A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types

Abstract

Summary The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. We have now developed a new ‘BACarray’ methodology, based on affinity purification of polysomal mRNAs from genetically defined cell populations. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. Even two morphologically indistinguishable subclasses of MSNs display vastly different translational profiles. Striatopallidal neurons are characterized by a strong and cell-specific release of intracellular Ca2+ in response to sphingosine 1-phosphate, consistent with their selective expression of Gpr6. In contrast, striatonigral neurons demonstrate a selective cell-specific increase in GABAA receptor subunits in response to chronic cocaine treatment. BACarray translational profiling is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.

Cell-specific translational profiling in acute kidney injury

“…TRAP followed a detailed protocol supplied by the Dr. N. Heintz laboratory at the Rockefeller University (New York, New York, USA) (19,20). Kidneys were removed 24 hours after surgery.…”

“…Recently, translating ribosome affinity purification (TRAP) has been developed to identify cell type-specific changes in mRNA populations at the whole-organ level (19,20). A driver for development of TRAP technology has been the need to obtain molecular signatures for adult neuronal types where recovery of intact cells is not possible through tissue dissociation and FACS.…”

“…A driver for development of TRAP technology has been the need to obtain molecular signatures for adult neuronal types where recovery of intact cells is not possible through tissue dissociation and FACS. In TRAP, an EGFP-tagged form of the L10a ribosomal subunit (EGFP-L10a) is activated within a cell type of interest (19,20). The profile of ribosome-bound mRNAs in the cell type is determined by affinity purification of ribosomes from a whole-organ lysate with anti-EGFP antibodies, followed by microarray profiling or sequencing of stripped mRNAs.…”

“…The neural TRAP studies have used large BACs carrying cell type-specific regulatory information to drive an EGFP-L10a expression cassette within neural cell types of interest (19,20). Recently, reports in Drosophila, Xenopus laevis, and zebrafish emphasize the utility of the approach (21)(22)(23).…”

Cellular and Behavioral Outcomes of Dorsal Striatonigral Neuron Ablation: New Insights into Striatal Functions

“…In wild-type cultured hippocampal neurons expressing DTR-GFP fusion, administration of DT triggered apoptosis of GFP-positive cells (Supplementary Figure S1A). To specifically ablate striatonigral MSNs, we chose to drive the expression of the DTR-GFP construct under the control of a highly enriched striatonigral MSNs gene, Slc35d3 (Heiman et al, 2008;Lobo et al, 2006), using the BAC-based strategy (Supplementary Figure S1B). Transgenic mice were generated by injecting the recombined BAC into the pronuclei of hybrid C57Bl/ 6 Â CB1 F1 fertilized oocytes, which were then implanted into pseudopregnant females.…”