Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2^G2019Smodel of Parkinson’s disease

Abstract

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson’s disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019Sknock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Evidence for low affinity of GABA at the vesicular monoamine transporter VMAT2 – implications for transmitter co-release from dopamine neurons

“…In contrast, co-release of GABA from DA neurons is less well understood. Though there seem to be small subsets of DA neurons expressing GAD1/2 (Azcorra et al, 2023;Gaertner et al, 2024) and even VGAT (Conrad et al, 2024) the majority does not. Rather, it was suggested that GABA is taken up from outside by the plasmalemmal GABA transporter GAT1 (Melani et al, 2022;Tritsch et al, 2014) and/or synthesized de novo by ALDH1a1 (Kim et al, 2015) prior to being loaded into vesicles by VMAT2 (Melani et al, 2022;Tritsch et al, 2012).…”

“…This finding was rather surprising because GABA is a zwitterionic amino acid and structurally very different from classical VMAT2 substrates that have an aromatic ring and a positive charge (Peter et al, 1994;Yelin et al, 1995;Zheng et al, 2006). In contrast to glutamate, which is presumably present in all cells with active gene expression, GABA is typically produced through decarboxylation of glutamate in cells containing GAD1 or GAD2, which are little expressed in DA neurons (Azcorra et al, 2023;Gaertner et al, 2024;Tritsch et al, 2014). But how do DA neurons get GABA in the first place?…”