Abstract: α-Synuclein (α-Syn) pathology is inextricably linked to the pathogenesis of Parkinson’s disease (PD), yet we have very limited understanding why specific neuronal populations are vulnerable to α-Syn-related PD pathology while others are much more resistant. Likewise, we have very limited knowledge of whether neurons may transition from a resistant state to a vulnerable state, how neuronal vulnerability manifests itself during disease development, how it defines distinct stages of α-Syn pathology, and how other non-neuronal cells (astroglia, microglia, infiltrating immune cells) contribute to these processes. 4DPD-Omics is a consortium of experts in α-Syn biochemistry, neuroanatomy and neuropathology, translational α-Syn disease models, spatial and single cell omics profiling, bioinformatics and systems biology who are ideally positioned to perform an unprecedented, integrated, spatiotemporal transcriptome and proteome analysis of neuronal and non-neuronal responses to α-Syn pathology. The consortium has already collaboratively generated rich sets of bulk transcriptomics and proteomics data in preclinical models and post mortem human samples with α-Syn pathology. Using these data and subsequent bioinformatics and functional interrogations, the 4DPD-Omics consortium members have identified mitochondrial dysfunction, rewiring of metabolic networks, defects in mitophagy/autophagy, proinflammatory processes and altered synaptic vesicle trafficking as key mechanisms contributing to α-Syn pathology. However, it remains unresolved which cell types display these changes, how these processes are altered over time in the context of development and spreading of targeted α-Syn pathology and neurodegeneration, and which signalling pathways trigger these alterations. 4DPD-Omics will avail of wellcharacterised preclinical mouse models developed by consortium partners (striatal injections of α-Syn fibrils with progressive disease-like pathology), as well as clinically and genetically annotated human post mortem samples of defined Braak stages (0-VI), to perform an in-depth cell-specific spatial transcriptomics and proteomics analysis. We will avail of spatial transcriptomics, mass spectrometry, and CellDIVE multiplexing platforms to determine whole transcriptome/proteome changes at spatial tissue level and single cell level in neuronal and non-neuronal cell populations over time, and correlate these alterations to the progression and spreading of α-Syn pathology. We will create a 4D dynamic atlas of the distribution of specific cell subpopulations, correlate their transcriptome/proteome changes to their vulnerability to pathogenic processes, and characterise the spatial cellular landscape in relation to α-Syn pathology. Integrative bioinformatics analyses, including network modelling and master regulator analyses, will identify biomarkers, pathways, and signatures in cellular sub-types susceptible and resistant to α-Syn pathology that may precede appearance of the pathological phenotype. Utilising systems biology approaches such as genome scale modelling, previously identified disease pathways will be systematically investigated in the context of disease progression and the cell types in which they occur, and mapped to master regulators. Identified signalling pathways and master regulators will be validated in clinical samples using multiplexing approaches that enable the quantification of up to 50 markers in ±10,000 individual cells within a single tissue section/region, providing unprecedented insights into disease patterns in humans at single cell resolution. Collectively, by generating spatial tissue profiles and more than 10,000,000 single cell profiles and by analysing the spatiotemporal responses of vulnerable and less vulnerable neurons and non-neuronal cells to α-Syn pathology, we will identify biomarkers of disease progression and endophenotypes in PD patients, representing novel cell-specific targets and strategies for drug development.
