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.

Dissection, in vivo imaging and analysis of the mouse epitrochleoanconeus muscle.

Analysis of rodent muscles affords an opportunity to glean key insights into neuromuscular development and the detrimental impact of disease-causing genetic mutations. Muscles of the distal leg, for instance the gastrocnemius and tibialis anterior, are commonly used in such studies with mice and rats. However, thin and flat muscles, which can be dissected, processed and imaged without major disruption to muscle fibres and nerve-muscle contacts, are more suitable for accurate and detailed analyses of the peripheral motor nervous system. One such wholemount muscle is the predominantly fast twitch epitrochleoanconeus (ETA), which is located in the upper forelimb, innervated by the radial nerve, and contains relatively large and uniformly flat neuromuscular junctions (NMJs). To facilitate incorporation of the ETA into the experimental toolkit of the neuromuscular disease field, here, we describe a simple method for its rapid isolation (<5 min), supported by high-resolution videos and step-by-step images. Furthermore, we outline how the ETA can be imaged in live, anaesthetised mice, to enable examination of dynamic cellular processes occurring at the NMJ and within intramuscular axons, including transport of organelles, such as mitochondria and signalling endosomes. Finally, we present reference data on wild-type ETA fibre-type composition in young adult, male C57BL6/J mice. Comparative neuroanatomical studies of different muscles in rodent models of disease can generate critical insights into pathogenesis and pathology; dissection of the wholemount ETA provides the possibility to diversify the repertoire of muscles analysed for this endeavour.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport.

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative. This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

PTPN23 binds the dynein adaptor BICD1 and is required for endocytic sorting of neurotrophin receptors.

Signalling by target-derived neurotrophins is essential for the correct development of the nervous system and its maintenance throughout life. Several aspects concerning the lifecycle of neurotrophins and their receptors have been characterised over the years, including the formation, endocytosis and trafficking of signalling-competent ligand-receptor complexes. However, the molecular mechanisms directing the sorting of activated neurotrophin receptors are still elusive. Previously, our laboratory identified Bicaudal-D1 (BICD1), a dynein motor adaptor, as a key factor for lysosomal degradation of brain-derived neurotrophic factor (BDNF)-activated TrkB (also known as NTRK2) and p75NTR (also known as NGFR) in motor neurons. Here, using a proteomics approach, we identified protein tyrosine phosphatase, non-receptor type 23 (PTPN23), a member of the endosomal sorting complexes required for transport (ESCRT) machinery, in the BICD1 interactome. Molecular mapping revealed that PTPN23 is not a canonical BICD1 cargo; instead, PTPN23 binds the N-terminus of BICD1, which is also essential for the recruitment of cytoplasmic dynein. In line with the BICD1-knockdown phenotype, loss of PTPN23 leads to increased accumulation of BDNF-activated p75NTR and TrkB in swollen vacuole-like compartments, suggesting that neuronal PTPN23 is a novel regulator of the endocytic sorting of neurotrophin receptors.

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.

The many disguises of the signalling endosome.

Neurons are highly complex and polarised cells that must overcome a series of logistic challenges to maintain homeostasis across their morphological domains. A very clear example is the propagation of neurotrophic signalling from distal axons, where target-released neurotrophins bind to their receptors and initiate signalling, towards the cell body, where nuclear and cytosolic responses are integrated. The mechanisms of propagation of neurotrophic signalling have been extensively studied and, eventually, the model of a 'signalling endosome', transporting activated receptors and associated complexes, has emerged. Nevertheless, the exact nature of this organelle remains elusive. In this Review, we examine the evidence for the retrograde transport of neurotrophins and their receptors in endosomes, outline some of their diverse physiological and pathological roles, and discuss the main interactors, morphological features and trafficking destinations of a highly flexible endosomal signalling organelle with multiple molecular signatures.

Publisher Correction: IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A.

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a signalling network known as the unfolded protein response (UPR). Here, we identified filamin A as a major binding partner of the ER stress transducer IRE1α. Filamin A is an actin crosslinking factor involved in cytoskeleton remodelling. We show that IRE1α controls actin cytoskeleton dynamics and affects cell migration upstream of filamin A. The regulation of cytoskeleton dynamics by IRE1α is independent of its canonical role as a UPR mediator, serving instead as a scaffold that recruits and regulates filamin A. Targeting IRE1α expression in mice affected normal brain development, generating a phenotype resembling periventricular heterotopia, a disease linked to the loss of function of filamin A. IRE1α also modulated cell movement and cytoskeleton dynamics in fly and zebrafish models. This study unveils an unanticipated biological function of IRE1α in cell migration, whereby filamin A operates as an interphase between the UPR and the actin cytoskeleton.

Rab35 Functions in Axon Elongation Are Regulated by P53-Related Protein Kinase in a Mechanism That Involves Rab35 Protein Degradation and the Microtubule-Associated Protein 1B.

UNLABELLED: Rab35 is a key protein for cargo loading in the recycling endosome. In neuronal immortalized cells, Rab35 promotes neurite differentiation. Here we describe that Rab35 favors axon elongation in rat primary neurons in an activity-dependent manner. In addition, we show that the p53-related protein kinase (PRPK) negatively regulates axonal elongation by reducing Rab35 protein levels through the ubiquitin-proteasome degradation pathway. PRPK-induced Rab35 degradation is regulated by its interaction with microtubule-associated protein 1B (MAP1B), a microtubule stabilizing binding protein essential for axon elongation. Consistently, axon defects found in MAP1B knock-out neurons were reversed by Rab35 overexpression or PRPK inactivation suggesting an epistatic relationship among these proteins. These results define a novel mechanism to support axonal elongation, by which MAP1B prevents PRPK-induced Rab35 degradation. Such a mechanism allows Rab35-mediated axonal elongation and connects the regulation of actin dynamics with membrane trafficking. In addition, our study reveals for the first time that the ubiquitin-proteasome degradation pathway regulates a Rab GTPase. SIGNIFICANCE STATEMENT: Rab35 is required for axonal outgrowth. We define that its protein levels are negatively regulated by p53-related protein kinase (PRPK). We show that microtubule-associated protein 1B (MAP1B) interacts with PRPK, preventing PRPK-dependent Rab35 proteasome degradation. We demonstrate that Rab35 regulates Cdc42 activity in neurons. This is the first evidence showing that a Rab protein is regulated by degradation dependent on the ubiquitin-proteasome system.

Rab GTPase signaling in neurite outgrowth and axon specification.

Neurons are highly polarized cells that contain specialized subcellular domains involved in information transmission in the nervous system. Specifically, the somatodendritic compartment receives neuronal inputs while the axons convey information through the synapse. The establishment of asymmetric domains requires a specific delivery of components, including organelles, proteins, and membrane. The Rab family of small GTPases plays an essential role in membrane trafficking. Signaling cascades triggered by extrinsic and intrinsic factors tightly regulate Rab functions in cells, with Rab protein activation depending on GDP/GTP binding to establish a binary mode of action. This review summarizes the contributions of several Rab family members involved in trans-Golgi, early/late endosomes, and recycling endosomes during neurite development and axonal outgrowth. The regulation of some Rabs by guanine exchanging factors and GTPase activating proteins will also be addressed. Finally, discussion will be provided on how specific effector-mediated Rab activation modifies several molecules essential to neuronal differentiation. © 2016 Wiley Periodicals, Inc.

The MAP1B case: an old MAP that is new again.

The functions of microtubule-associated protein 1B (MAP1B) have historically been linked to the development of the nervous system, based on its very early expression in neurons and glial cells. Moreover, mice in which MAP1B is genetically inactivated have been used extensively to show its role in axonal elongation, neuronal migration, and axonal guidance. In the last few years, it has become apparent that MAP1B has other cellular and molecular functions that are not related to its microtubule-stabilizing properties in the embryonic and adult brain. In this review, we present a systematic review of the canonical and novel functions of MAP1B and propose that, in addition to regulating the polymerization of microtubule and actin microfilaments, MAP1B also acts as a signaling protein involved in normal physiology and pathological conditions in the nervous system.

Rab-mediated trafficking role in neurite formation.

Neuronal cells are characterized by the presence of two confined domains, which are different in their cellular properties, biochemical functions and molecular identity. The generation of asymmetric domains in neurons should logically require specialized membrane trafficking to both promote neurite outgrowth and differential distribution of components. Members of the Rab family of small GTPases are key regulators of membrane trafficking involved in transport, tethering and docking of vesicles through their effectors. RabGTPases activity is coupled to the activity of guanine nucleotide exchange factors or GEFs, and GTPase-activating proteins known as GAPs. Since the overall spatiotemporal distribution of GEFs, GAPs and Rabs governs trafficking through the secretory and endocytic pathways, affecting exocytosis, endocytosis and endosome recycling, it is likely that RabGTPases could have a major role in neurite outgrowth, elongation and polarization. In this review we summarize the evidence linking the functions of several RabGTPases to axonal and dendritic development in primary neurons, as well as neurite formation in neuronal cell lines. We focused on the role of RabGTPases from the trans-Golgi network, early/late and recycling endosomes, as well as the function of some Rab effectors in neuritogenesis. Finally, we also discuss the participation of the ADP-ribosylation factor 6, a member of the ArfGTPase family, in neurite formation since it seems to have an important cross-talk with RabGTPases.