Using RNA-Sequencing, In Vitro Calcium Imaging, and Proteomics to Investigate Novel Axon Degeneration Mediator, TMEM184B

Abstract

TMEM184B is a novel mediator of axon degeneration with unknown molecular function. Previous work has shown Tmem184b loss delays axon degeneration in somatosensory neurons. This thesis involves a multi-faceted approach to gain insight into TMEM184B’s molecular and cellular function: I have analyzed the transcriptome of Tmem184b-mutant somatosensory ganglia to determine how Tmem184b loss affects neuronal gene expression, and I employed immunoprecipitation coupled with tandem mass spectrometry (proteomics) to determine with which proteins TMEM184B directly interacts. I aimed to integrate these results to determine a cellular and molecular function of TMEM184B, and to determine how TMEM184B impacts axon degeneration. Interestingly, I found that Tmem184b loss did not transcriptionally alter key axon degeneration mediators, but it strongly downregulated Il31ra, a receptor with well-known roles in eczema and asthma. I also found that TMEM184B interacts with 2nd and 3rd order mitogen-activated protein kinases (MAPKs), including MAP3K4 (also known as MEKK4), a known upstream regulator of the SARM1 and MAPK axon degeneration cascade. More generically, however, TMEM184B interacts with key proteins involved in the endo-lysosomal pathway, including multiple subunits of the vesicular/vacuolar H+-ATPase complex. This interaction may dictate vesicular dynamics that affect the delivery of molecular signals from distal parts of sensorimotor neurons to the soma.