Bimodal regulation of axonal transport by the GDNF-RET signalling axis in healthy and diseased motor neurons

Abstract

Deficits in axonal transport are one of the earliest pathological outcomes in several models of amyotrophic lateral sclerosis (ALS), including SOD1G93A mice. Evidence suggests that rescuing these deficits prevents disease progression, stops denervation, and extends survival. Kinase inhibitors have been previously identified as transport enhancers, and are being investigated as potential therapies for ALS. For example, inhibitors of p38 mitogen-activated protein kinase and insulin growth factor receptor 1 have been shown to rescue axonal transport deficits in vivo in symptomatic SOD1G93A mice. In this work, we investigated the impact of RET, the tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), as a modifier of axonal transport. We identified the fundamental interplay between RET signalling and axonal transport in both wild-type and SOD1G93A motor neurons in vitro. We demonstrated that blockade of RET signalling using pharmacological inhibitors and genetic knockdown enhances signalling endosome transport in wild-type motor neurons and uncovered a divergence in the response of primary motor neurons to GDNF compared with cell lines. Finally, we showed that inhibition of the GDNF-RET signalling axis rescues in vivo transport deficits in early symptomatic SOD1G93A mice, promoting RET as a potential therapeutic target in the treatment of ALS.

BDNF-dependent modulation of axonal transport is selectively impaired in ALS

“…S1C). We then tested if this response was specific for BDNF or a general feature of neurotrophic factors, by stimulating FMN axons with GDNF, which is known to activate distinct signalling cascades via RET and GFRα receptors [ 8 , 12 ]. In contrast to BDNF, application of 25 ng of recombinant GDNF did not influence transport of H C T-555-positive signalling endosomes (Additional file 2 : Fig.…”

“…This was not due to overt differences in TrkB.FL, truncated TrkB or p75 NTR receptors. Moreover, the levels of two downstream effectors of BDNF-TrkB signalling, ERK1/2 and AKT, are unchanged between WT and SOD1 G93A cultures [ 8 ]. However, an important caveat of these analyses is that these mixed cultures contain several neuronal (e.g., α- and γ-MN, as well as cholinergic glutamatergic, glycinergic and GABA-ergic interneurons) and non-neuronal (e.g., different glia and fibroblasts) subtypes.…”

“…BDNF stimulation specifically enhances axonal transport in WT FMNs, an effect not observed with GDNF. This was surprising because GDNF is important for MN survival and development [ 12 , 30 ], is added to primary MN media [ 7 – 9 ], and enhances axonal transport of signalling endosomes in primary WT ventral horn neurons [ 8 ]. However, our observation that GDNF does not modulate axonal transport of signalling endosomes in vivo in WT FMNs, supports previous findings that specific neurotrophic factors elicit discrete signalling in different MN subtypes [ 22 , 30 , 31 ].…”

“…In this regard, γ-MNs require muscle spindle-derived GDNF for postnatal survival [ 32 ]. Moreover, we have recently reported that RET inhibition rescues in vivo deficits in axonal transport of signalling endosome in SOD1 G93A mice [ 8 ]. Altogether, this evidence indicates that neurotrophic factors elicit distinct effects on axonal transport in vivo.…”

“…As a result, perturbations in axonal transport have severe consequences for neuronal homeostasis and function [4]. Indeed, we have previously demonstrated that deficits in in vivo axonal transport occur pre-symptomatically (i.e., before MN loss) across diverse ALS mice [5][6][7][8][9].…”