Re-evaluating the Role of SARM1 as a Transcriptional Regulator Using Improved Genome-Edited Mouse Models

Abstract

SARM1 is an ancient and highly conserved protein, initially described as having functions in innate immunity. In the last decade, our understanding of SARM1 structure and function expanded rapidly. The majority of research studies now focus on the role of SARM1 as an executioner of axon degeneration, leaving non-neuronal roles for SARM1 less well-characterised. Specifically, how and where SARM1 contributes to immune responses remains to be clarified. Murine SARM1 has been reported to regulate the expression of chemokines in both neurons and macrophages. However, the mechanism by which and extent to which SARM1 contributes to transcriptional regulation are not yet fully understood.

Our laboratory previously reported that Ccl5 induction is impaired in macrophages from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice, or B6 congenic Sarm1-/-, relative to wild type controls. Here, using RNA sequencing I identify additional differentially expressed genes in the B6 congenic Sarm1-/- mice. However, these results were confounded by the presence of passenger genes in the genome of the B6 congenic Sarm1¬-/- mouse, derived from the 129 donor strain of mice used in their generation. To re-evaluate the transcriptional role of SARM1 in the absence of these passenger genes, three novel SARM1-deficient mice were generated by CRISPR/Cas9.

In contrast to results in the B6 congenic Sarm1¬-/- mouse, macrophages from the new SARM1-knockout mice showed similar transcription of all genes measured, including Ccl5, compared to wild type littermate controls. Hence I clarified that the differential gene expression previously observed in macrophages from B6 congenic Sarm1-/- mice is an artefact resulting from the presence of passenger genes, and is unrelated to the absence of SARM1. This adds to the body of literature implicating passenger genes in congenic mice as the legitimate cause of a phenotype previously ascribed to the targeted gene. Since the generation of these B6 congenic Sarm1-/- mice 15 years ago, they have been heavily relied on for the study of SARM1 function. The extent to which the passenger genes present in the genome of these mice may have influenced previous studies, and by extension, misinformed our understanding of SARM1 function has yet to be fully understood. The novel CRISPR/Cas9 SARM1-knockout mice described in thesis provide an improved model in which to further explore the roles for SARM1 and to re-evaluate SARM1 functions previously described in B6 congenic Sarm1-/- mice.

Additionally our laboratory has generated, to our knowledge, the first mouse expressing epitope-tagged SARM1 endogenously. Difficulty in detecting SARM1 protein expression outside of the neurons has been reported, and has led to some speculation that SARM1 expression may be limited to the nervous system. Using the mouse expressing epitope-tagged SARM1, I confirmed that SARM1 is abundantly expressed in the brain, where its roles are numerous and varied. SARM1 expression was not confined to the nervous system, as I showed that SARM1 expression is detectable in macrophages from this mouse.

Overall, this work clarifies that murine SARM1 does not function as a transcriptional regulator of Ccl5 expression in macrophages, though SARM1 expression is detectable in this cell type. This thesis cautions against the use of model animals in which the genome is contaminated by confounding passenger genes, and proposes CRISPR/Cas9 genome-edited animals as a more suitable alternative. Finally, newly generated SARM1-knockout and epitope-tagged mice are described and suggested as an improved new model to explore SARM1 function.