Abstract
Almost half of autosomal recessive early-onset parkinsonism has been associated with mutations in PARK2, coding for parkin, which plays an important role in mitochondria function and calcium homeostasis. Cyclic adenosine monophosphate (cAMP) is a major second messenger regulating mitochondrial metabolism, and it is strictly interlocked with calcium homeostasis. Parkin-mutant (Pt) fibroblasts, exhibiting defective mitochondrial respiratory/OxPhos activity, showed a significant higher value of basal intracellular level of cAMP, as compared with normal fibroblasts (CTRL). Specific pharmacological inhibition/activation of members of the adenylyl cyclase- and of the phosphodiesterase-families, respectively, as well as quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, indicate that the higher level of cAMP observed in Pt fibroblasts can contribute to a higher level of activity/expression by soluble adenylyl cyclase (sAC) and to low activity/expression of the phosphodiesterase isoform 4 (PDE4). As Ca2+ regulates sAC, we performed quantitative calcium-fluorimetric analysis, showing a higher level of Ca2+ in the both cytosol and mitochondria of Pt fibroblasts as compared with CTRL. Most notably, inhibition of the mitochondrial Ca2+ uniporter decreased, specifically the cAMP level in PD fibroblasts. All together, these findings support the occurrence of an altered mitochondrial Ca2+-mediated cAMP homeostasis in fibroblasts with the parkin mutation.
Highlights
Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, characterized by the selective death of dopaminergic neurons in the substantia nigra
We previously reported that fibroblasts harboring a heterozygous PARK2 mutation displayed a higher Cyclic adenosine monophosphate (cAMP) basal level, as compared to control cells associated with hyperphosphorylation of CREB [10]
We confirmed the presence of high cAMP level in fibroblasts harboring a heterozygous PARK2 mutation [10] and extended the analysis to a third patient harboring a different mutation in parkin gene [11]
Summary
Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, characterized by the selective death of dopaminergic neurons in the substantia nigra. Cells 2019, 8, 250 half of all the early-onset familial PD cases have been associated with mutations in PARK2, which codes for parkin, a multifunctional E3 ubiquitin ligase [1,2], which mediates ubiquitination of several target proteins [3]. Studies on in vitro and in vivo parkin-null models strongly suggest a role of parkin in the preservation of mitochondrial function. Parkin knockout mice exhibit mitochondrial dysfunction and oxidative damage [4,5]. Drosophila parkin null mutants display mitochondrial dysfunction and apoptotic muscle degeneration [6,7]. Functional assays in leukocytes [8], as well as the fibroblasts of patients with parkin mutations, consistently show mitochondrial impairment [9,10,11]
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