Shortened primary cilium length and dysregulated Sonic hedgehog signaling in Niemann-Pick C1 disease

The Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder due to mutations in the NPC1 gene, encoding a transmembrane protein related to the Sonic hedgehog receptor, Patched, and involved in intracellular trafficking of cholesterol. We have recently found that the proliferation of cerebellar granule neuron precursors is significantly reduced in Npc1-/- mice due to the downregulation of Shh expression. This finding prompted us to analyze the formation of the primary cilium, a non-motile organelle that is specialized for Shh signal transduction and responsible, when defective, for several human genetic disorders. In this study, we show that the expression and subcellular localization of Shh effectors and ciliary proteins are severely disturbed in Npc1-deficient mice. The dysregulation of Shh signaling is associated with a shortening of the primary cilium length and with a reduction of the fraction of ciliated cells in Npc1-deficient mouse brains and the human fibroblasts of NPC1 patients. These defects are prevented by treatment with 2-hydroxypropyl-β-cyclodextrin, a promising therapy currently under clinical investigation. Our findings indicate that defective Shh signaling is responsible for abnormal morphogenesis of the cerebellum of Npc1-deficient mice and show, for the first time, that the formation of the primary cilium is altered in NPC1 disease

Altered localization and functionality of TAR DNA Binding Protein 43 (TDP-43) in niemann- pick disease type C

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by the occurrence of visceral and neurological symptoms. At present, the molecular mechanisms causing neurodegeneration in this disease are unknown. Here we report the altered expression and/or mislocalization of the TAR-DNA binding protein 43 (TDP-43) in both NPC mouse and in a human neuronal model of the disease. We also report the neuropathologic study of a NPC patient's brain, showing that while TDP-43 is below immunohistochemical detection in nuclei of cerebellar Purkinje cells, it has a predominant localization in the cytoplasm of these cells. From a functional point of view, the TDP-43 mislocalization, that occurs in a human experimental neuronal model system, is associated with specific alterations in TDP-43 controlled genes. Most interestingly, treatment with N-Acetyl-cysteine (NAC) or beta-cyclodextrin (CD) can partially restore TDP-43 nuclear localization. Taken together, the results of these studies extend the role of TDP-43 beyond the Amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD)/Alzheimer disease (AD) spectrum. These findings may open novel research/therapeutic avenues for a better understanding of both NPC disease and the TDP-43 proteinopathy disease mechanism

Population-Based Study of New Mutations Causing Sandhoff Disease in Argentina

2pSandhoff Disease (SD) is a lysosomal storage disorder caused by mutations in the HEXB gene. A high incidence of SD has been described in an Argentine region called ‘‘Valle de Traslasierra.’’ Mutations c.445þ1G>A and p.S261Cfs12X were found in 98.7% and 1.3% of mutant alleles, respectively. In previous population-based studies, the carrier frequency has been estimated to be 1 in 16 to 29, all heterozygous with c.445þ1G>A. Recently, we detected new mutations in 5 Argentinian patients: c.1082þ5G>A, c.1242þ1G>A, c.1451G>A (p.Gly484Glu), c.1597C>T (p.Arg533Cys) and c.1601G>A (p.Cys534Tyr).Fil: Mugnaini, Julia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Hospital de Niños. Cátedra de Clínica Pediátrica; Argentina.Fil: Dardis, Andrea. Hospital Universitario ‘‘Santa Maria del la Misericordia’’. Centro Coordinador Regional de Enfermedades Raras, Udine; Italia.Fil: Dodelson de Kremer, Raquel. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Hospital de Niños. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Cátedra de Clínica Pediátrica; ArgentinaFil: Oller Ramírez, Ana María. Universidad Nacional de Córdoba. Facultad de Ciencias Medicas. Hospital de Niños. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Cátedra de Clínica Pediátrica; Argentina.Fil: Azar, Nydia Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Hospital de Niños. Cátedra de Clínica Pediátrica; ArgentinaFil: Becerra, Adriana. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Hospital de Niños. Cátedra de Clínica Pediátrica; Argentina.Fil; Zampieri, S. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Hospital de Niños. Cátedra de Clínica Pediátrica; Argentina.Fil: Guelbert, Norberto. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Centro de Estudio de las Metabolopatías Congénitas (CEMECO). Hospital de Niños. Cátedra de Clínica Pediátrica; Argentina.Bioquímica y Biología Molecula

CRISPR/Cas9 Editing for Gaucher Disease Modelling

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson’s disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.Instituto de Estudios Inmunológicos y Fisiopatológico

In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs

The transfer of genomic information into the primary RNA sequence can be altered by RNA editing. We have previously shown that genomic variants can be RNA-edited to wild-type. The presence of distinct “edited” iduronate 2-sulfatase (IDS) mRNA transcripts ex vivo evidenced the correction of a nonsense and frameshift variant, respectively, in three unrelated Hunter syndrome patients. This phenomenon was confirmed in various patient samples by a variety of techniques, and was quantified by single-nucleotide primer extension. Western blotting also confirmed the presence of IDS protein similar in size to the wild-type. Since preliminary experimental evidence suggested that the “corrected” IDS proteins produced by the patients were similar in molecular weight and net charge to their wild-type counterparts, an in vitro system employing different cell types was established to recapitulate the site-specific editing of IDS RNA (uridine to cytidine conversion and uridine deletion), and to confirm the findings previously observed ex vivo in the three patients. In addition, confocal microscopy and flow cytometry analyses demonstrated the expression and lysosomal localization in HEK293 cells of GFP-labeled proteins translated from edited IDS mRNAs. Confocal high-content analysis of the two patients’ cells expressing wild-type or mutated IDS confirmed lysosomal localization and showed no accumulation in the Golgi or early endosomes

Plasma neurofilament light (NfL) in patients affected by niemann–pick type C disease (NPCD)

Background: Niemann–Pick type C disease (NPCD) is an autosomal recessive lysoso-mal storage disorder caused by mutations in the NPC1 or NPC2 genes. The clinical presentation is characterized by visceral and neurological involvement. Apart from a small group of patients pre-senting a severe perinatal form, all patients develop progressive and fatal neurological disease with an extremely variable age of onset. Different biomarkers have been identified; however, they poorly correlate with neurological disease. In this study we assessed the possible role of plasma NfL as a neurological disease-associated biomarker in NPCD. (2) Methods: Plasma NfL levels were measured in 75 healthy controls and 26 patients affected by NPCD (24 NPC1 and 2 NPC2; 39 samples). (3) Results: Plasma NfL levels in healthy controls correlated with age and were significantly lower in pediatric patients as compared to adult subjects (p = 0.0017). In both pediatric and adult NPCD patients, the plasma levels of NfL were significantly higher than in age-matched controls (p < 0.0001). Most importantly, plasma NfL levels in NPCD patients with neurological involvement were significantly higher than the levels found in patients free of neurological signs at the time of sam-pling, both in the pediatric and the adult group (p = 0.0076; p = 0.0032, respectively). Furthermore, in adults the NfL levels in non-neurological patients were comparable with those found in age-matched controls. No correlations between plasma NfL levels and NPCD patient age at sampling or plasma levels of cholestan 3β-5α-6β-triol were found. (4) Conclusions: These data suggest a promising role of plasma NfL as a possible neurological disease-associated biomarker in NPCD.Fil: Dardis, Andrea. University Hospital of Udine; ItaliaFil: Pavan, Eleonora. University Hospital of Udine; ItaliaFil: Fabris, Martina. University Hospital of Udine; ItaliaFil: Da Riol, Rosalia Maria. University Hospital of Udine; ItaliaFil: Sechi, Annalisa. University Hospital of Udine; ItaliaFil: Fiumara, Agata. University of Catania; ItaliaFil: Santoro, Lucia. Polytechnic University of Marche; ItaliaFil: Ormazabal, Maximiliano Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Milanic, Romina. University Hospital of Udine; ItaliaFil: Zampieri, Stefania. University Hospital of Udine; ItaliaFil: Biasizzo, Jessica. University Hospital of Udine; ItaliaFil: Scarpa, Maurizio. University Hospital of Udine; Itali

Glucosylsphingosine Is a Highly Sensitive and Specific Biomarker for Primary Diagnostic and Follow-Up Monitoring in Gaucher Disease in a Non-Jewish, Caucasian Cohort of Gaucher Disease Patients

Gaucher disease (GD) is the most common lysosomal storage disorder (LSD). Based on a deficient β-glucocerebrosidase it leads to an accumulation of glucosylceramide. Standard diagnostic procedures include measurement of enzyme activity, genetic testing as well as analysis of chitotriosidase and CCL18/PARC as biomarkers. Even though chitotriosidase is the most well-established biomarker in GD, it is not specific for GD. Furthermore, it may be false negative in a significant percentage of GD patients due to mutation. Additionally, chitotriosidase reflects the changes in the course of the disease belatedly. This further enhances the need for a reliable biomarker, especially for the monitoring of the disease and the impact of potential treatments.Here, we evaluated the sensitivity and specificity of the previously reported biomarker Glucosylsphingosine with regard to different control groups (healthy control vs. GD carriers vs. other LSDs).Only GD patients displayed elevated levels of Glucosylsphingosine higher than 12 ng/ml whereas the comparison controls groups revealed concentrations below the pathological cut-off, verifying the specificity of Glucosylsphingosine as a biomarker for GD. In addition, we evaluated the biomarker before and during enzyme replacement therapy (ERT) in 19 patients, demonstrating a decrease in Glucosylsphingosine over time with the most pronounced reduction within the first 6 months of ERT. Furthermore, our data reveals a correlation between the medical consequence of specific mutations and Glucosylsphingosine.In summary, Glucosylsphingosine is a very promising, reliable and specific biomarker for GD

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder due to the deficient activity of the acid beta-glucosidase (GCase) enzyme, resulting in the progressive lysosomal accumulation of glucosylceramide (GlcCer) and its deacylated derivate, glucosylsphingosine (GlcSph). GCase is encoded by the GBA1 gene, located on chromosome 1q21 16 kb upstream from a highly homologous pseudogene. To date, more than 400 GBA1 pathogenic variants have been reported, many of them derived from recombination events between the gene and the pseudogene. In the last years, the increased access to new technologies has led to an exponential growth in the number of diagnostic laboratories offering GD testing. However, both biochemical and genetic diagnosis of GD are challenging and to date no specific evidence-based guidelines for the laboratory diagnosis of GD have been published. The objective of the guidelines presented here is to provide evidence-based recommendations for the technical implementation and interpretation of biochemical and genetic testing for the diagnosis of GD to ensure a timely and accurate diagnosis for patients with GD worldwide. The guidelines have been developed by members of the Diagnostic Working group of the International Working Group of Gaucher Disease (IWGGD), a non-profit network established to promote clinical and basic research into GD for the ultimate purpose of improving the lives of patients with this disease. One of the goals of the IWGGD is to support equitable access to diagnosis of GD and to standardize procedures to ensure an accurate diagnosis. Therefore, a guideline development group consisting of biochemists and geneticists working in the field of GD diagnosis was established and a list of topics to be discussed was selected. In these guidelines, twenty recommendations are provided based on information gathered through a systematic review of the literature and two different diagnostic algorithms are presented, considering the geographical differences in the access to diagnostic services. Besides, several gaps in the current diagnostic workflow were identified and actions to fulfill them were taken within the IWGGD. We believe that the implementation of recommendations provided in these guidelines will promote an equitable, timely and accurate diagnosis for patients with GD worldwide.Instituto de Estudios Inmunológicos y Fisiopatológico