Aleksandar Rakovic, PhD

Molecular Mechanisms of Parkinson Disease

Sarah Graf, Karin Wiegers, Franca Vulinovic, Aleksandar Rakovic, Britta Meier, Victor Krajka

Team: Aleksandar Rakovic (Group leader), Katharina Shurkewitsch (MD student), Jannik Prasuhn (MD student), Jonathan Ziegler (MD student), Christoph Mårtensson (MS student)

The main focus of the team “Molecular Mechanisms of Parkinson’s Disease” is the characterization of molecular pathways involved in the pathophysiology of Parkinson’s disease (PD) using human cellular models (human dermal fibroblasts, neuroblastoma cell lines and induced pluripotent stem cell (iPSC)-derived neurons). We investigate the interaction between the mitochondrial kinase PINK1 (PTEN-induced putative kinase 1) and the ubiquitin E3 ligase Parkin and their role in the removal of damaged mitochondria by autophagy (mitophagy). Previoiusly we discovered that PD-causing mutations in PINK1 impair recruitment of Parkin to damaged mitochondria and demonstrated the role of Parkin in the ubiquitination of outer mitochondrial membrane proteins as an initial step in mitophagy. Currently, we focus on depicting the exact role of the ubiquitin proteasome system and the lysosomal system in mitophagy.

Recently, we established a platform for genome editing using CRISPR/Cas9 system in various cellular models. For this, we are using episomal vectors for expressing both Cas9 protein and guide RNA (gRNAs) as well as in-vitro synthesized “capped” Cas9 mRNA and gRNAs for an integration-free approach.

In addition and in collaboration with Dr. Krishan Vishnolia (Institute of Integrative und Experimental Genomic (IIEG), University of Lübeck), we have generated PINK1 and Parkin knockout Zebrafish (Danio rerio) models of PD using CRISPR/Cas9 . Since 2014, we have been closely collaborating with Dr. Melissa Vos (Institute of Neurogenetics, University of Lübeck) on pink1 and parkin knockout Drosophila models.

Projects are funded by intramural grant support, the DFG, and by the Hermann and Lilly Schilling Foundation.

We collaborate with several national and international colleagues including Prof. Dimitri Krainc (Northwestern University, Feinberg School of Medicine, USA), Prof. Bart de Strooper (University of Leuven, Leuven, Belgium), the SysMed Consortium (Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg), Prof. Sonja Schrepfer (Hamburg Transplant and Stem Cell Immunobiology Laboratory, University of Hamburg, Germany).

(A) PINK1-/Parkin-mediated removal of depolarized mitochondria. Human dermal fibroblasts obtained from healthy control (Control) or from PD patient carrying a homozygous mutation in PINK1 (PINK1mut) stably overexpressing Parkin were either untreated (left lane) or treated with valinomycin for 12 h. Cells were fixed and immunostained with antibodies against Parkin (green) and a mitochondrial marker GRP75 (red). A complete loss of mitochondria (indicative of mitophagy) was detected only in controls but not in PINK1 mutants.
(B) CRISPR/Cas-mediated knockout of PINK1, Parkin, and DJ-1. Neuroblastoma cells were transiently transfected with episomal vectors expressing Cas9 and gRNAs against PINK1, Parkin, or DJ-1. Upon transfection, single cells were plated in separate wells to form monoclonal cell lines. The knockout was confirmed by Sanger sequencing, qPCR, and western blotting using antibodies against PINK1, Parkin, or DJ-1.
(C) Zebrafish embryos (1-cell stage) were injected with both Cas9GFP mRNA and gRNAs against zPINK1. Three days post fertilization, GFP-positive embryos were selected and further maintained. Seven days post fertilization, 10 larvae were sacrificed for DNA extraction followed by PCR using primers covering the gRNAs-targeted areas. Approximately 60% of the larvae were successfully gene-edited.