The Enzymatic Core of the Parkinson’s Disease-Associated Protein LRRK2 Impairs Mitochondrial Biogenesis in Aging Yeast

Abstract

Mitochondrial dysfunction is a prominent trait of cellular decline during aging and intimately linked to neuronal degeneration during Parkinson’s disease (PD). Various proteins associated with PD have been shown to differentially impact mitochondrial dynamics, quality control and function, including the leucine-rich repeat kinase 2 (LRRK2). Here, we demonstrate that high levels of the enzymatic core of human LRRK2, harboring GTPase as well as kinase activity, decreases mitochondrial mass via an impairment of mitochondrial biogenesis in aging yeast. We link mitochondrial depletion to a global downregulation of mitochondria-related gene transcripts and show that this catalytic core of LRRK2 localizes to mitochondria and selectively compromises respiratory chain complex IV formation. With progressing cellular age, this culminates in dissipation of mitochondrial transmembrane potential, decreased respiratory capacity, ATP depletion and generation of reactive oxygen species. Ultimately, the collapse of the mitochondrial network results in cell death. A point mutation in LRRK2 that increases the intrinsic GTPase activity diminishes mitochondrial impairment and consequently provides cytoprotection. In sum, we report that a downregulation of mitochondrial biogenesis rather than excessive degradation of mitochondria underlies the reduction of mitochondrial abundance induced by the enzymatic core of LRRK2 in aging yeast cells. Thus, our data provide a novel perspective for deciphering the causative mechanisms of LRRK2-associated PD pathology.

Membrane-tethering of cytochrome c accelerates regulated cell death in yeast

“…For discrimination between necrotic and early/late apoptotic cell death phenotypes, AnnexinV/PI co-staining was performed according to refs. 11,33 . Therefore,~2 × 10 7 cells were harvested, washed once in digestion buffer (35 mM K 3 PO 4 , 0.5 mM MgCl 2 , 1.2 M sorbitol; adjusted to pH 6.8) and resuspended in 330 µl of the same buffer containing 2.5 µl Lyticase (1000 U/ml) and 15 µl Glucoronidase/Arylsulfatase (4.5 U/ml).…”

“…Clonogenic survival was evaluated as described recently 33 . In aggregate, the number of cells in culture was quantified via a CASY cell counting device (Schärfe Systems) and 500 cells were plated on YEPD agar plates.…”

“…To monitor oxidative stress, the reactive oxygen species (ROS)-driven conversion of non-fluorescent dihydroethidium (DHE) to fluorescent ethidium (Eth) was quantified with flow cytometry and visualized with confocal laser scanning microscopy, adapted from ref. 33 . To that end,~2 × 10 6 cells were harvested in 96-well plates, resuspended in 250 µl PBS containing 2.5 mg/l DHE and incubated for 10 min at RT in the dark.…”

“…To investigate mitochondrial transmembrane potential (Δψ m ), the protocol described in ref. 33 was slightly adapted.~2 × 10 6 cells were resuspended in 250 µl PBS containing 5% glucose and 200 nM Mitotracker CMXRos. Cells were incubated, washed and analysed as described for DHE staining above.…”

“…Oxygen consumption of yeast was quantified with a Fire-Sting optical oxygen sensor system (Pyro Science) as described previously 33 . In brief, 2 ml of culture were transferred into glass tubes, hermetically sealed and directly used for analysis.…”