Calcium as principal regulator of cell death during Parkinson´s disease

Diverse Ca2+ signals govern a myriad of fundamental neuronal functions such as synaptic transmission, plasticity, regulated neurite outgrowth and synaptogenesis. Thus, impaired cellular Ca2+ homeostasis, unregulated Ca2+ fluxes and dysfunctional Ca2+signalling is implicated in a broad variety of neurodegenerative diseases, including Alzheimer`s disease, Parkinson`s disease, Huntington`s disease, Epilepsy and even the psychiatric disorder Schizophrenia. The causes and/or consequences of Ca2+ dyshomeostasis are manifold but eventually provoke the same outcome: neuronal dysfunction and death. This project focuses on the connection between impaired Ca2+ homeostasis and cell death during Parkinson`s disease (PD), a disabling neurodegenerative disorder strongly associated with age. Due to the extension of life expectancy, the incidence of PD is predicted to increase drastically within the next decades. The presence of intracellular inclusions called Lewy bodies represents a pathological hallmark of PD. The protein a-synuclein constitutes the major structural component of intracellular inclusions and is suggested to play a key role in the pathology of PD. Though an increasing body of evidence points towards a role for Ca2+ ions and Ca2+-dependent processes in a-synuclein-mediated neuronal death during PD, the pathological mechanisms behind remain elusive. Is the a-synuclein-induced rise in cytosolic Ca2+ a cause or a consequence of a-synuclein cytotoxicity and what are the molecular determinants involved? To further elucidate these questions, the amenable yeast model shall be applied, coding for many homologs of the machinery responsible for concerted Ca2+ homeostasis in mammalian cells but lacking its complexity and redundancy. Humanized yeast models based on heterologous expression of human native a-synuclein and clinical mutants not only faithfully recapitulated several features of PD, but also allowed to refine processes and identify novel players involved in a-synuclein-instigated cytotoxicity. Thus, this readily manipulable system provides an opportunity to decipher how ageing, Ca2+ homeostasis, mitochondrial (dys)function and oxidative stress as well as environmental and genetic (in particular a-synuclein) factors related to PD intertwine to impair and finally kill neurons. Our recent results obtained in this humanized yeast model suggest an interrelation between a-synuclein, basal cytosolic Ca2+ levels and cellular Ca2+ shock response as well as between a-synuclein toxicity and a specific Ca2+ ATPase (unpublished data). Using yeast clonogenic survival assays and flow cytometry- based quantification of apoptotic and necrotic markers combined with aequorin-based luminescence measurements to determine cytosolic as well as compartmentalized Ca2+ concentrations, the pathway(s) of a-synuclein-mediated toxicity intertwining with Ca2+ homeostasis will be further investigated. Auspicious results obtained applying the yeast system will be further validated in Drosophila melanogaster model for PD. In general, the intention is to elucidate the complex interplay between a-synuclein toxicity, ROS accumulation, mitochondrial impairment upon environmental toxins and cellular Ca2+ homeostasis. In combination, these diverse factors might lead into a vicious cycle that culminates in neuronal death. In a society with increasing average age, a better understanding of the molecular mechanisms that regulate or disturb cellular Ca2+ homeostasis during pathological and non-pathological ageing of neurons is desirable if not necessary.

This Elise-Richter project aimed at a better understanding of the connection between age-related deregulation of Ca2+ homeostasis and neuronal cell death during Parkinsons disease (PD). PD is a devastating neurodegenerative disorder characterized by the death of dopaminergic neurons in a specific brain region termed substania nigra. Several cellular processes are implicated in the development and progression of PD, among them impairment of Ca2+ homeostasis and autophagy, a term referring to cellular self-eating. Malfunctioning of the protein ?-synuclein is thought to play a main role in PD pathology. While ?-synuclein has been shown to cause an age-related increase in cytosolic Ca2+ levels, the mechanisms behind these disturbances of Ca2+ homeostasis remain elusive. Thus, the main goal of this project was the further elucidation of the effects of ?-synuclein on cellular Ca2+ transport, signalling, and storage and the identification of molecular determinants involved. We could show that ?-synuclein triggers an elevation of cytosolic Ca2+ levels that is followed by the generation of oxidative stress. Chemical inhibition of the ?-synuclein-induced cytosolic Ca2+ rise prevented the generation of oxidative stress and protected from cell death, indicting that the disturbances of Ca2+ homeostasis are causatively involved in PD-linked cellular demise. We could identify the Ca2+ pump PMR1 as an evolutionary conserved regulator of ?-synuclein-driven changes in Ca2+ homeostasis and cytotoxicity. In yeast, fly and nematode models for PD, the inactivation of this pump prevented both the elevation of Ca2+ levels and subsequent cell death. Furthermore, we used flies to study two additional features associated with PD pathology, namely the loss of dopaminergic neurons in the brain and an impairment of motor function. Remarkably, inactivation of PMR1 prevented the development of these PD-specific pathologies. Furthermore, we discovered an interesting new connection between Ca2+ signalling and autophagy, a process necessary for the efficient degradation of damaged cellular material. While we observed a block in autophagic processes upon high levels of ?-synuclein in our humanized yeast model for PD, autophagy could be re-installed via activation of specific components of the major cellular Ca2+ signalling pathway. These findings provide new insights into the mechanisms underlying PD-linked cellular demise and might point to new potential strategies in PD intervention.