Neuronal and astrocytic differentiation from Sanfilippo C syndrome iPSCs for disease modeling and drug development

Sanfilippo syndrome type C (mucopolysaccharidosis IIIC) is an early-onset neurodegenerative lysosomal storage disorder, which is currently untreatable. The vast majority of studies focusing on disease mechanisms of Sanfilippo syndrome were performed on non-neural cells or mouse models, which present obvious limitations. Induced pluripotent stem cells (iPSCs) are an efficient way to model human diseases in vitro. Recently developed transcription factor-based differentiation protocols allow fast and efficient conversion of iPSCs into the cell type of interest. By applying these protocols, we have generated newneuronal and astrocyticmodels of Sanfilippo syndrome using our previously established disease iPSC lines. Moreover, our neuronal model exhibits disease-specific molecular phenotypes, such as increase in lysosomes and heparan sulfate. Lastly, we tested an experimental, siRNA-based treatment previously shown to be successful in patients' fibroblasts and demonstrated its lack of efficacy in neurons. Our findings highlight the need to use relevant human cellular models to test therapeutic interventions and shows the applicability of our neuronal and astrocyticmodels of Sanfilippo syndrome for future studies on disease mechanisms and drug development

Calorie restriction rescues mitochondrial dysfunction in Adck2-Deficient skeletal muscle

ADCK2 haploinsufficiency-mediated mitochondrial coenzyme Q deficiency in skeletal muscle causes mitochondrial myopathy associated with defects in beta-oxidation of fatty acids, aged-matched metabolic reprogramming, and defective physical performance. Calorie restriction has proven to increase lifespan and delay the onset of chronic diseases associated to aging. To study the possible treatment by food deprivation, heterozygous Adck2 knockout mice were fed under 40% calorie restriction (CR) and the phenotype was followed for 7 months. The overall glucose and fatty acids metabolism in muscle was restored in mutant mice to WT levels after CR. CR modulated the skeletal muscle metabolic profile of mutant mice, partially rescuing the profile of WT animals. The analysis of mitochondria isolated from skeletal muscle demonstrated that CR increased both CoQ levels and oxygen consumption rate (OCR) based on both glucose and fatty acids substrates, along with mitochondrial mass. The elevated aerobic metabolism fits with an increase of type IIa fibers, and a reduction of type IIx in mutant muscles, reaching WT levels. To further explore the effect of CR over muscle stem cells, satellite cells were isolated and induced to differentiate in culture media containing serum from animals in either ad libitum or CR diets for 72 h. Mutant cells showed slower differentiation alongside with decreased oxygen consumption. In vitro differentiation of mutant cells was increased under CR serum reaching levels of WT isolated cells, recovering respiration measured by OCR and partially beta-oxidation of fatty acids. The overall increase of skeletal muscle bioenergetics following CR intervention is paralleled with a physical activity improvement, with some increases in two and four limbs strength tests, and weights strength test. Running wheel activity was also partially improved in mutant mice under CR. These results demonstrate that CR intervention, which has been shown to improve age-associated physical and metabolic decline in WT mice, also recovers the defective aerobic metabolism and differentiation of skeletal muscle in mice caused by ADCK2 haploinsufficiency.This work was supported by Junta de Andalucía grant BIO-177, the Instituto de Salud Carlos III FIS grant FIS PI20/00541, CIBERER (U729)-ISCIII, the FEDER Funding Program from the European Union and the Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T. This work was supported by the Spanish Ministry of Education, Culture and Sports through fellowship FPU16/03264 to JH-C, and the Association Française contre les Myopathies (AFM) through fellowship grant #22450 to CV-G. This work was funded in part by the Intramural Research Program of the National Institute on Aging, NIH. This research was also supported by the Instituto de Salud Carlos III (PI19/01310) (Co-funded by the European Union) and by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) (2017: SGR 1428) and the CERCA

Leigh syndrome is the main clinical characteristic of PTCD3 deficiency

Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1G>C];[1918C>G] and c.[710del];[902C>T]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.© 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology

Clinical presentation and proteomic signature of patients with TANGO2 mutations

Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C&gt;T/p.Arg88*; c.220A&gt;C/p.Thr74Pro; c.380+1G&gt;A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11-13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged-red/cytochrome c oxidase-negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10 ) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum-Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions.</p

Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients

Guia para a construção de espaços culturais inclusivos

A criação de espaços culturais e artísticos inclusivos requer uma revisão da forma como estes são concebidos, desenhados e organizados com o objetivo de dar as boasvindas a toda a gente e garantir o acesso e participação igualitários aos serviços, atividades e conteúdos que oferecem. Desta maneira, museus, centros culturais e artísticos ou espaços de exposições contribuem para a promoção do direito a aceder e participar na vida cultural e artística, tal como expresso em várias convenções internacionais, incluindo a Declaração Universal dos Direitos Humanos.info:eu-repo/semantics/publishedVersio

A crowdsourcing database for the copy-number variation of the Spanish population

Background: Despite being a very common type of genetic variation, the distribution of copy-number variations (CNVs) in the population is still poorly understood. The knowledge of the genetic variability, especially at the level of the local population, is a critical factor for distinguishing pathogenic from non-pathogenic variation in the discovery of new disease variants. Results: Here, we present the SPAnish Copy Number Alterations Collaborative Server (SPACNACS), which currently contains copy number variation profiles obtained from more than 400 genomes and exomes of unrelated Spanish individuals. By means of a collaborative crowdsourcing effort whole genome and whole exome sequencing data, produced by local genomic projects and for other purposes, is continuously collected. Once checked both, the Spanish ancestry and the lack of kinship with other individuals in the SPACNACS, the CNVs are inferred for these sequences and they are used to populate the database. A web interface allows querying the database with different filters that include ICD10 upper categories. This allows discarding samples from the disease under study and obtaining pseudo-control CNV profiles from the local population. We also show here additional studies on the local impact of CNVs in some phenotypes and on pharmacogenomic variants. SPACNACS can be accessed at: http://csvs.clinbioinfosspa.es/spacnacs/. Conclusion: SPACNACS facilitates disease gene discovery by providing detailed information of the local variability of the population and exemplifies how to reuse genomic data produced for other purposes to build a local reference database