IDENTIFYING PARKINSON’S DISEASE PENETRANCE-MODIFYING FACTORS IN THE POPULATION-BASED COOPERATIVE HEALTH RESEARCH IN SOUTH TYROL (CHRIS) COHORT ​

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. Mutations in PRKN (Parkin), an E3 ubiquitin ligase mostly known for its role in facilitating the selective clearance of dysfunctional mitochondria (mitophagy) (Celardo et al., 2014), are the most common cause of autosomal recessive PD (Kitada et al., 1998). Carriers of these mutations show an earlier age of onset and slower disease progression compared to idiopathic PD (Klein & Schlossmacher, 2007). PRKN mutations have been identified in numerous families with diverse genetic backgrounds (Hedrich et al., 2004), encompassing copy number variations, small deletions/insertions, and single nucleotide polymorphisms (Kasten et al., 2018). Single, heterozygous mutations may increase the susceptibility to disease symptoms with significantly lower penetrance (Huttenlocher et al., 2015; Weissbach, Konig, et al., 2017). Given that up to 4% of the population carries heterozygous PRKN mutations, it is crucial to identify mechanisms/factors in non-PD manifesting heterozygous PRKN mutation carriers that influence the penetrance and could represent diagnostic tools or novel targets for potential neuroprotective or disease modifying therapeutic interventions. Specifically, I investigated circulating cell-free mitochondrial DNA (ccf-mtDNA), interleukin 6 (IL-6), c-reactive protein (CRP), and complement system activation and tested for association with mutation carrier status. I also determined the mtDNA mutational load, transcription/replication-associated 7S DNA, mtDNA copy number, and mitochondrial metabolism in blood and cell models derived from carriers of PRKN mutations. Non-PD manifesting heterozygous PRKN mutation carriers showed elevated IL-6 levels, altered complement system activity, and increased release of mtDNA into the cytosol. They also exhibited mtDNA alterations, along with changes in mtDNA copy number over time. Furthermore, an increased flux of the tricarboxylic acid (TCA) cycle was detected. A combination of these parameters might represent markers for PD development in prodromal/non-disease manifesting individuals carrying a heterozygous PRKN mutation. Mitochondrial stomatin-like protein 2 (SLP-2) interacts with Parkin and can alleviate mitochondrial dysfunction in PRKN PD patients. A moderate increase of SLP-2 expression was shown to correct mitochondrial defects caused by Parkin deficiency in cellular and in vivo models (Zanon et al., 2017). The use of the SINEUP technology, which is based on a new class of synthetic long non-coding RNAs, was proposed for treating haploinsufficiencies but also to increase the expression of neuroprotective proteins to compensate for a disease phenotype (Espinoza et al., 2021). It represents an ideal tool for upregulating protein levels without causing unphysiological overexpression with potential toxic effects. I used this approach to increase SLP-2 levels in a PRKN-deficient cellular model. Six molecules targeting SLP-2 mRNA were designed, and after confirming SINEUP activity by quantifying SLP-2 protein levels, the most promising molecules were further analyzed for their functional effects. Specifically, I evaluated the ability of these SINEUP-SLP-2 molecules to rescue the mitochondrial network fragmentation in PRKN knockout SH-SY5Y cells and found two molecules able to rescue the phenotype. This approach, therefore, offers an innovative method to upregulate SLP-2 expression within physiological levels, rescuing mitochondrial function and showing significant potential as a therapeutic strategy.