Dysregulation of autophagy by LRRK2 and alpha-synuclein in Parkinsons´s disease models

Within the last decade, deregulation of autophagy, a cellular bulk degradation process essentially involved in the removal of aggregated proteins and damaged mitochondria, has emerged as a decisive factor in the pathology of numerous neurodegenerative diseases, including Parkinsons disease (PD). Dysfunction of leucine-rich repeat kinase 2 (LRRK2) and alpha-synuclein (Syn), two proteins that upon mutation cause familial PD, have both been shown to differentially influence autophagic processes. Vice versa, modulation of autophagy altered the cellular consequences of their toxic gain-of-function. However, exact mechanisms remain yet to be elucidated. Both insufficient as well as excessive autophagy have been linked to LRRK2- and Syn-associated cellular demise, but the specific aspects of potentially distinct subtypes of autophagic processes that explain these apparently opposing effects remain largely elusive. Within proposed research, two simple model systems shall be applied to further elucidate LRRK2-mediated cytotoxicity and the involvement of distinct autophagic pathways and players, namely the yeast S. cerevisiae and the fruit fly D. melanogaster. While humanized yeast models have been successfully applied to study the effects of Syn, not only recapitulating PD-associated molecular characteristics but also allowing for the identification of phylogenetically conserved pathways influencing Syn-cytotoxicity, very little is known about the effects of heterologous LRRK2-expression in yeast. This project aims at (i) the establishment of an aging LRRK2-yeast model to elucidate the effects of LRRK2 (as well as enzymatic subdomains and pathogenic mutants) on cytotoxicity, mitochondrial function and autophagy in young versus old cells and (ii) a further understanding of autophagy as a common denominator of LRRK2- and Syn-linked cellular demise during PD. First, quantitative proteomics and genetic screens in yeast will identify autophagic regulators and pathways causally involved in LRRK2 and/or Syn- induced cytotoxicity. These candidates will then be further investigated in Drosophila. Finally, the most promising ones will be tested in mammalian cell culture and in brain tissue samples form PD patients to demonstrate potential relevance for neuronal decay in the human PD brain. Genetic tractability and the possibility of large-scale determination of survival, oxidative stress and autophagic levels combined with a high degree of conservation in respect to essential cellular processes governing aging, autophagy and cell death render yeast a valuable tool to investigate the consequences of neurotoxic proteins. Drosophila broadens the possibility of molecular studies towards in-depth functional analysis of a sophisticated, centralized nervous system. Both models in combination with a subsequent validation in the mammalian system will probably provide insights into the molecular mechanisms and importantly the distinctions underlying cytoprotective and detrimental autophagy upon LRRK2 and/or Syn dysfunction. This may well serve as a basis for pharmacological approaches selectively targeting over-activated or impaired autophagy during distinct PD-scenarios.

Parkinson's disease is a multifactorial neurodegenerative disorder strongly associated with age as well as with environmental and genetic risk factors. Among the cellular processes implicated in the progressive cell death of neurons during Parkinson's disease are defects in the functionality of mitochondria, the main suppliers of cellular energy, and compromised autophagy, referring to cellular "self-eating" and recycling of unused and damaged material. This project focused on the two proteins LRRK2 and -Synuclein, which are both implicated in sporadic as well as hereditary Parkinson's disease. In the course of this project, we could provide new insights into cell death triggered by these two proteins and unravel overlapping and distinct mechanisms involved in LRRK2 and -Synuclein-mediated cellular demise. Our results show that high levels of LRRK2 cause an overall impairment of mitochondrial function, leading to the aggregation of mitochondria and a general decrease in mitochondrial mass. This culminates in a decline of ATP, the cellular energy currency, and thus progressive energy depletion. Interestingly, we found that this was not caused by increased degradation of mitochondria, which is often the case in scenarios of mitochondrial damage, but rather by an early inhibition of mitochondrial biogenesis. Nutritional and genetic interventions that were able to prevent this depletion of mitochondrial mass and that induced enforced biogenesis of mitochondria improved overall cellular fitness and survival. In respect to -Synuclein, we found that a decrease in the activity of enzymes breaking down damaged material in the so-called vacuole, the cellular waste bin and recycling factory, represents a pre-lethal event in cellular degeneration. -Synuclein caused the mislocalization of one of the main degradative enzymes of the vacuole, leading to insufficient breakdown and recycling of cargo delivered to the vacuole via autophagy. Remarkably, artificial high levels of this degradative enzyme did restore the turnover of delivered cargo, and at the same time prevented not only cell death induced by -Synuclein but also death caused by LRRK2. In sum, this project provided important new insights into the mechanisms of Parkinson's disease-associated cell death, both in respect to dysregulation of autophagy and to mitochondrial dysfunction.