The PINK1—Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells

Abstract

Mutations in the PINK1 and PRKN genes are the most common cause of early-onset familial Parkinson disease. These genes code for the PINK1 and Parkin proteins, respectively, which are involved in the degradation of dysfunctional mitochondria through mitophagy. An early step in PINK1 –Parkin mediated mitophagy is the ubiquitination of the mitofusin proteins MFN1 and -2. The ubiquitination of MFN1 and -2 in patient samples may therefore serve as a biomarker to determine the functional effects of PINK1 and PRKN mutations, and to screen idiopathic patients for potential mitophagy defects. We aimed to characterise the expression of the PINK1 –Parkin mitophagy machinery in peripheral blood mononuclear cells (PBMCs) and assess if these cells could serve as a platform to evaluate mitophagy via analysis of MFN1 and -2 ubiquitination. Mitophagy was induced through mitochondrial depolarisation by treatment with the protonophore CCCP and ubiquitinated MFN proteins were analysed by western blotting. In addition, PINK1 and PRKN mRNA and protein expression levels were characterised with reverse transcriptase quantitative PCR and western blotting, respectively. Whilst CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable and PINK1 mRNA levels were remarkably low in control PBMCs. Our findings suggest that the PINK1 –Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable biosample for analysis of mitophagy function in Parkinson disease patients.

Monogenic Parkinson’s Disease: Genotype, Phenotype, Pathophysiology, and Genetic Testing

“…Mutations in PRKN and PINK1 likely disturb PINK1/parkin-mediated mitophagy, which is the selective degradation of mitochondria, a function essential for mitochondrial homeostasis [ 65 ]. In brief, parkin is a E3 ubiquitin ligase that ubiquinates outer mitochondrial membrane proteins such as mitofusin 1 and 2 [ 66 ]. PINK1 phosphorylates parkin and maintains its mitochondrial stabilization and translocation, mediating parkin activation [ 2 , 66 ].…”

“…In brief, parkin is a E3 ubiquitin ligase that ubiquinates outer mitochondrial membrane proteins such as mitofusin 1 and 2 [ 66 ]. PINK1 phosphorylates parkin and maintains its mitochondrial stabilization and translocation, mediating parkin activation [ 2 , 66 ].…”

TRIM25 mutation (p.C168*), coding for an E3 ubiquitin ligase, is a cause of early‐onset autosomal dominant dementia with amyloid load and parkinsonism

“…E3 ubiquitin ligases are therefore involved in the degradation of misfolded proteins that, when aggregated, are associated with neurodegenerative diseases. 7,26,27 Examples of this are mutations in parkin and malin (both E3 ubiquitin ligases), which are associated with development of juvenile recessive Parkinson's disease 9 and Lafora disease, 10,11 respectively.…”

“…TRIM25 codes for an RNA-binding protein (i.e., tripartite motifcontaining protein 25) acting as an E3 ubiquitin ligase, 5 enzymes which are involved in the selective recognition and ubiquitination of proteins with multiple outcomes such as degradation through the proteasome system 6,7 or activation of signaling pathways. 8 Mutations in other E3 ubiquitin ligases are known to be associated with neurodegenerative diseases such as juvenile recessive Parkinson's disease (Parkin) 9 or Lafora disease (Malin). 10,11 The study of this family adds a new gene to the panel of genetic causes of neurodegenerative diseases and suggests a novel underlying mechanism for brain amyloidosis.…”

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

“…In that regard, a very recent lab resource paper reported the generation of CRISPR/Cas9-edited homozygous PRKN , PINK1 and double PINK1 / PRKN knockout iPSC lines which can be differentiated into neuronal and glial cells for studying PD and related neuropathological conditions (Chen et al 2022 ). The PINK1 and PRKN genes encode proteins involved in mitophagy-mediated dysfunctional mitochondria degradation, and mutations in these genes are among the most common causes of early-onset familial PD (Bradshaw et al 2021 ). VPS13C is another notable PD risk factor belonging to a gene family (the VPS13 gene family) responsible for encoding lipid transfer proteins which localize in several distinct contact sites located in-between membranous proteins.…”