Stefania Zampieri

Oxidative stress in NPC1 deficient cells: protective effect of allopregnanolone

Niemann‐Pick C disease (NPC) is an autosomal recessive neurodegenerative disorder caused by the abnormal function of NPC1 or NPC2 proteins, leading to an accumulation of unesterified cholesterol and glycosphingolipids (GSLs) in the lysosomes. The mechanisms underlying the pathophysiology in NPC disease are not clear. Oxidative damage is implicated in the pathophysiology of different neurological disorders and the effect of GSL accumulation on the intracellular redox state has been documented. Therefore, we determined whether the intracellular redox state might contribute to the NPC disease pathophysiology. Because the treatment of NPC mice with allopregnanolone (ALLO) increases their lifespan and delays the onset of neurological impairment, we analysed the effect of ALLO on the oxidative damage in human NPC fibroblasts. Concentrations of reactive oxygen species (ROS) and lipid peroxidation were higher in fibroblasts from NPC patients than in fibroblasts from normal subjects. Fibroblasts from NPC patients were more susceptible to cell death through apoptosis after an acute oxidative insult. This process is mediated by activation of the NF‐κB signalling pathway. Knockdown of NPC1 mRNA both in normal fibroblasts and in human SH‐SY5Y neuroblastoma cells caused increased ROS concentrations. ALLO treatment of fibroblasts from NPC patients or NPC1 knockdown cells reduced the levels of ROS and lipid peroxidation and prevented peroxide‐induced apoptosis and NF‐kB activation. Thus, these findings suggest that oxidative stress might contribute to the NPC disease and ALLO might be beneficial in the treatment of the disease, at least in part, due to its ability to restore the intracellular redox state.

Molecular analysis of NPC1 and NPC2 gene in 34 Niemann–Pick C Italian Patients: identification and structural modeling of novel mutations

Niemann–Pick C, the autosomal recessive neuro-visceral disease resulting from a failure of cholesterol trafficking within the endosomal–lysosomal pathway, is due to mutations in NPC1 or NPC2 genes. We characterized 34 unrelated patients including 32 patients with mutations in NPC1 gene and two patients in NPC2 gene. Overall, 33 distinct genotypes were encountered. Among the 21 unpublished NPC1 alleles, 15 were due to point mutations resulting in 13 codon replacements (p.C100S, p.P237L, p.R389L, p.L472H, p.Y634C, p.S636F, p.V780G, p.Q921P, p.Y1019C, p.R1077Q, p.L1102F, p.A1187V, and p.L1191F) and in two premature stop codons (p.R934X and p.Q447X); a new mutant carried two in cis mutations, p.[L648H;M1142T] and four other NPC1 alleles were small deletions/insertions leading both to frame shifts and premature protein truncations (p.C31WfsX26, p.F284LfsX26, p.E1188fsX54, and p.T1205NfsX53). Finally, the new intronic c.464-2A>C change at the 3′ acceptor splice site of intron 4 affected NPC1 messenger RNA processing. We also found a new NPC2 mutant caused by a change of the first codon (p.M1L). The novel missense mutations were further investigated by two bioinformatics approaches. Panther proein classification system computationally predicted the detrimental effect of all new missense mutations occurring at evolutionary conserved positions. The other bioinformatics approach was based on prediction of structural alterations induced by missense mutations on the NPC1 atomic models. The in silico analysis predicted protein malfunctioning and/or local folding alteration for most missense mutations. Moreover, the effects of the missense mutations (p.Y634C, p.S636F, p.L648H, and p.V780G) affecting the sterol-sensing domain (SSD) were evaluated by docking simulation between the atomic coordinates of SSD model and cholesterol.

Functional characterization of the common c.-32-13T>G mutation of GAA gene: identification of potential therapeutic agents

Glycogen storage disease type II is a lysosomal storage disorder due to mutations of the GAA gene, which causes lysosomal alpha-glucosidase deficiency. Clinically, glycogen storage disease type II has been classified in infantile and late-onset forms. Most late-onset patients share the leaky splicing mutation c.-32-13T>G. To date, the mechanism by which the c.-32-13T>G mutation affects the GAA mRNA splicing is not fully known. In this study, we demonstrate that the c.-32-13T>G mutation abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2 and that several splicing factors affect exon 2 inclusion, although the only factor capable of acting in the c.-32-13 T>G context is the SR protein family member, SRSF4 (SRp75). Most importantly, a preliminary screening using small molecules described to be able to affect splicing profiles, showed that resveratrol treatment resulted in a significant increase of normal spliced GAA mRNA, GAA protein content and activity in cells transfected with a mutant minigene and in fibroblasts from patients carrying the c-32-13T>G mutation. In conclusion, this work provides an in-depth functional characterization of the c.-32-13T>G mutation and, most importantly, an in vitro proof of principle for the use of small molecules to rescue normal splicing of c.-32-13T>G mutant alleles.

Splicing mutations in glycogen-storage disease type II: evaluation of the full spectrum of mutations and their relation to patients' phenotypes

Glycogen-storage disease type II is an autosomal recessive-inherited disorder due to the deficiency of acid α-glucosidase. A large number of mutations in the acid α-glucosidase gene have been described to date. Among them, ∼15% are variations that may affect mRNA splicing process. In this study, we have for the first time comprehensively reviewed the available information on splicing mutations of the acid α-glucosidase gene and we have evaluated their possible impact on the splicing process using different in silico approaches. Out of the 39 different GAA-sequence variations described, an in silico analysis using seven different programs showed that 97% of them are predicted to have an impact on the splicing process. Moreover, this analysis showed a quite good correlation between the impact of the mutation on the splicing process and the clinical phenotype. In addition, we have performed the functional characterization of three novel sequence variants found in Italian patients and still uncharacterized. Using a minigene system, we have confirmed their pathogenic nature. In conclusion, this study has shown that in silico analysis represents a useful tool to select mutations that affect the splicing process of the acid α-glucosidase gene and provides an updated picture of all this kind of mutations reported till now.

Functional analysis of 11 novel GBA alleles

Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid β-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or ‘de novo’ mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.

SMPD1 Mutation Update: Database and Comprehensive Analysis of Published and Novel Variants

Niemann–Pick Types A and B (NPA/B) diseases are autosomal recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase (ASM) because of the mutations in the SMPD1 gene. Here, we provide a comprehensive updated review of already reported and newly identified SMPD1 variants. Among them, 185 have been found in NPA/B patients. Disease‐causing variants are equally distributed along the SMPD1 gene; most of them are missense (65.4%) or frameshift (19%) mutations. The most frequently reported mutation worldwide is the p.R610del, clearly associated with an attenuated NP disease type B phenotype. The available information about the impact of 52 SMPD1 variants on ASM mRNA and/or enzymatic activity has been collected and whenever possible, phenotype/genotype correlations were established. In addition, we created a locus‐specific database easily accessible at http://www.inpdr.org/genes that catalogs the 417 SMPD1 variants reported to date and provides data on their in silico predicted effects on ASM protein function or mRNA splicing. The information reviewed in this article, providing new insights into the genotype/phenotype correlation, is extremely valuable to facilitate diagnosis and genetic counseling of families affected by NPA/B.

A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient’s skin

BackgroundNiemann Pick C (NPC) disease is a neurovisceral lysosomal storage disorder due to mutations in NPC1 or NPC2 genes, characterized by the accumulation of endocytosed unesterified cholesterol, gangliosides and other lipids within the lysosomes/late endosomes. Even if the neurodegeneration is the main feature of the disease, the analysis of the molecular pathways linking the lipid accumulation and cellular damage in the brain has been challenging due to the limited availability of human neuronal models.ObjectiveThe aim of this study was to develop a human neuronal model of NPC disease by inducing neuronal differentiation of multipotent adult stem cells (MASC) isolated from NPC patients.MethodsStem cells were isolated from 3 NPC patients and 3 controls both from skin biopsies and previously established skin fibroblast cultures. Cells were induced to differentiate along a neuronal fate adapting methods previously described by Beltrami et al, 2007. The surface immunophenotype of stem cells was analyzed by FACS. Stem cell and neuronal markers expression were evaluated by immunofluorescence. Intracellular accumulation of cholesterol and gangliosides were assessed by filipin staining and immunofluorescence, respectively. A morphometric analysis was performed using a Neurite outgrowth image program.ResultsAfter 3 passages in selective medium, MASC isolated either from skin biopsies or previously established skin fibroblast cultures displayed an antigenic pattern characteristic of mesenchymal stem cells and expressed the stem cell markers Oct-4, Nanog, Sox-2 and nestin. A massive lysosomal accumulation of cholesterol was observed only in cells isolated from NPC patients. After the induction of neural differentiation, remarkable morphologic changes were observed and cells became positive to markers of the neuronal lineage NeuN and MAP2. Differentiated cells from NPC patients displayed characteristic features of NPC disease, they showed intracellular accumulation of unesterified cholesterol and GM2 ganglioside and presented morphological differences with respect to cells derived from healthy donors.In conclusion, we generated a human neuronal model of NPC disease through the induction of differentiation of stem cells obtained from patient’s easily accessible sources. The strategy described here may be applied to easily generate human neuronal models of other neurodegenerative diseases.

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by the lysosomal/late endosomal (LE) accumulation of unesterified cholesterol and other lipids due to a defect in the intracellular lipid trafficking. About 95% of patients present mutations in the NPC1 gene. Among the 290 mutations reported in the NPC1 gene, about 70% are missense. However, little information is available regarding the impact of missense mutations on NPC1 protein stability and function. In this study, we in vitro characterized the pathogenic effect of 7 NPC1 missense mutations. In all cases, the basal levels of mutant NPC1 expression were reduced with respect to wild type. Treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity, with the proteasome inhibitor MG132 or glycerol 10%, a chemical chaperone known to stabilize misfolded proteins, resulted in a significant increase of NPC1 protein levels in all cell lines, indicating that these mutants are subjected to proteasomal degradation due to protein misfolding The increment of NPC1 mutant protein induced by the proteasome inhibitor was associated with a localization of NPC1 protein within lysosomal/LE compartment. In cell lines carrying mutations p.N1156S, p.L1191F, p.V1165M, and p.I1061T, the increment of NPC1 mutant protein resulted in an improvement of the intracellular trafficking of cholesterol and GM1. These findings showed that it is possible to correct the NPC cellular phenotype by increasing the amount of endogenous NPC1 mutated protein, suggesting that at least some NPC1 mutations might be potentially rescued by small molecules-based chaperone therapy.

Cerebrospinal fluid β‐glucocerebrosidase activity is reduced in parkinson's disease patients

Reduced β‐glucocerebrosidase activity was observed in postmortem brains of both GBA1 mutation carrier and noncarrier Parkinsons disease patients, suggesting that lower β‐glucocerebrosidase activity is a key feature in the pathogenesis of PD. The objectives of this study were to confirm whether there is reduced β‐glucocerebrosidase activity in the CSF of GBA1 mutation carrier and noncarrier PD patients and verify if other lysosomal enzymes show altered activity in the CSF.