Temporal examination of novel transcription factors in the cell body response of sensory neurons to injury

Abstract

Primary sensory neurons in dorsal root ganglia (DRG) undergo a cell body response after injury, where the neurons activate genetic growth programs with the goal of regenerating new axons. Transcriptional regulators are key to this response and the role of cell stress mediated transcription factors including FOXO3a, Luman and Zhangfei (ZF) are not fully understood. FOXO3a is proapoptotic and implicated in many neuronal pathologies. Recently, Luman, a regulator of the unfolded protein response, was identified as a retrograde injury signal essential for intrinsic regenerative axon growth, while ZF is a known inhibitor of Luman in other cell types. This thesis focused on the cell body response of DRG neurons to injury and whether expression patterns of these stress related transcription factors were affected by axotomy. A rat unilateral spinal nerve transection time course was employed and temporal protein and mRNA changes evaluated. Ipsilateral observations were as follows: FOXO3a protein decreased in injured neurons, while mRNA levels remained relatively constant, suggesting changes were secondary to post-translational modifications; while there was an initial decline in ZF expression post-injury, both ZF and Luman protein and mRNA were upregulated in ipsilateral neurons in a biphasic manner. Brain-derived neurotrophic factor (BDNF) is a known regulator of the regeneration response in DRG neurons. Its impact on these factors was determined by reducing endogenous BDNF with small interfering RNAs (siBDNF) or applying brief electrical stimulation to injured nerves, the latter upregulating BDNF. SiBDNF diminished injury triggered FOXO3a mRNA and ZF protein alterations, while stimulation enhanced the responses of somal FOXO3a and axonal Luman. A striking finding was that unilateral injury resulted in a mostly parallel, albeit lower biphasic response in contralateral DRG for all three transcription factors, with similar impacts on FOXO3a expression observed in cervical DRG remote from injury. Such dramatic contralateral biphasic changes are novel and support the existence of a systemic injury response. The findings of this thesis expand on the importance of transcription factors in the cell body response of DRG neurons, the impact of BDNF on regeneration and enforces the reality of contralateral and systemic effects to injury that cannot be ignored.