Comprehensive-targeted lipidomic analysis in Niemann-Pick C disease.

Niemann-Pick C disease (NPC) is a lysosomal disease caused by mutations in NPC1 or NPC2 genes responsible for intracellular accumulation of free cholesterol and glycosphingolipids in a variety of tissues. We collected plasma samples from 15 NPC1 patients and 15 age-matched controls to analyze the impairment of lipid metabolism. Comprehensive-targeted quantitative lipidomic analysis was per-formed by Ion Mobility Mass Spectrometry, while oxysterols and lyso-sphingolipids, the classical NPC biomarkers, were analyzed by LC-MS/MS. Lipidomic analysis allowed the quantitation of ~1100 lipid species, belonging to 13 different classes. Statistical analysis of collected data showed a significant differentiation between NPC patients and controls. Lipid profiling showed an elevation of arachidonic acid and total diacylglycerols. Conversely, sphingomyelins, phosphatidylethano-lamines, phosphatidylcholines, cholesterylesters, and lactosylceramides were decreased. Indeed, the lipid imbalance was consistent with the increased concentrations of oxysterols and lyso-sphingolipids. Our study revealed a novel disease biosignature suggesting new potential diagnostic biomarkers. The alteration in key lipids molecules involved in inflammatory pathways and in oxidative stress regulation, provides new insights in the complex pathophysiology of the disease, still largely un-known.

Neurodegeneration in Niemann-Pick Type C Disease: An Updated Review on Pharmacological and Non-Pharmacological Approaches to Counteract Brain and Cognitive Impairment.

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.

Unbiased yeast screens identify cellular pathways affected in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a rare lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Mutations in the NPC1 gene lead to the majority of clinical cases (95%); however, the function of NPC1 remains unknown. To gain further insights into the biology of NPC1, we took advantage of the homology between the human NPC1 protein and its yeast orthologue, Niemann-Pick C-related protein 1 (Ncr1). We recreated the NCR1 mutant in yeast and performed screens to identify compensatory or redundant pathways that may be involved in NPC pathology, as well as proteins that were mislocalized in NCR1-deficient yeast. We also identified binding partners of the yeast Ncr1 orthologue. These screens identified several processes and pathways that may contribute to NPC pathogenesis. These included alterations in mitochondrial function, cytoskeleton organization, metal ion homeostasis, lipid trafficking, calcium signalling, and nutrient sensing. The mitochondrial and cytoskeletal abnormalities were validated in patient cells carrying mutations in NPC1, confirming their dysfunction in NPC disease.

Current Challenges in Understanding the Cellular and Molecular Mechanisms in Niemann-Pick Disease Type C1.

Rare diseases are a heterogeneous group of very different clinical syndromes. Their most common causes are defects in the hereditary material, and they can therefore be passed on to descendants. Rare diseases become manifest in almost all organs and often have a systemic expressivity, i.e., they affect several organs simultaneously. An effective causal therapy is often not available and can only be developed when the underlying causes of the disease are understood. In this review, we focus on Niemann-Pick disease type C1 (NPC1), which is a rare lipid-storage disorder. Lipids, in particular phospholipids, are a major component of the cell membrane and play important roles in cellular functions, such as extracellular receptor signaling, intracellular second messengers and cellular pressure regulation. An excessive storage of fats, as seen in NPC1, can cause permanent damage to cells and tissues in the brain and peripheral nervous system, but also in other parts of the body. Here, we summarize the impact of NPC1 pathology on several organ systems, as revealed in experimental animal models and humans, and give an overview of current available treatment options.

Adenosine A2A receptor stimulation restores cell functions and differentiation in Niemann-Pick type C-like oligodendrocytes.

Niemann Pick type C (NPC) disease is a rare neurovisceral disorder. Mutations in npc1 gene induce an intracellular accumulation of unesterified cholesterol in the endosomal/lysosomal system causing cell death. We recently showed that stimulation of adenosine A2A receptors (A2AR) restores cholesterol accumulation in late endosomes/lysosomes in human NPC fibroblasts and neural cell lines transiently transfected with NPC1 siRNA, suggesting that these receptors might be targeted to contrast the disease. Since NPC1 disease is characterized by dysmyelination and maturational arrest of oligodendrocyte progenitors (OPs), in this study, we investigated whether A2AR stimulation could promote oligodendrocyte differentiation and myelin formation, thus overcoming these important neurological abnormalities. We developed a NPC1 pharmacological model, in which primary cultures of OPs are exposed to a cholesterol transport inhibitor to induce a NPC1-like phenotype characterized by several typical features such as (i) cholesterol accumulation, (ii) altered mitochondrial morphology and membrane potential, (iii) defect of autophagy and (iv) maturation arrest. The A2AR agonist CGS21680 normalized all NPC1-like features. The ability of CGS21680 of rescuing OP from maturational arrest and promoting their differentiation to mature OL, suggests that A2AR stimulation might be exploited to correct dysmyelination in NPC1, further supporting their therapeutic potential in the disease.

Characterization of the Subventricular-Thalamo-Cortical Circuit in the NP-C Mouse Brain, and New Insights Regarding Treatment.

Gliosis in Niemann-Pick type C (NP-C) disease is characterized by marked changes in microglia and astrocytes. However, the gliosis onset and progression in NP-C has not been systematically studied, nor has the mechanism underlying this finding. Here, we found early gliosis in the subventricular zone (SVZ) of NP-C mice. Neural progenitor damage by Npc1 mutation suppressed vascular endothelial growth factor (VEGF) expression and further induced microglia activation followed by astrogliosis. Interestingly, excessive astrogliosis in the SVZ induced neural progenitor retention and/or migration into thalamus via astrocyte-derived VEGF, resulting in acceleration of thalamic and cortical gliosis through thalamo-cortical pathways. Transplantation of VEGF-overexpressing neural stem cells into the SVZ improved whole-brain pathology of NP-C mice. Overall, our data provide a new pathological perspective on NP-C neural pathology, revealing abnormalities in the subventricular-thalamo-cortical circuit of NP-C mouse brain and highlighting the importance of the SVZ microenvironment as a therapeutic target for NP-C disease.

The complexity of a monogenic neurodegenerative disease: More than two decades of therapeutic driven research into Niemann-Pick type C disease.

Niemann-Pick type C (NP-C) disease is a rare and fatal neurodegenerative disease typified by aberrations in intracellular lipid transport. Cholesterol and other lipids accumulate in the late endosome/lysosome of all diseased cells thereby causing neuronal and visceral atrophy. A cure for NP-C remains elusive despite the extensive molecular advances emanating from the identification of the primary genetic defect in 1997. Penetration of the blood-brain barrier and efficacy in the viscera are prerequisites for effective therapy, however the rarity of NP-C disease is the major impediment to progress. Disease diagnosis is challenging and establishment of appropriate test populations for clinical trials difficult. Fortunately, disease models that span the diversity of microbial and metazoan life have been utilized to advance the quest for a therapy. The complexity of lipid storage in this disorder and in the model systems, has led to multiple theories on the primary disease mechanism and consequently numerous and varied proposed interventions. Here, we conduct an evaluation of these studies.

Early Hippocampal i-LTP and LOX-1 Overexpression Induced by Anoxia: A Potential Role in Neurodegeneration in NPC Mouse Model.

Niemann-Pick type C disease (NPCD) is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol within the late endo-lysosomal compartment of cells. In the central nervous system, hypoxic insults could result in low-density lipoprotein (LDL) oxidation and Lectin-like oxidized LDL receptor-1 (LOX-1) induction, leading to a pathological hippocampal response, namely, ischemic long-term potentiation (i-LTP). These events may correlate with the progressive neural loss observed in NPCD. To test these hypotheses, hippocampal slices from Wild Type (WT) and NPC1-/- mice were prepared, and field potential in the CA1 region was analyzed during transient oxygen/glucose deprivation (OGD). Moreover, LOX-1 expression was evaluated by RT-qPCR, immunocytochemical, and Western blot analyses before and after an anoxic episode. Our results demonstrate the development of a precocious i-LTP in NPC1-/- mice during OGD application. We also observed a higher expression of LOX-1 transcript and protein in NPC1-/- mice with respect to WT mice; after anoxic damage to LOX-1 expression, a further increase in both NPC1-/- and WT mice was observed, although the protein expression seems to be delayed, suggesting a different kinetic of induction. These data clearly suggest an elevated susceptibility to neurodegeneration in NPC1-/- mice due to oxidative stress. The observed up-regulation of LOX-1 in the hippocampus of NPC1-/- mice may also open a new scenario in which new biomarkers can be identified.

[Research advances in diagnosis and therapy of Niemann-Pick disease type C].

Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal lipid storage disease associated with impaired intracellular cholesterol trafficking. A wide spectrum of clinical phenotype has been described, with a possible onset at all ages of life from the neonatal period to adulthood, more often in childhood. Typically, hepatosplenomegaly, dystaxia, dysphagia, dysarthria and dementia are presented in NPC patients. Neurologic symptoms vary according to the onset age, but prolonged neonatal cholestasis, splenomegaly, cataplexy and vertical supranuclear gaze palsy are more specific signs to the diagnosis of the disease. Impaired cholesterol trafficking and unesterified cholesterol accumulation in the late endosomes and lysosomals, as a results of mutations in NPC1 or NPC2 genes, are initial for the disease, and defective cellular autophagy, defective lysosomal calcium homeostasis and oxidative stress may all play roles in the physiological processes. The definite diagnosis requires demonstration of unesterified cholesterol accumulated in fibroblasts cultured from skin biopsies or of pathogenic mutation of NPC1/NPC2 genes. Miglustat, the only available treatment approved to date, can alleviate neurological symptoms and slow disease progression when administered earlier.

Role of cathepsin D in U18666A-induced neuronal cell death: potential implication in Niemann-Pick type C disease pathogenesis.

Cathepsin D is an aspartyl protease that plays a crucial role in normal cellular functions and in a variety of neurodegenerative disorders, including Niemann-Pick type C (NPC) disease, which is characterized by intracellular accumulation of cholesterol and glycosphingolipids in many tissues, including the brain. There is evidence that the level and activity of cathepsin D increased markedly in vulnerable neurons in NPC pathology, but its involvement in neurodegeneration remains unclear. In the present study, using mouse hippocampal cultured neurons, we evaluated the significance of cathepsin D in toxicity induced by U18666A, a class II amphiphile, which triggers cell death by impairing the trafficking of cholesterol, as observed in NPC pathology. Our results showed that U18666A-mediated toxicity is accompanied by an increase in cathepsin D mRNA and enzyme activity but a decrease in the total peptide content. The cytosolic level of cathepsin D, on the other hand, was increased along with cytochrome c and activated caspase-3 in U18666A-treated neurons. The cathepsin D inhibitor, pepstatin A, partially protected neurons against toxicity by attenuating these signaling mechanisms. Additionally, down-regulation of cathepsin D level prevented, whereas overexpression of the protease increased, vulnerability of cultured N2a cells to U18666A-induced toxicity. We also showed that extracellular cathepsin D from U18666A-treated neurons or application of exogenous enzyme can induce neurotoxicity by activating the autophagic pathway. These results suggest that increased release/activation of cathepsin D can trigger neurodegeneration and possibly development of NPC pathology. Thus, targeting cathepsin D level/activity may provide a new therapeutic opportunity for the treatment of NPC pathology.

Genotype/phenotype of 6 Chinese cases with Niemann-Pick disease type C.

UNLABELLED: Niemann-Pick disease type C (NP-C), caused by mutations of either NPC1 or NPC2 gene, is an inherited lysosomal lipid storage disorder that is difficult to be diagnosed and treated. NP-C is rarely reported in China and so far very few literatures are available for Chinese clinical workers. To better characterize this disease in China and improve genetic counseling, mutational analyses of NPC1 gene were carried out in 6 unrelated Chinese patients. METHODS: Clinical data of the probands from 2007 to 2010 were collected and analyzed. All exons of NPC1 were analyzed by direct sequencing. RESULTS: The six cases, four males and two females, included three cases of late infantile subtype and three cases of juvenile subtype. Case one and case six had siblings who suffered from the same disease. The onset of clinical symptoms varied from three to ten years old, and they included progressive cognitive and language impairment, and motion retrogradation. All were caught by focal or generalized seizures from one to four years after the onset. Vertical supranuclear gaze palsy, dysarthria, dysphagia, internal rotation and adduction of bilateral hands and splenomegaly occurred gradually during the disease progression. Five patients had laughter-cataplexy. MRI indicated mild brain atrophy. Sea blue cells and Niemann-Pick cells were presented in bone marrow smears. Activity of acid sphingomyelinase was normal or only slightly lower than controls. Supporting and symptomatic treatments could improve some of the clinical signs. We identified 10 different NPC1 mutations were identified in 12/12 alleles, 3 of which are described for the first time. All mutations were missense mutations, which located throughout the gene with five clustering in the cysteine-rich luminal domain. Homozygous mutation of S865L correlated with a relatively severe juvenile neurological form. CONCLUSIONS: NP-C is a rare autosomal recessive lysosomal storage disease that affects intellectual development of children, causing dementia, vegetative state and eventual death. The awareness of NP-C should be raised in the Chinese population. The typical clinical features of this disease include vertical supranuclear gaze palsy, seizures and cataplexy. Laboratory features include the presence of sea blue cells and Niemann-Pick cells in bone marrow smears. NPC1 mutation can be identified in most of these patients and most of them are missense mutations.

Clues to neuro-degeneration in Niemann-Pick type C disease from global gene expression profiling.

BACKGROUND: Niemann-Pick Type C (NPC) disease is a neurodegenerative disease that is characterized by the accumulation of cholesterol and glycosphingolipids in the late endocytic pathway. The majority of NPC cases are due to mutations in the NPC1 gene. The precise function of this gene is not yet known. METHODOLOGY/PRINCIPAL FINDINGS: Using cDNA microarrays, we analyzed the genome-wide expression patterns of human fibroblasts homozygous for the I1061T NPC1 mutation that is characterized by a severe defect in the intracellular processing of low density lipoprotein-derived cholesterol. A distinct gene expression profile was identified in NPC fibroblasts from different individuals when compared with fibroblasts isolated from normal subjects. As expected, NPC1 mutant cells displayed an inappropriate homeostatic response to accumulated intracellular cholesterol. In addition, a number of striking parallels were observed between NPC disease and Alzheimer's disease. CONCLUSIONS/SIGNIFICANCE: Many genes involved in the trafficking and processing of amyloid precursor protein and the microtubule binding protein, tau, were more highly expressed. Numerous genes important for membrane traffic and the cellular regulation of calcium, metals and other ions were upregulated. Finally, NPC fibroblasts exhibited a gene expression profile indicative of oxidative stress. These changes are likely contributors to the pathophysiology of Niemann-Pick Type C disease.

[Cholesterol is an important molecule in the processes of the synaptic plasticity and degeneration of neurons].

The importance of homeostasis of neural tissue to neuron functioning, the synaptic plasticity of the hippocampus, and laboratory animals' behavior was demonstrated by the authors earlier. A range of experimental data evidences that cholinergic neurotransmission, ionotropic and metabotropic receptors, excessive tau phosphorylation, alterations in amyloid-beta biochemistry, oxidative reactions, and other features of neurodegenerative processes depend on the precise regulation of cerebral cholesterol metabolism. Such results suggest that disturbances in cholesterol homeostasis are the common primary cause of the sporadic and familial forms of Alzheimer's disease, Down syndrome, Niemann-Pick disease type C, and explain the similarity of neurodegenerative signs in different degenerative diseases of the nervous system. The present work was introduced at an annual conference of American Society for Neuroscience, and is available as a scientific report at www.neurobiologyoflipids.org/content/3/7/.