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

Abstract

14Mutations in the PINK1 and PRKN genes are the most common cause of early-onset familial 15 Parkinson disease. These genes code for the PINK1 and Parkin proteins, respectively, which are 16 involved in the degradation of dysfunctional mitochondria through mitophagy. An early step in 17 PINK1 -Parkin mediated mitophagy is the ubiquitination of the mitofusin proteins MFN1 and -2. The 18 ubiquitination of MFN1 and -2 in patient samples may therefore serve as a biomarker to determine 19 the functional effects of PINK1 and PRKN mutations, and to screen idiopathic patients for potential 20 mitophagy defects. We aimed to characterise the expression of the PINK1 -Parkin mitophagy 21 machinery in peripheral blood mononuclear cells (PBMCs) and assess if these cells could serve as a 22 platform to evaluate mitophagy via analysis of MFN1 and -2 ubiquitination. Mitophagy was induced 23 through mitochondrial depolarisation by treatment with the protonophore CCCP and ubiquitinated 24 MFN proteins were analysed by western blotting. In addition, PINK1 and PRKN mRNA and protein 25 expression levels were characterised with reverse transcriptase quantitative PCR and western 26 blotting, respectively. Whilst CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-27 SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce 28 ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable 29 levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable 30 and PINK1 mRNA levels remarkably low in control PBMCs. Our findings suggest that the PINK1 -31Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable 32 biosample for analysis of mitophagy function in Parkinson disease patients. 33 34

Targeting mitophagy in Parkinson's disease

“…Mitochondrial function is affected in peripheral blood mononuclear cells (PBMCs) of sporadic PD patients ( Table 3 ) ( 227 , 228 ); however, it is unclear whether PINK1–Parkin mitophagy can be measured in these cells. PINK1 knockout has been shown to alter bioenergetics in PBMCs from rat ( 229 ), but a recent study suggests that the PINK1–Parkin pathway is not active in human PBMCs, and PINK1 may in fact not be expressed in these cells ( 230 ). Whether the PINK1–Parkin pathway is active in human PBMCs or not will determine their usefulness for analyzing mitophagy.…”

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.…”

Phytochemicals: Targeting Mitophagy to Treat Metabolic Disorders

“…Additionally, Parkin can also promote the ubiquitination of the mitochondrial fusion proteins mitofusin 1 (MFN1) and mitofusin 2 (MFN2), the mitochondrial adapter protein Miro1, translocase of outer mitochondrial membrane 20 (TOM20), and voltage-dependent anion channel (VDAC) to induce mitophagy ( Nardin et al, 2016 ; Bradshaw et al, 2020 ). Normally, there are two major mechanisms involved in Parkin-dependent mitophagy: the first mechanism is that Parkin ubiquitinates MFN1 and MFN2, and further degraded by the proteasome, resulting in mitochondrial fission.…”