Spatiotemporal Proteomic and Transcriptomic Landscape of DAT+ Dopaminergic Neurons Development and Function

Dopaminergic (DA) neurons marked by the dopamine transporter (DAT) have multiple physiological functions and are involved in the regulation of mental and neurological diseases, prompting in-depth studies into their development and functions. This research explores the spatiotemporal proteomic and transcriptomic changes in DAT+ DA neurons within key brain regions involved in DA signaling—the nucleus accumbens (NAc), substantia nigra (SNc), and ventral tegmental area (VTA). Utilizing cutting-edge multi-omics techniques, such as ultrasensitive trace sample proteomics and SMART_x0002_seq2 for transcriptomics, we examine the DA neuronal system at critical postnatal milestones: postnatal day 7 (P7), postnatal day 30 (P30), and postnatal day 60 (P60). The study reveals unique molecular profiles within DA neuron populations, showcasing their varied functional roles and developmental progression. Immunofluorescence mapping illustrates these molecular distributions, validating the quantitative data and highlighting the dynamic molecular structure of DA neurons. Our findings notably highlight a marked increase over time in Aldh1a1 expression, an essential enzyme for retinoic acid production, suggesting its evolving role in neuronal development and specific functions. This comprehensive analysis offers a profound molecular perspective on DAT+ DA neuron development, enhancing our understanding of their functional diversity and potential relevance in DA-related diseases. Overall design: we outlined a stepwise approach for multi-omics profiling of brain dopaminergic neuron development. We used mice at different ages to represent various developmental stages: P7, representing infancy (n=5); P30, juvenile phase (n=5); and P60, adulthood (n=5). We focused on three specific brain regions rich in DAT+ neurons within these developmental groups: the NAc, SNc, and VTA. After dissecting and isolating the brain region tissues (Fig. 1C), we dissociated the tissue samples to obtain single-cell suspensions and then performed flow cytometry to isolate the DAT+ neuron populations.Once isolated, the DAT+ neurons from each region and developmental stage were simultaneously analyzed through proteomic and transcriptomic techniques