatment washout visit assessments in all trials was -0.05, SD 1.98, i.e., minimal, indicating no significant differences, learning effects or other systematic biases. For the CGI-I responses and change in SARA scores, Area Under the Curve (AUC) values were 0.82, 0.71, and 0.77 for the Investigator's, Caregiver's, and Patient's CGI-I respectively, indicating strong agreement. Further qualitative analyses of the patient/caregiver interviews demonstrated a 1-point or greater change on SARA to be a clinically meaningful improvement which is directly relevant to the patient's everyday functioning and quality of life. Changes captured by the SARA were also paralleled by improvement in a broad range of neurological signs and symptoms and beyond cerebellar ataxia. Qualitative and quantitative data demonstrate the reliability and responsiveness of the SARA score as a valid measure of neurological signs and symptoms in LSDs with CNS involvement, such as NPC and GM2 Gangliosidoses. A 1-point change represents a clinically meaningful transition reflecting the gain or loss of complex function.

Bafilomycin A1 Inhibits HIV-1 Infection by Disrupting Lysosomal Cholesterol Transport.

The productive replication of human immunodeficiency virus type 1 (HIV-1) involves intricate interactions between viral proteins and host cell machinery. However, the contributions of the lysosomal pathways for HIV-1 replication are not fully understood. The goal of this study was to determine the impact of lysosome-targeting compounds on HIV-1 replication and identify the cellular changes that are linked to HIV-1 inhibition using cell culture models of HIV-1 infection. Here, we demonstrate that the treatment of cells with various pharmacological agents known to inhibit lysosomal functions interfere with HIV-1 replication. The vacuolar ATPase (V-ATPase) inhibitor bafilomycin A1 exerted a potent inhibition of HIV-1 replication. Bafilomycin A1 inhibition of HIV-1 was independent of coreceptor tropism of HIV-1. Our data suggest that bafilomycin A1 inhibits HIV-1 at the post-integration steps of the virus life cycle, which include viral gene expression, virus assembly, and/or egress. Analysis of the cellular alterations following bafilomycin A1 treatment indicates that bafilomycin A1 causes a disruption in lysosome structure and functions. Treatment of cells with bafilomycin A1 caused an accumulation of unesterified cholesterol in lysosomes along with the expansion of the lysosomal compartments. Interestingly, the overexpression of the lysosomal cholesterol transporter Niemann-Pick type C 1 (NPC1) partially relieved bafilomycin A1 inhibition of HIV-1. Collectively, our data suggest that bafilomycin A1 inhibits HIV-1 replication in part by disrupting the lysosomal cholesterol trafficking pathway.

Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

Advances in research on potential therapeutic approaches for Niemann-Pick C1 disease.

Niemann-Pick disease type C1 (NP-C1) is a rare and devastating recessive inherited lysosomal lipid and cholesterol storage disorder caused by mutations in the NPC1 or NPC2 gene. These two proteins bind to cholesterol and cooperate in endosomal cholesterol transport. Characteristic clinical manifestations of NP-C1 include hepatosplenomegaly, progressive neurodegeneration, and ataxia. While the rarity of NP-C1 presents a significant obstacle to progress, researchers have developed numerous potential therapeutic approaches over the past two decades to address this condition. Various methods have been proposed and continuously improved to slow the progression of NP-C1, although they are currently at an animal or clinical experimental stage. This overview of NP-C1 therapy will delve into different theoretical treatment strategies, such as small molecule therapies, cell-based approaches, and gene therapy, highlighting the complex therapeutic challenges associated with this disorder.

Dysregulation of muscle cholesterol transport in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons, with a typical lifespan of 3-5 years. Altered metabolism is a key feature of ALS that strongly influences prognosis, with an increase in whole-body energy expenditure and changes in skeletal muscle metabolism, including greater reliance on fat oxidation. Dyslipidemia has been described in ALS as part of the metabolic dysregulation, but its role in the pathophysiology of the disease remains controversial. Among the lipids, cholesterol is of particular interest as a vital component of cell membranes, playing a key role in signal transduction and mitochondrial function in muscle. The aim of this study was to investigate whether motor dysfunction in ALS might be associated with dysregulation of muscle cholesterol metabolism. We determined cholesterol content and analyzed the expression of key determinants of the cholesterol metabolism pathway in muscle biopsies from thirteen ALS patients and ten asymptomatic ALS-mutation gene carriers compared to sixteen controls. Using human control primary myotubes, we further investigated the potential contribution of cholesterol dyshomeostasis to reliance on mitochondrial fatty acid. We found that cholesterol accumulates in the skeletal muscle of ALS patients and that cholesterol overload significantly correlates with disease severity evaluated by the Revised ALS Functional Rating Scale. These defects are associated with overexpression of the genes of the lysosomal cholesterol transporters Niemann-Pick type C1 (NPC1) and 2 (NPC2), which are required for cholesterol transfer from late endosomes/lysosomes to cellular membranes. Most notably, a significant increase in NPC2 mRNA levels could be detected in muscle samples from asymptomatic ALS-mutation carriers, long before disease onset. We found that filipin-stained unesterified cholesterol accumulated in the lysosomal compartment in ALS muscle samples, suggesting dysfunction of the NPC1/2 system. Accordingly, we report here that experimental NPC1 inhibition or lysosomal pH alteration in human primary myotubes was sufficient to induce the overexpression of NPC1 and NPC2 mRNA. Finally, acute NPC1 inhibition in human control myotubes induced a shift towards a preferential use of fatty acids, thus reproducing the metabolic defect characteristic of ALS muscle. We conclude that cholesterol homeostasis is dysregulated in ALS muscle from the presymptomatic stage. Targeting NPC1/2 dysfunction may be a new therapeutic strategy for ALS to restore muscle energy metabolism and slow motor symptom progression.

Npc1 deficiency impairs microglia function via TREM2-mTOR signaling in Niemann-Pick disease type C

Niemann-Pick disease Type C (NPC) is a neurodegenerative disease mainly caused by the mutation in NPC1 gene, leading to massive accumulation of unesterified cholesterol in the late endosome/lysosome of cells. Impaired phenotype of microglia is a hallmark in Npc1 mutant mice (Npc1−/− mice). However, the mechanism of Npc1 in regulating microglial function is still unclear. Here, we showed that the reactive microglia in the neonatal Npc1−/− mice indicated by the increased lysosome protein CD68 and phagocytic activity were associated with disrupted TREM2-mTOR signaling in microglia. Furthermore, in Npc1-deficient BV2 cells, genetic deletion of Trem2 partially restored microglial function, probably via restored mTOR signaling. Taken together, our findings indicated that loss of Npc1 in microglia caused changes of their morphologies and the impairment of lysosomal function, which were linked to the TREM2-mTOR signaling pathway.