The tyrosine phosphatases LAR and PTPRδ act as receptors of the nidogen-tetanus toxin complex.

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecular composition of its receptor complex remains unclear. Here, we show that the receptor-type protein tyrosine phosphatases LAR and PTPRδ interact with the nidogen-TeNT complex, enabling its neuronal uptake. Binding of LAR and PTPRδ to the toxin complex is mediated by their immunoglobulin and fibronectin III domains, which we harnessed to inhibit TeNT entry into motor neurons and protect mice from TeNT-induced paralysis. This function of LAR is independent of its role in regulating TrkB receptor activity, which augments axonal transport of TeNT. These findings reveal a multi-subunit receptor complex for TeNT and demonstrate a novel trafficking route for extracellular matrix proteins. Our study offers potential new avenues for developing therapeutics to prevent tetanus and dissecting the mechanisms controlling the targeting of physiological ligands to long-distance axonal transport in the nervous system.

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice.

Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.

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

Axonal transport ensures long-range delivery of essential cargoes between proximal and distal compartments, and is needed for neuronal development, function, and survival. Deficits in axonal transport have been detected at pre-symptomatic stages in the SOD1G93A and TDP-43M337V mouse models of amyotrophic lateral sclerosis (ALS), suggesting that impairments in this critical process are fundamental for disease pathogenesis. Strikingly, in ALS, fast motor neurons (FMNs) degenerate first whereas slow motor neurons (SMNs) are more resistant, and this is a currently unexplained phenomenon. The main aim of this investigation was to determine the effects of brain-derived neurotrophic factor (BDNF) on in vivo axonal transport in different α-motor neuron (MN) subtypes in wild-type (WT) and SOD1G93A mice. We report that despite displaying similar basal transport speeds, stimulation of wild-type MNs with BDNF enhances in vivo trafficking of signalling endosomes specifically in FMNs. This BDNF-mediated enhancement of transport was also observed in primary ventral horn neuronal cultures. However, FMNs display selective impairment of axonal transport in vivo in symptomatic SOD1G93A mice, and are refractory to BDNF stimulation, a phenotype that was also observed in primary embryonic SOD1G93A neurons. Furthermore, symptomatic SOD1G93A mice display upregulation of the classical non-pro-survival truncated TrkB and p75NTR receptors in muscles, sciatic nerves, and Schwann cells. Altogether, these data indicate that cell- and non-cell autonomous BDNF signalling is impaired in SOD1G93A MNs, thus identifying a new key deficit in ALS.

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

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.

Genetic dissection of down syndrome-associated alterations in APP/amyloid-ß biology using mouse models.

Individuals who have Down syndrome (caused by trisomy of chromosome 21), have a greatly elevated risk of early-onset Alzheimer's disease, in which amyloid-ß accumulates in the brain. Amyloid-ß is a product of the chromosome 21 gene APP (amyloid precursor protein) and the extra copy or 'dose' of APP is thought to be the cause of this early-onset Alzheimer's disease. However, other chromosome 21 genes likely modulate disease when in three-copies in people with Down syndrome. Here we show that an extra copy of chromosome 21 genes, other than APP, influences APP/Aß biology. We crossed Down syndrome mouse models with partial trisomies, to an APP transgenic model and found that extra copies of subgroups of chromosome 21 gene(s) modulate amyloid-ß aggregation and APP transgene-associated mortality, independently of changing amyloid precursor protein abundance. Thus, genes on chromosome 21, other than APP, likely modulate Alzheimer's disease in people who have Down syndrome.

IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons.

Signalling endosomes are essential for trafficking of activated ligand-receptor complexes and their distal signalling, ultimately leading to neuronal survival. Although deficits in signalling endosome transport have been linked to neurodegeneration, our understanding of the mechanisms controlling this process remains incomplete. Here, we describe a new modulator of signalling endosome trafficking, the insulin-like growth factor 1 receptor (IGF1R). We show that IGF1R inhibition increases the velocity of signalling endosomes in motor neuron axons, both in vitro and in vivo. This effect is specific, since IGF1R inhibition does not alter the axonal transport of mitochondria or lysosomes. Our results suggest that this change in trafficking is linked to the dynein adaptor bicaudal D1 (BICD1), as IGF1R inhibition results in an increase in the de novo synthesis of BICD1 in the axon of motor neurons. Finally, we found that IGF1R inhibition can improve the deficits in signalling endosome transport observed in a mouse model of amyotrophic lateral sclerosis (ALS). Taken together, these findings suggest that IGF1R inhibition may be a new therapeutic target for ALS.

The evolution of the axonal transport toolkit.

Neurons are highly polarized cells that critically depend on long-range, bidirectional transport between the cell body and synapse for their function. This continual and highly coordinated trafficking process, which takes place via the axon, has fascinated researchers since the early 20th century. Ramon y Cajal first proposed the existence of axonal trafficking of biological material after observing that dissociation of the axon from the cell body led to neuronal degeneration. Since these first indirect observations, the field has come a long way in its understanding of this fundamental process. However, these advances in our knowledge have been aided by breakthroughs in other scientific disciplines, as well as the parallel development of novel tools, techniques and model systems. In this review, we summarize the evolution of tools used to study axonal transport and discuss how their deployment has refined our understanding of this process. We also highlight innovative tools currently being developed and how their addition to the available axonal transport toolkit might help to address key outstanding questions.

Inhibiting p38 MAPK alpha rescues axonal retrograde transport defects in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the degeneration of upper and lower motor neurons. Defects in axonal transport have been observed pre-symptomatically in the SOD1G93A mouse model of ALS, and have been proposed to play a role in motor neuron degeneration as well as in other pathologies of the nervous system, such as Alzheimer's disease and hereditary neuropathies. In this study, we screen a library of small-molecule kinase inhibitors towards the identification of pharmacological enhancers of the axonal retrograde transport of signalling endosomes, which might be used to normalise the rate of this process in diseased neurons. Inhibitors of p38 mitogen-activated protein kinases (p38 MAPK) were identified in this screen and were found to correct deficits in axonal retrograde transport of signalling endosomes in cultured primary SOD1G93A motor neurons. In vitro knockdown experiments revealed that the alpha isoform of p38 MAPK (p38 MAPKα) was the sole isoform responsible for SOD1G93A-induced transport deficits. Furthermore, we found that acute treatment with p38 MAPKα inhibitors restored the physiological rate of axonal retrograde transport in vivo in early symptomatic SOD1G93A mice. Our findings demonstrate the pathogenic effect of p38 MAPKα on axonal retrograde transport and identify a potential therapeutic strategy for ALS.