Modifiers of Parkin and PINK1 mutation penetrance in Parkinson’s disease: Endogenous human models

• Applicant: Dr. Aleksandar Rakovic
• Subject Area: Molecular and Cellular Neurology and Neuropathology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 287074911

Mutations in PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin lead to recessive forms of Parkinson’s disease (PD). It has been shown that PINK1 and Parkin work together in a common pathway maintaining mitochondrial function and control removal of damaged mitochondria through mitophagy. Interestingly, heterozygous mutations in PINK1 and Parkin increase the risk to develop PD and may even be viewed to act in a dominant fashion with highly reduced penetrance. This suggests the existence of differentially expressed protective genes or pathways between affected and unaffected mutation carriers. Since PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, it is imperative to investigate potential genetic modifiers influencing the penetrance of heterozygous Parkin and PINK1 mutations in these neurons in a cell-type specific manner. The induced pluripotent stem cell (iPSC) technology has opened up a novel avenue to generate PD patient- (and healthy control)-derived dopaminergic neuronal cultures. Several protocols have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neuronal cultures, however, they all contain only a small proportion of TH-positive neurons. In addition, there is substantial variability in the percentage of TH-positive neurons between differentiations and even between samples within the same differentiation. These differences occur independently of the mutational status. To overcome this limitation, we established an efficient method to generate human iPSC TH-mCherry reporter lines using CRISPR/Cas9-based genome editing. Upon differentiation into heterogeneous dopaminergic neuronal cultures, only mCherry-positive (TH-positive) cells will be isolated from the rest of the culture using fluorescence- activated cell sorting to obtain homogenous, TH-positive population of neurons (Objective 1). Next, we aim to identify genetic modifiers influencing the penetrance of heterozygous Parkin and PINK1 mutations employing a multi-omic transcriptome and proteome expression analysis on pure iPSC- derived dopaminergic cultures obtained from affected and unaffected heterozygous mutation carriers followed by an integrated computational network analysis (Objective 2). Finally, we aim to validate modifiers identified in Objective 2 and elucidate their mechanistic role using well-established PD cellular phenotypes such as mitochondrial dysfunction, oxidative stress, and dopamine metabolism as readouts (Objective 3). We will provide cutting-edge technologies, i.e., CRISPR/Cas9-based genome editing (Objective 4) to several other projects of the Research Unit. Data generated in this project will be extensively exchanged with other projects focusing on PINK1- and Parkin-associated PD within the ProtectMove consortium. Methodological aspects of the multi-omics analysis will be discussed within Z2 and with P4.

• DFG Programme: Research Units
• Subproject of: FOR 2488: Reduced penetrance in hereditary movement disorders: Elucidating mechanisms of endogenous disease protection
• International Connection: Luxembourg
• Partner Organisation: Fonds National de la Recherche
• Cooperation Partner: Patrick May, Ph.D.