Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis.

BACKGROUND: Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The pathological mechanisms, underlying NPC1 are not yet completely understood. Especially the contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. As an analysis of affected cells is unfeasible in NPC1-patients, we recently developed an in vitro model system, based on cells derived from NPC1-patient specific iPSCs. Here, we asked if this model system recapitulates gliosis, observed in non-human model systems and NPC1 patient post mortem biopsies. We determined the amount of reactive astrocytes and the regulation of the intermediate filaments GFAP and vimentin, all indicating gliosis. Furthermore, we were interested in the assembly and phosphorylation of these intermediate filaments and finally the impact of the activation of protein kinase C (PKC), which is described to ameliorate the pathogenic phenotype of NPC1-deficient fibroblasts, including hypo-phosphorylation of vimentin and cholesterol accumulation. METHODS: We analysed glial cells derived from NPC1 patient specific induced pluripotent stem cells, carrying different NPC1 mutations. The amount of reactive astrocytes was determined by means of immuncytochemical stainings and FACS-analysis. Semi-quantitative western blot was used to determine the amount of phosphorylated GFAP and vimentin. Cholesterol accumulation was analysed by Filipin staining and quantified by Amplex Red Assay. U18666A was used to induce NPC1 phenotype in unaffected cells of the control cell line. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC. RESULTS: Immunocytochemical detection of GFAP, vimentin and Ki67 revealed that NPC1 mutant glial cells undergo gliosis. We found hypo-phosphorylation of the intermediate filaments GFAP and vimentin and alterations in the assembly of these intermediate filaments in NPC1 mutant cells. The application of U18666A induced not only NPC1 phenotypical accumulation of cholesterol, but characteristics of gliosis in glial cells derived from unaffected control cells. The application of phorbol 12-myristate 13-acetate, an activator of protein kinase C resulted in a significantly reduced number of reactive astrocytes and further characteristics of gliosis in NPC1-deficient cells. Furthermore, it triggered a restoration of cholesterol amounts to level of control cells. CONCLUSION: Our data demonstrate that glial cells derived from NPC1-patient specific iPSCs undergo gliosis. The application of U18666A induced comparable characteristics in un-affected control cells, suggesting that gliosis is triggered by hampered function of NPC1 protein. The activation of protein kinase C induced an amelioration of gliosis, as well as a reduction of cholesterol amount. These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced PKC activity due to the phenotypical cholesterol accumulation observed in NPC1. In addition, our data support the involvement of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.

Methyl-ß-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK.

The drug 2-hydroxypropyl-ß-cyclodextrin (HPßCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-ß-cyclodextrin (MßCD), a potent analog of HPßCD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through MßCD binding to its ß-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK ß1 or ß2 subunit) expression and an AMPK inhibitor abolished MßCD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of MßCD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.

Assessment of plasma chitotriosidase activity, CCL18/PARC concentration and NP-C suspicion index in the diagnosis of Niemann-Pick disease type C: a prospective observational study.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare, autosomal recessive neurodegenerative disease caused by mutations in either the NPC1 or NPC2 genes. The diagnosis of NP-C remains challenging due to the non-specific, heterogeneous nature of signs/symptoms. This study assessed the utility of plasma chitotriosidase (ChT) and Chemokine (C-C motif) ligand 18 (CCL18)/pulmonary and activation-regulated chemokine (PARC) in conjunction with the NP-C suspicion index (NP-C SI) for guiding confirmatory laboratory testing in patients with suspected NP-C. METHODS: In a prospective observational cohort study, incorporating a retrospective determination of NP-C SI scores, two different diagnostic approaches were applied in two separate groups of unrelated patients from 51 Spanish medical centers (n = 118 in both groups). From Jan 2010 to Apr 2012 (Period 1), patients with ≥2 clinical signs/symptoms of NP-C were considered 'suspected NP-C' cases, and NPC1/NPC2 sequencing, plasma chitotriosidase (ChT), CCL18/PARC and sphingomyelinase levels were assessed. Based on findings in Period 1, plasma ChT and CCL18/PARC, and NP-C SI prediction scores were determined in a second group of patients between May 2012 and Apr 2014 (Period 2), and NPC1 and NPC2 were sequenced only in those with elevated ChT and/or elevated CCL18/PARC and/or NP-C SI ≥70. Filipin staining and 7-ketocholesterol (7-KC) measurements were performed in all patients with NP-C gene mutations, where possible. RESULTS: In total across Periods 1 and 2, 10/236 (4%) patients had a confirmed diagnosis o NP-C based on gene sequencing (5/118 [4.2%] in each Period): all of these patients had two causal NPC1 mutations. Single mutant NPC1 alleles were detected in 8/236 (3%) patients, overall. Positive filipin staining results comprised three classical and five variant biochemical phenotypes. No NPC2 mutations were detected. All patients with NPC1 mutations had high ChT activity, high CCL18/PARC concentrations and/or NP-C SI scores ≥70. Plasma 7-KC was higher than control cut-off values in all patients with two NPC1 mutations, and in the majority of patients with single mutations. Family studies identified three further NP-C patients. CONCLUSION: This approach may be very useful for laboratories that do not have mass spectrometry facilities and therefore, they cannot use other NP-C biomarkers for diagnosis.

Reducing GBA2 Activity Ameliorates Neuropathology in Niemann-Pick Type C Mice.

The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two ß-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1-/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and the activity of secretory phospholipase A2 (sPLA2 ) using two Chinese hamster ovary (CHO)-K1 cell mutants, LY-B and LY-A cells, deficient in sphingolipid synthesis. In LY-B cells, deficiency of sphingolipids enhanced the release of AA induced by bee venom sPLA2-III or human sPLA2-V. These alterations were reversed by replenishment of exogenous sphingomyelin (SM). In LY-A cells, deficiency of SM increased the release of AA induced by sPLA2. In CHO-K1 cells, decrease and increase of SM level in the plasma membrane by pharmacological methods increased and inhibited the release of AA, respectively. SM inhibited the activity of sPLA2 in vitro. Niemann-Pick disease type C (NPC) is a lysosomal storage disorder caused by mutation of either the NPC1 or NPC2 gene, and is characterized by accumulation of cholesterol and sphingolipids including SM in late endosomes/lysosomes. Increased levels of AA and sPLA2 activity are involved in various neurodegenerative diseases. In CHO cells lacking NPC1 (A101 cells), SM level was lower in the plasma membrane, while it was higher in late endosomes/lysosomes. The release of AA induced by sPLA2 was increased in A101 cells than that in parental cells (JP17 cells), which was attenuated by adding exogenous SM. In addition, sPLA2 -III-induced cytotoxicity in A101 cells was much higher than that in JP17 cells. These results suggest that SM in the plasma membrane plays important roles in regulating sPLA2 activity and the enzyme-induced cytotoxicity in A101 cells.

A novel mouse model of a patient mucolipidosis II mutation recapitulates disease pathology.

Mucolipidosis II (MLII) is a lysosomal storage disorder caused by loss of N-acetylglucosamine-1-phosphotransferase, which tags lysosomal enzymes with a mannose 6-phosphate marker for transport to the lysosome. In MLII, the loss of this marker leads to deficiency of multiple enzymes and non-enzymatic proteins in the lysosome, leading to the storage of multiple substrates. Here we present a novel mouse model of MLII homozygous for a patient mutation in the GNPTAB gene. Whereas the current gene knock-out mouse model of MLII lacks some of the characteristic features of the human disease, our novel mouse model more fully recapitulates the human pathology, showing growth retardation, skeletal and facial abnormalities, increased circulating lysosomal enzymatic activities, intracellular lysosomal storage, and reduced life span. Importantly, MLII behavioral deficits are characterized for the first time, including impaired motor function and psychomotor retardation. Histological analysis of the brain revealed progressive neurodegeneration in the cerebellum with severe Purkinje cell loss as the underlying cause of the ataxic gait. In addition, based on the loss of Npc2 (Niemann-Pick type C 2) protein expression in the brain, the mice were treated with 2-hydroxypropyl-ß-cyclodextrin, a drug previously reported to rescue Purkinje cell death in a mouse model of Niemann-Pick type C disease. No improvement in brain pathology was observed. This indicates that cerebellar degeneration is not primarily triggered by loss of Npc2 function. This study emphasizes the value of modeling MLII patient mutations to generate clinically relevant mouse mutants to elucidate the pathogenic molecular pathways of MLII and address their amenability to therapy.

Diagnosis of Niemann-Pick disease type C with 7-ketocholesterol screening followed by NPC1/NPC2 gene mutation confirmation in Chinese patients.

BACKGROUND: It has been reported that oxidation product of cholesterol, 7-ketocholesterol, increases in plasma of patients with NP-C. Previously, we established a rapid test to determine the plasma 7-ketocholesterol level and found it elevated significantly in patients with acid sphingomyelinase deficient NPD and NP-C disease. METHODS: Individuals randomly referred to our outpatient clinics in the past two years for hepatosplenomegaly or isolated splenomegaly, who have been excluded as acid sphingomyelinase deficient NPD or Gaucher disease, and individuals with newborn cholestasis, psychomotor regression/retardation, were screened for plasma 7-ketocholesterol level. Individuals with high 7-ketocholesterol level were then analyzed for NPC1 and NPC2 gene mutation to confirm the accuracy of NP-C diagnosis. RESULTS: By screening the plasma 7-ketocholesterol of suspect individuals, 12 out of 302 (4%) had shown remarkable high levels compared with reference. All these twelve individuals were subsequently confirmed to be NP-C by DNA analysis of NPC1 and NPC2 genes, with the early infantile form (n = 7), the late infantile form (n = 1), the juvenile form (n = 1) and the adult form (n = 1). Furthermore, two NP-C patients without observable neuropsychiatric disability were picked up through this procedure. Only one patient had NP-C due to NPC2 gene mutations, with the rest due to NPC1 gene mutations. We found that in NP-C patients AST was usually mildly elevated and ALT was in a normal range when jaundice was not present. In total, 22 mutant alleles were identified in the NPC1 gene, including six novel small deletions/insertions, e.g., c.416_417insC, c.1030delT, c.1800delC, c.2230_2231delGT, c.2302_2303insG, and c.2795dupA; seven novel exonic point mutations, c.1502A>T (p.D501V), c.1553G>A (p.R518Q), c.1832A>G (p.D611G), c.2054T>C (p.I685T), c.2128C>T(p.Q710X), c.2177G>C (p.R726T), c.2366G>A (p.R789H), and one novel intronic mutation c.2912-3C>G. Small deletions/insertions constituted nearly half of the mutant alleles (10/22, 45%), indicating a unique mutation spectrum in this cohort of Chinese NP-C patients. CONCLUSION: Our data confirm in a clinical setting that screening plasma 7-ketocholesterol is an efficient and practical diagnostic tool to identify NP-C patients from suspect individuals. Patients without neuropsychological involvement could also be identified by this method therefore allowing an opportunity for earlier treatment.

Inhibition of GM3 synthase attenuates neuropathology of Niemann-Pick disease Type C. by affecting sphingolipid metabolism.

In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy.

Miglustat: a review of its use in Niemann-Pick disease type C.

Miglustat (Zavesca®, Brazaves®), a small iminosugar molecule that reversibly inhibits glycosphingolipid synthesis, is the only disease-specific drug approved for the treatment of progressive neurological manifestations of Niemann-Pick disease type C (NP-C) in adult and paediatric patients. NP-C is a rare, autosomal-recessive lipid storage disorder characterized by impaired intracellular lipid trafficking and progressive neurological symptoms leading to premature death. In a randomized clinical trial, long-term extension studies and a retrospective observational cohort study, treatment with oral miglustat stabilized key neurological manifestations of NP-C (including horizontal saccadic eye movement peak velocity, ambulation, manipulation, language and swallowing) in paediatric and adult patients with the disease. The therapeutic effects of miglustat in stabilizing or slowing disease progression have been confirmed in other reports in the clinical experience setting. The primary tolerability issues associated with miglustat are mild to moderate gastrointestinal effects (e.g. diarrhoea, flatulence and abdominal pain/discomfort) and weight loss, which usually occur during initial therapy and are generally manageable. In the absence of a cure, miglustat is a valuable agent to reduce the progression of clinically relevant neurological symptoms in paediatric and adult patients with NP-C, which is considered a significant achievement in the treatment of this disease.

Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.