Shared Regulators of Axon Degeneration and Synaptic Structure

Abstract

The functioning of the nervous system rests on connections between neurons and their synaptic targets. Often these targets can be a great distance from the neuron, which leaves the connecting axon vulnerable to many stressors, including injury, that can damage this link. When an axon is severed, it undergoes Wallerian degeneration, where it breaks apart and is cleared by immune cells. Our lab previously identified a role for the transmembrane protein Raw in promoting this type of degeneration, presumably through its role in restraining cJun N-Terminal Kinase (JNK) signaling. In raw mutants, Wallerian degeneration is significantly delayed, so that the severed axon remains intact long after injury. Despite evidence that this delayed degeneration is dependent on altered gene expression via the Fos transcription factor, it was unclear how Raw regulated axon destruction.

To determine how Raw promotes degeneration, I tested for possible interactions between Raw and the prodegenerative enzyme dSarm. dSarm acts as a metabolic tipping point in axon degeneration and has been the focus of intense research in the past decade. When activated, dSarm rapidly breaks down the electron carrier NAD+ to drive catastrophic energy loss, which ultimately results in axon degeneration and cell death. Much work has examined how dSarm is regulated allosterically, but less is known about how it may be regulated through expression or localization. I found that loss of Raw leads to decreased levels of dSarm in the axon, and that the delayed degeneration seen in a raw knockdown is rescued by dSarm overexpression.This indicates that Raw likely promotes degeneration via Sarm1.

During my work with Raw, I also discovered its role in restraining neuromuscular junction (NMJ) overgrowth and regulating synaptic protein levels. When Raw is lost, the number of branches and the surface area of the NMJ increase greatly, while individual bouton definition is largely lost. Interestingly, dSarm is also known to influence NMJ structure, but as a promoting regulator rather than an inhibitory one. As Raw appears to regulate degeneration through dSarm, I checked if the NMJ growth seen in a raw knockdown was also dSarm dependent, but found no evidence of a direct relationship through either mutation or Cas9-mediated knockout of dSarm. Given the role of Raw as a JNK regulator, and JNK’s importance in NMJ growth, I next tested whether Raw and dSarm may be acting through JNK to regulate the NMJ. I found that dSarm promotes, while Raw inhibits, JNK activation in motoneurons and that their influences on NMJ structure are dependent on the JNK target Fos. While this strongly suggests that the overgrowth caused by manipulations of Raw and dSarm is due to JNK activation, I instead found that not only does JNK inhibition not rescued the overgrowth caused by raw knockdown, but JNK inhibition alone results in NMJ overgrowth. I also discovered that overexpression of JNK rescues the overgrowth caused by dSarm overexpression, despite the fact that both manipulations cause overgrowth on their own. This introduces a role for JNK in restraining NMJ overgrowth, which contrasts greatly with its well-documented role as a promoter of NMJ growth. Overall, these data indicate that Raw and dSarm have opposing influences on a multifaceted JNK signaling pathway that impacts gene transcription, axon degeneration, and NMJ structure.