Analysis of Metabolic Changes in Endogenous Metabolites and Diagnostic Biomarkers for Various Diseases Using Liquid Chromatography and Mass Spectrometry.

Analysis of endogenous metabolites in various diseases is useful for searching diagnostic biomarkers and elucidating the molecular mechanisms of pathophysiology. The author and collaborators have developed some LC/tandem mass spectrometry (LC/MS/MS) methods for metabolites and applied them to disease-related samples. First, we identified urinary conjugated cholesterol metabolites and serum N-palmitoyl-O-phosphocholine serine as useful biomarkers for Niemann-Pick disease type C (NPC). For the purpose of intraoperative diagnosis of glioma patients, we developed the LC/MS/MS analysis methods for 2-hydroxyglutaric acid or cystine and found that they could be good differential biomarkers. For renal cell carcinoma, we searched for various biomarkers for early diagnosis, malignancy evaluation and recurrence prediction by global metabolome analysis and targeted LC/MS/MS analysis. In pathological analysis, we developed a simultaneous LC/MS/MS analysis method for 13 steroid hormones and applied it to NPC cells, we found 6 types of reductions in NPC model cells. For non-alcoholic steatohepatitis (NASH), model mice were prepared with special diet and plasma bile acids were measured, and as a result, hydrophilic bile acids were significantly increased. In addition, we developed an LC/MS/MS method for 17 sterols and analyzed liver cholesterol metabolites and found a decrease in phytosterols and cholesterol synthetic markers and an increase in non-enzymatic oxidative sterols in the pre-onset stage of NASH. We will continue to challenge themselves to add value to clinical practice based on cutting-edge analytical chemistry methodology.

Overview of clinical, molecular, and therapeutic features of Niemann-Pick disease (types A, B, and C): Focus on therapeutic approaches.

Niemann-Pick disease (NPD) is another type of metabolic disorder that is classified as lysosomal storage diseases (LSDs). The main cause of the disease is mutation in the SMPD1 (type A and B) or NPC1 or NPC2 (type C) genes, which lead to the accumulation of lipid substrates in the lysosomes of the liver, brain, spleen, lung, and bone marrow cells. This is followed by multiple cell damage, dysfunction of lysosomes, and finally dysfunction of body organs. So far, about 346, 575, and 30 mutations have been reported in SMPD1, NPC1, and NPC2 genes, respectively. Depending on the type of mutation and the clinical symptoms of the disease, the treatment will be different. The general aim of the current study is to review the clinical and molecular characteristics of patients with NPD and study various treatment methods for this disease with a focus on gene therapy approaches.

Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.

S-Adenosyl-l-methionine restores brain mitochondrial membrane fluidity and GSH content improving Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

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.

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.

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.

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.

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.

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.

Familial Alzheimer's disease associated with heterozygous NPC1 mutation.

INTRODUCTION: NPC1 mutations are responsible for Niemann-Pick disease type C (NPC), a rare autosomal recessive neurodegenerative disease. Patients harbouring heterozygous NPC1 mutations may rarely show parkinsonism or dementia. Here, we describe for the first time a large family with an apparently autosomal dominant late-onset Alzheimer's disease (AD) harbouring a novel heterozygous NPC1 mutation. METHODS: All the five living siblings belonging to the family were evaluated. We performed clinical evaluation, neuropsychological tests, assessment of cerebrospinal fluid markers of amyloid deposition, tau pathology and neurodegeneration (ATN), structural neuroimaging and brain amyloid-positron emission tomography. Oxysterol serum levels were also tested. A wide next-generation sequencing panel of genes associated with neurodegenerative diseases and a whole exome sequencing analysis were performed. RESULTS: We detected the novel heterozygous c.3034G>T (p.Gly1012Cys) mutation in NPC1, shared by all the siblings. No other point mutations or deletions in NPC1 or NPC2 were found. In four siblings, a diagnosis of late-onset AD was defined according to clinical characterisation and ATN biomarkers (A+, T+, N+) and serum oxysterol analysis showed increased 7-ketocholesterol and cholestane-3ß,5α,6ß-triol. DISCUSSION: We describe a novel NPC1 heterozygous mutation harboured by different members of a family with autosomal dominant late-onset amnesic AD without NPC-associated features. A missense mutation in homozygous state in the same aminoacidic position has been previously reported in a patient with NPC with severe phenotype. The alteration of serum oxysterols in our family corroborates the pathogenic role of our NPC1 mutation. Our work, illustrating clinical and biochemical disease hallmarks associated with NPC1 heterozygosity in patients affected by AD, provides relevant insights into the pathogenetic mechanisms underlying this possible novel association.