Deciphering how axons terminate growth and forms synapses is essential if we are to understand how a nervous
system is built. Such knowledge could provide opportunities to treat neurodevelopmental disorders and will be
needed if we are to harness the robust and resilient nature of the developing nervous system to design novel
therapies for treating neurodegenerative diseases, such as Alzheimer’s disease (AD). Our long-term goal is to
understand the molecular and cellular mechanisms that govern axon termination and synapse formation
in vivo using the nematode C. elegans.
The Pam/Highwire/RPM-1 (PHR) proteins are ubiquitin ligases and signaling hubs that are important
conserved regulators of axon termination, synapse formation and axon degeneration. Emerging links between
PHR signaling and neurodevelopmental disorders and neurodegenerative diseases (including AD) have further
heightened interest in understanding PHR signaling networks. Here, we use the latest high-sensitivity mass
spectrometry technology; rapid automated protein extraction and purification; and novel ubiquitination ‘traps’ to
decipher the signaling network of the C. elegans PHR protein, RPM-1. This has revealed two putative RPM-1
ubiquitination substrates and provided numerous footholds for deciphering how RPM-1 is regulated.
Our first aim focuses on an autophagy initiating kinase as a novel RPM-1 ubiquitination substrate.
CRISPR/Cas9 editing and genetics test if RPM-1 ubiquitin ligase activity affects the stability and turnover of this
kinase to influence axon and synapse development. We also evaluate how RPM-1 effects on this kinase affect
autophagosome formation in neurons. Outcomes will provide insight into whether PHR proteins regulate
autophagy, and address how autophagy is inhibited in the nervous system in vivo. Our interest in these questions
is further fueled by the prominent role autophagy plays in neurodegenerative diseases, including AD.
Our second aim will evaluate another novel RPM-1 ubiquitination substrate, a kinase with prominent roles
in synapse development, synaptic plasticity and AD. We will determine how RPM-1 inhibits this kinase, and
whether this affects axon termination and synapse formation. We also aim to address which downstream
mechanisms this kinase utilizes to affect axon and synapse development. Despite the importance of this kinase
in nervous system health and disease, how it is inhibited remains unknown in any organism.
Finally, proteomics provided several entry points into understanding how RPM-1 might be regulated. In our
third aim, we focus on three particularly compelling entry points. 1) The most prominent RPM-1 binding protein
identified. 2) Components of an entire receptor signaling system identified as RPM-1 binding proteins. 3)
Numerous residues in RPM-1 that are phosphorylated in vivo. We will evaluate how these mechanisms affect
RPM-1 localization, and RPM-1 functions in axon and synapse development. Our interest in these questions is
driven by a simple theme: How PHR proteins are regulated, in any system, remains dark biology.