Proteomic insights into the biology of dopaminergic neurons

Abstract

Dopaminergic neurons, primarily located in the substantia nigra, hypothalamus, and ventral tegmental area of the brain, play crucial roles in motor control, reward, motivation, and cognition. Alterations in their function are associated with numerous neurological and psychiatric disorders, such as Parkinson's disease, but also Schizophrenia, substance use disorders, and bipolar disorder. Recent advances in mass spectrometry-based proteomics have enabled the comprehensive profiling of protein expression, turnover, subcellular localization, and post-translational modifications at an unprecedented depth of analysis. This review summarizes the developments in proteomic approaches taken to study dopaminergic neurons. We cover findings from global and spatial proteomics studies that revealed brain region-specific protein signatures, as well as dynamic turnover of proteins and the importance of mitochondrial and synaptic proteins for the health and vulnerability of dopaminergic neurons. Combined with advanced molecular cell biology tools, such as growth in microfluidic devices, fluorescent-activated synaptosome sorting, and enzymatic proximity labeling, modern proteomics allows for investigation of synaptic and subcellular proteomes. Despite these advancements, the complexity of the human brain and its cell-specific characteristics remain a challenge. The continuing integration of advanced proteomic techniques with other -omics will eventually yield improved and mechanistic understanding of dopaminergic neurons in health and disease.

Keywords: dopaminergic neurons; iPSCs; protein turnover; proteomics; synaptosomes.

FIGURE 1

Dopamine pathways and subcellular compartments of DA neurons. (A) Schematic of the different brain regions where dopaminergic neurons are located. The four main dopamine pathways (nigrostriatal, mesocortical, mesolimbic, and tuberoinfundibular) are also depicted in different colors (red, purple, blue, and green respectively). (B) DA neuron with a zoom-in of one synapse. The key steps in the synthesis of dopamine and its release can be seen. Papers that are discussed across the review related to axonal proteomics or synaptosomal proteomics are also annotated in the figure.

FIGURE 2

Proteomic workflows. (A) The workflows for quantification strategies in proteomics. The upper panel depicts a standard workflow for LFQ, wherein proteins are extracted from a sample and subsequently digested into peptides. Thereafter, the peptides are measured using LC-MS/MS. Each sample is measured independently. The middle panel shows SILAC strategy. In this approach, samples are cultivated in SILAC media, allowing the proteins to incorporate the isotopic labels during their synthesis. Following this, the proteins are extracted, mixed in a 1:1 ratio, and digested into peptides for measurement. The lower panel outlines the workflow for TMT or Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling. In this process, proteins are digested, and the peptides are subsequently labeled. After labeling, the peptides are mixed and measured. Quantitative information is derived from reporter ions in the MS/MS spectrum. (B) The strategies for sample preparation. The upper panel depicts the preparation of samples for post-translational modification (PTM) studies. Proteins are extracted and digested into peptides. Following this, a PTM enrichment step is conducted, during which unmodified peptides are removed. The samples are subsequently measured. In the upper-middle panel, the apex or BioID workflow is presented. The protein of interest, tagged with an enzyme, is activated by the addition of a reagent, resulting in the enzyme biotinylating proteins in its immediate vicinity. Cells are lysed, and the biotinylated proteins are enriched using streptavidin beads. Thereafter, proteins are digested into peptides and further analyzed with LC-MS/MS. The FASS workflow is depicted in the lower-middle panel. Initially, synaptosomes are enriched and their subpopulations are sorted using fluorescent tagging of synaptic markers. Subsequently, proteins are digested and measured. In the lower panel, the LCM workflow is illustrated, where a tissue sample is stained with various antibodies, and a specific region is microdissected and isolated using a laser. This is followed by a standard proteomic workflow. The papers discussed in the review are indicated adjacent to the workflow utilized.