The Genetics of the Neuromuscular Junction: Mechanisms and Disease Models

PROJECT SUMMARY
Re-training supplement request for R37NS054154 under NOT-OD-23-170
The overall goal of R37NS054154 is to understand the molecular mechanisms through which dominant
mutations in tRNA synthetase genes cause Charcot-Marie-Tooth type 2D and related inherited peripheral
neuropathies. Towards this, we will 1) examine the biochemical interactions of the mutant synthetases and their
cognate tRNAs, 2) explore why only alpha motor neurons and a subset of sensory neurons are affected by these
mutations, and 3) test the therapeutic potential of inhibiting the integrated stress response, which is activated by
these mutations. Much of this work has been done in mouse models of these diseases, but to extend these
studies into a human system, we have engineered GARS/CMT2D mutations into a healthy control iPSC line,
KOLF2, which we can differentiate into motor neurons in vitro. We have encountered two challenges with this
experimental system. First, the motor neurons are still immature in culture. We have assessed this in part by
testing the extent to which they turn on eEF1A2 and turn off eEF1A1. This developmental switch occurs in the
first few weeks of life in mice, correlating with the onset of neuropathy in our Gars mice, and we believe it to be
central to the cell-type specificity of tRNA synthetase mutations, since eEF1As directly interact with tRNA
synthetases and charged tRNAs. Thus, we need to develop sophisticated hiPSC co-culture systems to generate
more mature motor neurons that switch more completely to eEF1A2. Second, inhibiting the integrated stress
response in our mouse models is highly efficacious, but the rapid response to inhibition and neurophysiological
improvements suggest this benefit is coming from improved transmission at neuromuscular junctions. However,
whether NMJs are perturbed in the human disease is unknown. The best model for testing this before moving
to patients is an hiPSC-derived nerve-muscle co-culture system where we can establish that motor neuron-
muscle connectivity is indeed perturbed and that treatment with integrated stress response inhibitors improves
function. Establishing this would make ISR inhibitors more translational and increase interest in CMT from
companies developing such drugs. These more sophisticate co-culture systems and particularly those for
evaluating NMJ connectivity, are beyond the current expertise of the Burgess lab. We therefore propose this re-
training supplement request to train Dr. Timothy Hines in these approaches. Dr. Hines is an accomplished senior
postdoc who will transition to an Associate Research Scientist position at JAX to do this work. We have
assembled a team of local and external mentors to train Dr. Hines in these methods. With these skills, Dr. Hines
will be well-positioned to find an independent position and secure NIH funding in the future. This supplement
will benefit Dr. Hines’ professional development and the parent grant by making the experiments more
translationally relevant.

Grant Number: 3R37NS054154-16S1
NIH Institute/Center: NIH
Principal Investigator: Robert Burgess