A Systems biology approach to elucidate the contribution of alpha-synuclein to early in vitro phenotypes of Parkinson’s disease

Although Parkinson's disease (PD was first described more than two hundred years ago, the clinical treatment options remain limited to symptom alleviation. Consequently, understanding the underlying molecular mechanisms is vital for the development of new therapeutic strategies. Most cases of PD are associated with toxic aggregations of the alpha-synuclein (α-syn) protein. However, the physiological and pathological mechanisms of α-syn aggregation are not entirely understood. One main reason for this knowledge gap is the lack of models that properly recapitulate the pathology in a human-midbrain-like context. Organoid models have emerged as an attractive model system that covers key aspects of in vivo tissue and organ complexity. Here, we present an optimized organoid protocol, which recapitulates features of the human midbrain. These human midbrain organoids (hMOs) present reduced levels of cell death in the core, while exhibiting reduced variability and increased viability. Their smaller size also allowed the implementation of a time-efficient image analysis technique. By using the protocol mentioned above, we generated hMOs from patient-derived induced pluripotent stem cells (iPSCs harboring a triplication of the SNCA gene (3xSNCA. 3xSNCAexhibited twice the levels of α-syn protein compared to wild type (WT) hMOs. Transcriptionalanalysis of 3xSNCA hMOs showed upregulation of PD- and SNCA-associated genes, as wellas transcriptional deregulations in neurogenesis, cell death, proliferation, and synapse formation. The analysis of cellular phenotypes in patient-specific hMOs supported these genetic observations. 3xSNCA hMOs presented reduced proliferation, cell death and reduced synapse count in mature organoids. Furthermore, 3xSNCA hMOs showed a reduced total number of neurons and impaired astrocytic differentiation. In addition, analysis of transcriptional and metabolomic data showed deregulation in metabolic pathways. To further analyze and explain our results, we used the latest human metabolic reconstruction (Recon3D) to generate an in silico model. The results presented here are a systematic analysis of patient-specific phenotypes in midbrain organoids from individuals with a triplication in the SNCA gene, which represent a starting point for further approaches to develop therapies