Plexin-Semaphorin Signaling Modifies Neuromuscular Defects in a Drosophila Model of Peripheral Neuropathy

Abstract

Dominant mutations in GARS, encoding the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and Charcot-Marie-Tooth disease type 2D (CMT2D). This genetic disorder exemplifies a recurring paradigm in neurodegeneration, in which mutations in essential genes cause selective degeneration of the nervous system. Recent evidence suggests that the mechanism underlying CMT2D involves extracellular neomorphic binding of mutant GlyRS to neuronally-expressed proteins. Consistent with this, our previous studies indicate a non-cell autonomous mechanism, whereby mutant GlyRS is secreted and interacts with the neuromuscular junction (NMJ). In this Drosophila model for CMT2D, we have previously shown that mutant gars expression decreases viability and larval motor function, and causes a concurrent build-up of mutant GlyRS at the larval neuromuscular presynapse. Here, we report additional phenotypes that closely mimic the axonal branching defects of Drosophila plexin transmembrane receptor mutants, implying interference of plexin signaling in gars mutants. Individual dosage reduction of two Drosophila Plexins, plexin A (plexA) and B (plexB) enhances and represses the viability and larval motor defects caused by mutant GlyRS, respectively. However, we find plexB levels, but not plexA levels, modify mutant GlyRS association with the presynaptic membrane. Furthermore, increasing availability of the plexB ligand, Semaphorin-2a (Sema2a), alleviates the pathology and the build-up of mutant GlyRS, suggesting competition for plexB binding may be occurring between these two ligands. This toxic gain-of-function and subversion of neurodevelopmental processes indicate that signaling pathways governing axonal guidance could be integral to neuropathology and may underlie the non-cell autonomous CMT2D mechanism.

Developmental demands contribute to early neuromuscular degeneration in CMT2D mice

“…We therefore suggest that the polyinnervation phenotype is an epiphenomenon resulting from aberrant interaction of mutant GlyRS with (a) key trans-synaptic protein(s). There is precedent for this in a CMT2D Drosophila model; musclesecreted mutant GlyRS aberrantly accumulates at NMJs prior to degeneration 16 and coincides with neurodevelopmental wiring defects 28 . This phenocopies an ectopic motor neuron branching caused by loss-of-function mutations in plexA and plexB, which encode neuronal transmembrane proteins that bind secreted semaphorins to facilitate axonal guidance, retrograde signalling and synaptic plasticity 50,51 .…”

“…This observation led to the finding that plexin B serves as an erroneous binding partner for mutant GlyRS at the NMJ; however, while mutant GlyRS interferes with the fidelity of axon guidance pathways causing wiring defects, this was de-coupled from the NMJ pathologies, i.e. was an epiphenomenon 28 .…”

“…Neuromuscular degeneration is replicated in a Drosophila melanogaster model for GARS1 neuropathy and is dependent on the toxic accumulation of mutant GlyRS at the NMJ 16 . Suggestive of a non-cell autonomous mechanism, this pathological build-up requires muscle-secreted, but not neuron-derived, GlyRS, and appears to be mediated by deviant interaction with the neuronal receptor, Plexin B 28 .…”

Altered sensory neuron development in CMT2D mice is site-specific and linked to increased GlyRS levels

“…Moreover, all assessed GlyRS mutants possess a similar conformational opening that excavates neomorphic surfaces usually buried within the structure of the wildtype enzyme (He et al, 2011(He et al, , 2015. Given that GlyRS is secreted from several different cell types (Park et al, 2012;Grice et al, 2015;He et al, 2015;Park et al, 2018), these uncovered protein regions can mediate aberrant deleterious interactions both inside and outside the cell (He et al, 2015;Sleigh et al, 2017a;Mo et al, 2018), likely accounting for non-cell autonomous aspects of pathology (Grice et al, 2015(Grice et al, , 2018. While some of these misinteractions are with neuronally-enriched proteins, the pathomechanisms underlying neuronal selectivity in CMT2D remain unresolved.…”

Post-synaptic morphology of mouse neuromuscular junctions is linked to muscle fibre type

“…The neuromuscular junction (NMJ) is a peripheral synapse formed between lower motor neurons and skeletal muscle fibres that has been studied in many different species including humans (Cramer and Van Essen, 1995;Campbell and Ganetzky, 2012;Grice et al, 2018;Hammond et al, 2016;Jones et al, 2017;Kuno et al, 1971;Nyström, 1968;Prakash et al, 1996;Sakowski et al, 2012). Upon action potential-mediated depolarisation of the pre-synaptic neuronal membrane, the neurotransmitter acetylcholine (ACh) is released into the NMJ synaptic cleft, where it binds to ACh receptors (AChRs) found in high density on post-synaptic muscle membranes in direct apposition to motor nerve terminals.…”