Neuronal subtype-specific growth cone and soma purification from mouse and other mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes

• Published in: Nature protocols Vol. 17; no. 2; pp. 222 - 251
• Main Authors: Engmann, Anne K., Hatch, John J., Nanda, Prakruti, Veeraraghavan, Priya, Ozkan, Abdulkadir, Poulopoulos, Alexandros, Murphy, Alexander J., Macklis, Jeffrey D.
• Format: Journal Article
• Language: English
• Published: 12.01.2022
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-021-00638-7

During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit “wiring”. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from rodent central nervous system (CNS) in vivo. Using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is purified by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting (FSPS) and established fluorescence-activated cell sorting (FACS) of neurons, and are suitable for downstream analysis of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes about 3 hours, plus sorting time, and about 1–2 hours for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, being dependent on the turnaround time of the core facility involved. This enables isolation of growth cones and paired somata from any neuronal subtype of the CNS that can be fluorescently labeled by fluid shear forces, ultracentrifugation, fluorescent small particle sorting, and fluorescence-activated cell sorting.