PT - JOURNAL ARTICLE AU - Chiara Diquigiovanni AU - Christian Bergamini AU - Rebeca Diaz AU - Irene Liparulo AU - Francesca Bianco AU - Luca Masin AU - Antonia Tranchina AU - Francesco Buscherini AU - Titia Anita Wischmeijer AU - Tommaso Pippucci AU - Emanuela Scarano AU - Duccio Maria Cordelli AU - Romana Fato AU - John Milton Lucocq AU - Marco Seri AU - Silvia Paracchini AU - Elena Bonora TI - In Troyer syndrome Spartin loss induces Complex I impairments and alters pyruvate metabolism AID - 10.1101/488239 DP - 2018 Jan 01 TA - bioRxiv PG - 488239 4099 - http://biorxiv.org/content/early/2018/12/06/488239.short 4100 - http://biorxiv.org/content/early/2018/12/06/488239.full AB - Growth delay and retardation are complex phenotypes which can results by a range of factors including genetics variants. We identified a novel homozygous frameshift mutation, c.892dupA, in SPART gene, in two brothers with short stature and psychomotor retardation, born from healthy consanguineous parents. Mutations in SPART are the cause of Troyer syndrome, an autosomal recessive form of spastic paraplegia resulting in muscle weakness, short stature and cognitive defects. SPART encodes for Spartin, a protein with different cellular functions, such as endosomal trafficking and mitochondrial stability.We evaluated the effects of Spartin loss by transiently silencing SPART in human neural stem cells (hNSCs) and by generating an SH-SY5Y cell line model carrying the c.892dupA mutation via CRISPR/Cas9. In both models, we observed an altered neuronal growth and an increase in neurite outgrowth. In the SH-SY5Y cell line carrying the c.892dupA mutation, Spartin absence led to an altered distribution of mitochondria, and to a severe decrease in the NADH-dehydrogenase activity of mitochondrial Complex I. These impairments determined an energetic failure with a decrease in ATP synthesis due to a halt in mitochondrial oxidative phosphorylation, increased reactive oxygen species production, and alteration in intracellular Ca2+ homeostasis. Transient re-expression of Spartin in mutant cells restored an intracellular Ca2+ level. Mutant cells presented a significant increase in extracellular pyruvate, which may result from increased glycolysis due to impaired Complex I activity. Consistently, Spartin loss led to an over-activation of Signal Transducer and Activator of Transcription 3 (STAT3) factor, a key regulator of glycolysis.These data demonstrate that Spartin loss leads to a profound bioenergetics imbalance with defective OXPHOS activity, and this altered metabolism might underlie Troyer syndrome and neurodevelopmental delays.AcCoAAcetyl-coAALSAmyotrophic Lateral SclerosisBMPBone Morphogenic ProteinCATCatalaseComplex INADH dehydrogenaseComplex I+IIINADH-cytochrome c reductase activityComplex II+IIIsuccinate-cytochrome c reductase activityCSCitrate SynthaseDAPI4′,6-diamidino-2-phenylindoleDBDecylbenzoquinoneDCFDA2′,7′–dichlorofluorescin diacetateDDSDeciphering ConsortiumDMEMDulbecco’s modified Eagle’s mediumDTNB5,5′-dithiobis-2-nitrobenzoic acidECMExtra-Cellular MatrixExAcExome Aggregation databaseFCCPcarbonyl cyanide-4-(trifluoromethoxy) phenylhydrazoneGHGrowth HormonegnomADGenome Aggregation databasegRNAguide RNAGRP75Glucose-Regulated Protein 75hESChuman embryonic stem cellhNSChuman neural stem cellhpfhours post fertilizationHPLCHigh Performance Liquid ChromatographyHSPHereditary Spastic ParaplegiaIMSInter-Membrane SpaceIUGRIntra-Uterine Growth RestrictionMIMMitochondrial Inner MembraneMITMicrotubule Interacting and TraffickingMOMMitochondrial Outer MembraneMPCMitochondrial Pyruvate CarrierMTGMitotracker GreenmΔψMitochondrial transmembrane potentialOXPHOSoxidative phosphorylationPDHPyruvate DehydrogenasepSpCas9n(BB)-2A-GFPCas9 from S. pyogenes with 2A-EGFPpSpgRNAS. pyogenes Cas9 guide RNAROHRuns Of HomozygosityROSreactive oxygen speciesSDstandard deviationsSDS-PAGEsodium dodecyl sulfate polyacrylamide electrophoresisSEMStandard Error of MeanSNPsSingle Nucleotide PolymorphismsSOD1Superoxide DismutaseSOD2Manganese Superoxide DismutasessODNsingle stranded-oligonucleotideTBSTris Buffered SalineTMRMTetramethylrhodamine methyl esterUPD7Uniparental disomy 7WESWhole Exome Sequencing