Periodic Reporting for period 1 - PARNANT (Pathogenetic pathways in age-related neurodegenerations as novel therapeutic targets for Parkinson’s disease) Reporting period: 2019-09-02 to 2021-09-01 Summary of the context and overall objectives of the project This project laid the foundation for the identification of new therapeutic targets for Parkinson’s disease (PD). PD, which was first described in detail in An Essay on the Shaking Palsy by James Parkinson, is strongly associated with progressive loss of dopaminergic neurons during aging and affects more than 10 million people worldwide over 65 years of age. The pathological hallmark of PD is the Lewy body, consisting largely of aggregated alpha-Synuclein. The abnormal distribution of alpha-Synuclein correlates with neurodegeneration, which is seen in several PD models, including a roundworm Caenorhabditis elegans, a powerful model organism for aging research that has helped us to understand the development of proteinopathy. Only 10% of diagnosed PD patients have familial history with identified genetic variations affecting common cellular mechanisms responsible for cell protection during stress. Pathogenic mutations have been identified in genes responsible for protein and organelle quality control machineries amongst others, such as Parkin. On the other hand, sporadic PD does not have a clear mechanism of development and, therefore, it is considered a complex disease with multifactorial etiology. Several genome-wide association studies (GWAS) have identified several tens of risk signals associated with sporadic PD that are surrounded by hundreds of susceptibility genes. To date, there is little to no published functional validation of genes in these loci.As the incidence of PD increases with age (with estimated 4% prevalence over 85 years of age), the rapidly growing lifespan of human population is a huge economic and social burden for society. Current therapeutic interventions only alleviate symptoms but do not halt disease progression or induce neuro-restoration. Identifying novel molecular targets and searching for therapeutic agents that block neurodegeneration and promote neuronal restoration is a key challenge in the field. This project aimed to use a roundworm C. elegans, as an animal model, to identify genes modulating pathology associated with Parkinson’s disease (Objective 1), and subsequently elucidate the conservation of the newly identified genetic components in human cell-based model systems (Objective 2). Firstly, a new model (eraIs1) was constructed with a fluorescent alpha-synuclein construct. The obtained data using eraIs1, identified 28 C. elegans genes that modulated neurotoxicity caused by human alpha-Synuclein and genes that regulated the abundance of Parkin (the major cause of autosomal recessive juvenile parkinsonism). Subsequent evaluation of some of these genes on PD pathogenesis in human cell model systems suggested that genes regulating intracellular calcium levels could modulate alpha-Synuclein pathology. These data indicate that they might play a role in the pathogenesis of PD. This project thus provided both a new C. elegans model for PD gene screening, and identified new potential therapeutic targets for aging-related neurodegenerative diseases, and should therefore also bring forth an understanding of pathology in PD. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The first objective of the project was to identify the evolutionarily conserved genes that modulate toxicity of an alpha-Synuclein utilizing C. elegans model system (eraIs1). The first aim was to perform, for the first time, a systematic large-scale functional genetic screen of PD susceptibility genes, identified in human GWAS, for a modulation of neurodegeneration triggered by expressed human alpha-Synuclein. The screen of nearly a hundred PD risk genes, elicited by RNA interference, revealed 28 genes and their associated pathways that can modulate neuronal pathology of an alpha-Synuclein reporter accompanied with neurodegeneration in the eraIs1C. elegans model. The second aim was to identify genetic pathways mediating neuroprotection/degeneration in C. elegans. Various neuronal and protein aggregation phenotypes upon different stresses have been determined both upon inactivation as well as overactivation of some of the genes. The obtained data revealed 4 genes that regulate calcium levels in the cells, that might therefore constitute a pathogenetic pathway in PD. These results demonstrated that hypothesis-free identification of candidate PD genes through genome-wide association studies can be followed up functionally in a relatively high-throughput way with the eraIs1 model, to reveal new roles of evolutionary conserved genes in neuronal maintenance upon proteotoxic stress. In addition to these experiments, the regulation of Parkin, whose deficiency is associated with the most common young-onset PD, has been investigated. Through protein interactor and genetic screens to identify genes that regulate Parkin abundance this study showed that excessive Parkin accumulation in the roundworm C. elegans resemble some characteristics of aging-related diseases, including changes of the autophagy-lysosomal dynamics along with alpha-Synuclein processing. These data provided possible new insights for cellular pathology of some Parkin-related diseases, such as Parkinson's disease (as well as cancer and heart disease). The achieved objective 1 aims together provided new potential therapeutic targets for synucleinopathies and other neurodegenerative diseases associated with aging.The second objective of the project was to evaluate the conserved modulatory role of the identified genes on PD pathology using a human cell model system. The obtained data suggested that the identified genes regulating calcium levels are also mis-regulated in PD, and their overexpression can modulate alpha-Synuclein levels in human neuronal models. These interesting preliminary data lay the basis for many possible follow up studies. The results of the Objective 1 have been summarized in two manuscripts, while the results of the Objective 2 have generated preliminary data for ongoing projects that will be published in the following years. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This project identified several new genes that may play a role in PD pathogenesis and thus may serve as new therapeutic targets for the aging-related neurodegenerative diseases. Obtained data lay the basis for follow up studies that should first evaluate therapeutic potential of the identified genes in the human cell models, then use those cell models for a compound library screening, and eventually perform clinical trials.Moreover, using C. elegans at the Host institute will serve as a new tool to screen for risk genes of several diseases, including neurological and cardiovascular diseases, identified from the genetic diagnosis and large population screening data from the South Tyrol. The established animal model facility strengthens the collaboration between Bioinformatics and “wet-lab” Research within the Institute and provides a unique interdisciplinary and competitive approach for biomedical science in South Tyrol. This project thus fulfilled the research gap in the province and should lead to collaborative projects to evaluate newly identified neuroprotective pathways in vertebrate models in the near future and eventually with local stakeholders for clinical trials involving patients with the disease.