Abstract
Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson’s disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.
Highlights
Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder after Alzheimer’s disease and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc)
To determine whether the loss of Parkin protein had an impact on mitochondrial function, fibroblasts were stained with Mitotracker Red CMXRos, which accumulates in mitochondria with an intact membrane potential, in the absence and in the presence of the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP)
In order to verify that the observed reduced staining was due to mitochondrial depolarization and not to a reduced number of mitochondria, the ATP Synthase beta signal was quantified by immunofluorescence and normalized by cell surface (Figure 2B)
Summary
Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder after Alzheimer’s disease and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). About 5–10% of PD patients suffer from familial forms of the disease, which are characterized by a clear genetic etiology (Shulman et al, 2011). Several mutations have been described to affect the function of these genes, causing both autosomal dominant (e.g., PARK1, PARK8) and autosomal recessive (e.g., PARK2, PARK7, PARK6) forms of familial PD (Lunati et al, 2018). The PARK2 gene encodes Parkin, an E3 ubiquitin ligase. Mutations in this gene have been linked to autosomal recessive juvenile PD. This PD form is characterized by an age-of-onset between childhood and 45 years of age (West and Maidment, 2004)
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