IntroductionADCY5 mutations have been recently identified as an important cause of early-onset hyperkinetic movement disorders. The phenotypic spectrum associated with mutations in this gene is expanding. However, the ADCY5 mutational frequency in cohorts of paediatric patients with hyperkinetic movement disorders has not been evaluated.MethodsWe performed a screening of the entire ADCY5 coding sequence in 44 unrelated subjects with genetically undiagnosed childhood-onset hyperkinetic movement disorders, featuring chorea alone or in combination with myoclonus and dystonia. All patients had normal CSF analysis and brain imaging and were regularly followed-up in tertiary centers for paediatric movement disorders.ResultsWe identified five unrelated subjects with ADCY5 mutations (11% of the cohort). Three carried the p. R418W mutation, one the p. R418Q and one the p. R418G mutation. Mutations arose de novo in four cases, while one patient inherited the mutation from his similarly affected father. All patients had delayed motor and/or language milestones with or without axial hypotonia and showed generalized chorea and dystonia, with prominent myoclonic jerks in one case. Episodic exacerbations of the baseline movement disorder were observed in most cases, being the first disease manifestation in two patients. The disease course was variable, from stability to spontaneous improvement during adolescence.ConclusionMutations in ADCY5 are responsible for a hyperkinetic movement disorder that can be preceded by episodic attacks before the movement disorder becomes persistent and is frequently misdiagnosed as dyskinetic cerebral palsy. A residual degree of neck hypotonia and a myopathy-like facial appearance are frequently observed in patients with ADCY5 mutations.
Mutations in PARK2, encoding Parkin, cause an autosomal recessive form of juvenile Parkinson Disease (JPD). The aim of the present study was to investigate the impact of PARK2 mutations on mitochondrial function and morphology in human skin fibroblasts. We analyzed cells obtained from four patients clinically characterized by JPD, harboring recessive mutations in PARK2. By quantitative PCR we found a reduction (<50%) of PARK2 transcript in all patients but one; however Western Blot analysis demonstrated the virtual absence of Parkin protein in all mutant fibroblasts. Respiration assays showed an increment of oxygen consumption, which was uncoupled to ATP cellular levels. This finding was probably due to presence of altered mitochondrial membrane potential (ΔΨm), confirmed by JC-1 analysis. The mitochondrial network was comparable between mutant and control cells but, interestingly, a “chain-like” network was found only in mutant fibroblasts. Dissipation of ΔΨm usually leads to mitochondrial fragmentation in healthy cells and eventually to mitophagy; however, this behavior was not observed in patients' fibroblasts. The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy. This condition should increase the oxidative stress and lead to cellular dysfunction and death. Our results suggest that PARK2 mutations cause mitochondrial impairment, in particular reduction in ATP cellular levels and alteration of ΔΨm, even in non-neuronal cells and confirm the hypothesis that Parkin holds a pivotal role in pro-fission events.
The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
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