Unique molecular features and cellular responses differentiate two populations of motor cortical Layer 5b neurons in a preclinical model of ALS.

A major paradox of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is that many genes linked to disease are ubiquitously expressed, yet only discrete subtypes of cells degenerate. Therapeutic advancement depends on understanding unique molecular responses of vulnerable cells in the context of disease-causing mutations. Here, we describe two bacTRAP lines, Gprin3 and Colgalt2, that label distinct populations of projection neurons in layer 5b (L5b) of motor cortex. We demonstrate that these cells can be distinguished not only by their anatomical features, but by their underlying molecular properties. In the SOD1*G93A preclinical ALS model, both populations showed molecular responses in common, but Gprin3 corticospinal population showed additional, unique responses in disease that may reflect their disease vulnerability since Gprin3 cells were lost during disease progression while Colgalt2 cells were spared. These results tease apart molecular pathways that may contribute to ALS pathology and provide a framework for elucidating selective vulnerability. The translating ribosome affinity purification (TRAP) method was used to collect polysome-bound mRNAs were collected from two subpopulations of layer 5b pyramidal neurons using Gprin3-bacTRAP mice or Colgalt2-bacTRAP mice. TRAP IPs were compared to whole tissue mRNAs (input) or TRAP IP mRNAs were compared between cell types (Gprin3 and Colgalt2), or between transgenic ALS mice (SOD1*G93A) and wildtype littermates within each cell type (Gprin3 and Colgalt2). We collected 2-5 replicates for each experimental group.