Spatiotemporal transcriptomics reveals distinct responses of ALDH1A1-positive and ALDH1A1-negative midbrain dopaminergic neurons to alpha-synuclein overexpression

Parkinson’s disease is characterized by the progressive and preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta , and intraneuronal alpha-synuclein (αSyn) accumulation. Dopaminergic neurons (DANs) are anatomically and molecularly heterogeneous, but the impact of αSyn pathology on distinct subpopulations is not well defined. One midbrain DAN sub-population expresses Aldehyde Dehydrogenase 1A1 (ALDH1A1), an enzyme that detoxifies aldehyde by-products of dopamine metabolism, and has been associated with differential vulnerability. Here, we applied GeoMx spatial transcriptomics to profile ALDH1A1-positive (ALDH1A1 ⁺ ) and ALDH1A1-negative (ALDH1A1 ⁻ ) DAN subpopulations in the mouse midbrain in situ at 3- and 8-weeks following adeno-associated virus (AAV)-mediated αSyn overexpression. Analyzing 10,532 genes, we identified robust transcriptional differences between ALDH1A1 ⁺ and ALDH1A1 ⁻ DANs under control conditions, supporting their characterization as distinct molecular subpopulations. In AAV-αSyn-injected mice, we observed increased Snca expression and a reduction in ALDH1A1 ⁻ DANs in the ipsilateral substantia nigra . αSyn overexpression induced subpopulation-specific and time-dependent transcriptional responses, with dysregulation in ALDH1A1 ⁻ DANs characterized by early down-regulation of pathways related to synaptic function, neurotransmitter handling, and bioenergetics, including glycolysis. In contrast, ALDH1A1 ⁺ DANs displayed later up-regulation of genes enriched for Acetyl-CoA and cholesterol metabolism pathways, reflecting subpopulation-specific adaptations to αSyn overexpression. Analysis of human single nucleus RNA-sequencing data revealed partial conservation of the metabolic dysregulation signature. Together, our findings show that murine midbrain ALDH1A1 ⁺ and ALDH1A1 ⁻ DANs represent molecularly distinct subpopulations with divergent temporal responses to αSyn overexpression, emphasizing the importance of cell-type and disease-stage context in studies of Parkinson’s disease mechanisms.