Hou, Xu

Loss of either PINK1 or PRKN causes an early onset Parkinson’s disease (PD) phenotype. Functionally, PINK1 and PRKN work together to mediate stress-activated mitochondrial quality control. Upon mitochondrial damage, PINK1, a ubiquitin kinase and PRKN, a ubiquitin ligase, decorate damaged organelles with phosphorylated ubiquitin for sequestration and degradation in lysosomes, a process known as mitophagy. While several genetic mutations are established to result in loss of mitophagy function, many others have not been extensively characterized and are of unknown significance. Here, we analyzed a set of twenty variants, ten in each gene, focusing on understudied variants mostly from the Parkinson’s progressive marker initiative, with sensitive assays to define potential functional deficits. Our results nominate specific rare genetic PINK1 and PRKN variants that cause loss of enzymatic function in line with a potential causative role for PD. Additionally, we identify several variants with intermediate phenotypes and follow up on two of them by gene editing midbrain-derived neuronal precursor cells. Thereof derived isogenic neurons show a stability defect of the rare PINK1 D525N mutation, while the common PINK1 Q115L substitution results in reduced kinase activity. Our strategy to analyze variants with sensitive functional readouts will help aid diagnostics and disease treatment in line with current genomic and therapeutic advances.

Parkinson’s disease (PD) is generally considered a sporadic disorder, but a strong genetic background is often found. The aim of this study was to identify the underlying genetic cause of PD in two affected siblings and to subsequently assess the role of mutations in Cathepsin B (CTSB) in susceptibility to PD. A typical PD family was identified and whole-exome sequencing was performed in two affected siblings. Variants of interest were validated using Sanger sequencing. CTSB p.Gly284Val was genotyped in 2077 PD patients and 615 unrelated healthy controls from the Czech Republic, Ireland, Poland, Ukraine, and the USA. The gene burden analysis was conducted for the CTSB gene in an additional 769 PD probands from Mayo Clinic Florida familial PD cohort. CTSB expression and activity in patient-derived fibroblasts and controls were evaluated by qRT-PCR, western blot, immunocytochemistry, and enzymatic assay. The CTSB p.Gly284Val candidate variant was only identified in affected family members. Functional analysis of CTSB patient-derived fibroblasts under basal conditions did not reveal overt changes in endogenous expression, subcellular localization, or enzymatic activity in the heterozygous carrier of the CTSB variant. The identification of the CTSB p.Gly284Val may support the hypothesis that the CTSB locus harbors variants with differing penetrance that can determine the disease risk.

The mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PRKN (Parkin) together mediate the selective degradation of damaged mitochondria via the autophagy‐lysosome system (mitophagy). Complete loss of either gene function results in failure of this mitochondrial quality control and thus accumulation of damaged organelles. While homozygous or compound heterozygous mutations in PINK1 or PRKN are the most common causes of early‐onset Parkinson's disease (PD), the contribution of heterozygous variants to PD is debated. However, recently we identified a partial dominant‐negative effect of a heterozygous PINK1 missense mutations that is associated with a significant increased risk and an earlier onset compared to late‐onset, sporadic PD. This underscores that not all missense mutations are alike and suggests critical threshold functions of this stress‐activated, neuroprotective pathway. Here, we analyzed the underlying mechanisms in greater detail and found that the pathogenic PINK1 mutant, which can dimerize with wild‐type PINK1 protein, is aberrantly auto‐phosphorylated, impacting its enzymatic activity towards the substrates Ub and PRKN. Phosphorylation of both PINK1 targets however, is critical for PRKN recruitment as well as efficient labeling of mitochondria with phosphorylated poly‐Ub chains that serve as the mitophagy tag. In addition to cells from PD patients, we confirmed the altered phosphorylation pattern of mutant PINK1 under endogenous conditions in isogenic CRISPR/Cas9 genome engineered cells. Excitingly, while studying the pathogenic mechanisms in detail with a structure‐function approach, we identified a novel PINK1 variant that exerts even greater kinase activity than wild type PINK1. Our results further suggest that this hyperactive form of PINK1 likely modulates substrate receptivity thereby resulting in enhanced kinase activity and greater mitophagy rates. Going forward, it will be interesting to study if enhanced PINK1 signaling translates into greater neuroprotection. An in‐depth understanding of the regulation and enzyme functions of PINK1 may provide a rationale for a structure‐function based drug design in the future. Support or Funding Information This work was partially supported by funding from the Mayo Clinic Foundation, the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) [R01#NS085070] and the Michael J. Fox Foundation for Parkinson's Research to W.S. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Introduction The cytoprotective PTEN‐induced kinase 1 (PINK1)‐parkin RBR E3 ubiquitin protein ligase (PRKN) pathway selectively labels damaged mitochondria with phosphorylated ubiquitin (pS65‐Ub) for their autophagic removal (mitophagy). Because dysfunctions of mitochondria and degradation pathways are early features of Alzheimer's disease (AD), mitophagy impairments may contribute to the pathogenesis. Methods Morphology, levels, and distribution of the mitophagy tag pS65‐Ub were evaluated by biochemical analyses combined with tissue and single cell imaging in AD autopsy brain and in transgenic mouse models. Results Analyses revealed significant increases of pS65‐Ub levels in AD brain, which strongly correlated with granulovacuolar degeneration (GVD) and early phospho‐tau deposits, but were independent of amyloid beta pathology. Single cell analyses revealed predominant co‐localization of pS65‐Ub with mitochondria, GVD bodies, and/or lysosomes depending on the brain region analyzed. Discussion Our study highlights mitophagy alterations in AD that are associated with early tau pathology, and suggests that distinct mitochondrial, autophagic, and/or lysosomal failure may contribute to the selective vulnerability in disease.

The first clinical description of Parkinson’s disease (PD) will embrace its two century anniversary in 2017. For the past 30 years, mitochondrial dysfunction has been hypothesized to play a central role in the pathobiology of this devastating neurodegenerative disease. The identifications of mutations in genes encoding PINK1 (PTEN-induced kinase 1) and Parkin (E3 ubiquitin ligase) in familial PD and their functional association with mitochondrial quality control provided further support to this hypothesis. Recent research focused mainly on their key involvement in the clearance of damaged mitochondria, a process known as mitophagy. It has become evident that there are many other aspects of this complex regulated, multifaceted pathway that provides neuroprotection. As such, numerous additional factors that impact PINK1/Parkin have already been identified including genes involved in other forms of PD. A great pathogenic overlap amongst different forms of familial, environmental and even sporadic disease is emerging that potentially converges at the level of mitochondrial quality control. Tremendous efforts now seek to further detail the roles and exploit PINK1 and Parkin, their upstream regulators and downstream signaling pathways for future translation. This review summarizes the latest findings on PINK1/Parkin-directed mitochondrial quality control, its integration and cross-talk with other disease factors and pathways as well as the implications for idiopathic PD. In addition, we highlight novel avenues for the development of biomarkers and disease-modifying therapies that are based on a detailed understanding of the PINK1/Parkin pathway.