Axon Terminals Research Articles

In vivo imaging of axonal transport in peripheral nerves of rodent forelimbs.

Axonal transport is the essential process by which neurons actively traffic a variety of cargoes between the cell soma and axon terminals. Accordingly, dysfunctional axonal transport is linked to many nervous system conditions. Therefore, being able to image and quantify this dynamic process in live neurons of animal disease models is beneficial for understanding neuropathology and testing new therapies at the preclinical level. As such, intravital approaches have been developed to assess cargo movement in the hindlimb sciatic nerves of live, anaesthetised mice. Here, we describe an adapted method for in vivo imaging of axonal transport in intact median and ulnar nerves of the rodent forelimb. Injection of a fluorescently labelled and non-toxic fragment of tetanus neurotoxin (HCT) into the mouse forepaw permits the identification of signalling endosomes in intact axons of median and ulnar nerves. Through immunofluorescent analysis of forelimb lumbrical muscles and median/ulnar nerves, we confirmed that HCT is taken up at motor nerve terminals and predominantly locates to motor axons. We then showed that the baseline trafficking of signalling endosomes is similar between the median/ulnar nerves and the sciatic nerve in adult wild-type mice. Importantly, this adapted method can be readily tailored for assessment of additional cargoes, such as mitochondria. By measuring transport in forelimb and hindlimb nerves, comparative anatomical and functional analyses can be performed in rodent disease models to aid our understanding of peripheral nerve disease pathogenesis and response to injury.

Where is the mind within the brain? Transient selection of subnetworks by metabotropic receptors and G protein-gated ion channels

Perhaps the most important question posed by brain research is: How the brain gives rise to the mind. To answer this question, we have primarily relied on the connectionist paradigm: The brain’s entire knowledge and thinking skills are thought to be stored in the connections; and the mental operations are executed by network computations. I propose here an alternative paradigm: Our knowledge and skills are stored in metabotropic receptors (MRs) and the G protein-gated ion channels (GPGICs). Here, mental operations are assumed to be executed by the functions of MRs and GPGICs. As GPGICs have the capacity to close or open branches of dendritic trees and axon terminals, their states transiently re-route neural activity throughout the nervous system. First, MRs detect ligands that signal the need to activate GPGICs. Next, GPGICs transiently select a subnetwork within the brain. The process of selecting this new subnetwork is what constitutes a mental operation – be it in a form of directed attention, perception or making a decision. Synaptic connections and network computations play only a secondary role, supporting MRs and GPGICs. According to this new paradigm, the mind emerges within the brain as the function of MRs and GPGICs whose primary function is to continually select the pathways over which neural activity will be allowed to pass. It is argued that MRs and GPGICs solve the scaling problem of intelligence from which the connectionism paradigm suffers.

Current Advances in Gene Therapies of Genetic Auditory Neuropathy Spectrum Disorder.

Auditory neuropathy spectrum disorder (ANSD) refers to a range of hearing impairments characterized by an impaired transmission of sound from the cochlea to the brain. This defect can be due to a lesion or defect in the inner hair cell (IHC), IHC ribbon synapse (e.g., pre-synaptic release of glutamate), postsynaptic terminals of the spiral ganglion neurons, or demyelination and axonal loss within the auditory nerve. To date, the only clinical treatment options for ANSD are hearing aids and cochlear implantation. However, despite the advances in hearing-aid and cochlear-implant technologies, the quality of perceived sound still cannot match that of the normal ear. Recent advanced genetic diagnostics and clinical audiology made it possible to identify the precise site of a lesion and to characterize the specific disease mechanisms of ANSD, thus bringing renewed hope to the treatment or prevention of auditory neurodegeneration. Moreover, genetic routes involving the replacement or corrective editing of mutant sequences or defected genes to repair damaged cells for the future restoration of hearing in deaf people are showing promise. In this review, we provide an update on recent discoveries in the molecular pathophysiology of genetic lesions, auditory synaptopathy and neuropathy, and gene-therapy research towards hearing restoration in rodent models and in clinical trials.

Activity-regulated growth of motoneurons at the neuromuscular junction is mediated by NADPH oxidases.

Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo we used the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, we found a requirement for both NADPH oxidases, Nox and dual oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. Here, we show that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. Our data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, our results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.

Selective stimulation of nociceptive small fibers during intraepidermal electrical stimulation: Experiment and computational analysis.

Electrical stimulation of skin nociceptors is gaining attention in pain research and peripheral neuropathy diagnosis. However, the optimal parameters for selective stimulation are still difficult to determine because they require simultaneous characterization of the electrical response of small fibers (Aδ- and C-fibers). In this study, we measured the in vivo electrical threshold responses of small fibers to train-pulse stimulation in humans for the first time. We also examined selective stimulation via a computational model, which combines electrical analysis, and terminal fiber and synaptic models, including the first cutaneous pain C-fiber model. Selective stimulation of small fibers is performed by injecting train-pulse stimulation via coaxial electrodes with an intraepidermal needle tip at varying pulse counts and frequencies. The activation Aδ- or C-fibers was discriminated from the differences in reaction time. Aδ-fiber elicited a pinpricking sensation with a mean reaction time of 0.522 s, and C-fiber elicited a tingling sensation or slight burning itch with a mean reaction time of 1.243 s. The implemented multiscale electrical model investigates synaptic effects while considering stimulation waveform characteristics. Experimental results showed that perception thresholds decreased with the number of consecutive pulses and frequency up to convergence (five pulses or 70 Hz) during the selective stimulation of Aδ- and C-fibers. Considering the synaptic properties, the optimal stimulus conditions for selective stimulation of Aδ- vs. C-fibers were train of at least four pulses and a frequency of 40-70 Hz at a pulse width of 1 ms. The experimental results were modeled with high fidelity by incorporating temporal synaptic effects into the computational model. Numerical analysis revealed terminal axon thickness to be the most important biophysical factor affecting threshold variability. The computational model can be used to estimate perception thresholds while understanding the mechanisms underlying the selective stimulation of small fibers. The parameters derived here are important in exploring selective stimulation between Aδ- and C-fibers for diagnosing neuropathies.

Underlying mechanisms of long-term potentiation during the inhibition of the cannabinoid CB1 and GABAB receptors in the dentate gyrus of hippocampus

BackgroundThe release of various neurotransmitters and thereby the excitability of neuronal circuits are regulated by the endocannabinoid system in an activity-dependent manner. Hippocampal long-term potentiation (LTP) is augmented in cannabinoid type 1 (CB1) receptor-deficient mice. CB1 receptors exist on GABAergic axon terminals in the hippocampus. In our previous work, we showed that CB1 antagonists increased the population spike (PS) amplitude, field excitatory post-synaptic potential (fEPSP), and the LTP induction in the dentate gyrus (DG) of the rat hippocampus while the GABAB antagonist decreased these parameters. Determining the underlying mechanisms of the pre- and/or postsynaptic locus of LTP expression is of great importance. In this study, we investigated whether LTP alteration acutely caused by CB1 and GABAB receptor antagonists (AM251 and CGP55845, respectively) happens at the postsynaptic or presynaptic regions, or at both. Therefore, the paired-pulse ratio (PPR) was assessed prior to and following the LTP induction in the studied groups.MethodsMale Wistar rats were randomly assigned to the groups of control, AM251, CGP55845, CGP55845 + AM251. A high-frequency stimulation (HFS) of the perforant path (PP) was used to induce LTP in the DG region.ResultsStatistical analysis revealed that AM251 produced significant increase in excitatory postsynaptic potential (EPSP) slope and amplitude of PS. Conversely, administration of CGP55845 produced decrease in slope of EPSP. The current results indicated that the PPR was not influenced by LTP induction in the presence of AM251 or CGP55845 either alone or their combination.ConclusionsIt can be concluded that the site causing LTP expression is, at least in part, the postsynaptic site because PPR was not influenced by LTP induction in the presence of AM251 or CGP55845 either alone or their combination.

Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy.

Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 "cluster-like" formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.

Cyclic AMP response element-binding protein (CREB) transcription factor in astrocytic synaptic communication.

Astrocytes are known to actively participate in synaptic communication by forming structures called tripartite synapses. These synapses consist of presynaptic axon terminals, postsynaptic dendritic spines, and astrocytic processes where astrocytes release and receive transmitters. Although the transcription factor cyclic AMP response element (CRE)-binding protein (CREB) has been actively studied as an important factor for mediating synaptic activity-induced responses in neurons, its role in astrocytes is relatively unknown. Synaptic signals are known to activate various downstream pathways in astrocytes, which can activate the CREB transcription factor. Therefore, there is a need to summarize studies on astrocytic intracellular pathways that are induced by synaptic communication resulting in activation of the CREB pathway. In this review, we discuss the various neurotransmitter receptors and intracellular pathways that can induce CREB activation and CREB-induced gene regulation in astrocytes.

A genetically encoded sensor measures temporal oxytocin release from different neuronal compartments.

Oxytocin (OT), a peptide hormone and neuromodulator, is involved in diverse physiological and pathophysiological processes in the central nervous system and the periphery. However, the regulation and functional sequences of spatial OT release in the brain remain poorly understood. We describe a genetically encoded G-protein-coupled receptor activation-based (GRAB) OT sensor called GRABOT1.0. In contrast to previous methods, GRABOT1.0 enables imaging of OT release ex vivo and in vivo with suitable sensitivity, specificity and spatiotemporal resolution. Using this sensor, we visualize stimulation-induced OT release from specific neuronal compartments in mouse brain slices and discover that N-type calcium channels predominantly mediate axonal OT release, whereas L-type calcium channels mediate somatodendritic OT release. We identify differences in the fusion machinery of OT release for axon terminals versus somata and dendrites. Finally, we measure OT dynamics in various brain regions in mice during male courtship behavior. Thus, GRABOT1.0 provides insights into the role of compartmental OT release in physiological and behavioral functions.

Impact of Retinal Degeneration on Response of ON and OFF Cone Bipolar Cells to Electrical Stimulation.

In retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), the photoreceptors become stressed and start to degenerate in the early stages of the disease. Retinal prosthetic devices have been developed to restore vision in patients by applying electrical stimulation to the surviving retinal cells. However, these devices provide limited visual perception as the therapeutic interventions are generally considered in the later stages of the disease when only inner retinal layer cells are left. A potential treatment option for retinal degenerative diseases in the early stages can be stimulating bipolar cells, which receive presynaptic signals from photoreceptors. In this work, we constructed computational models of healthy and degenerated (both ON and OFF-type) cone bipolar cells (CBCs) with realistic morphologies extracted from connectomes of the healthy and early-stage degenerated rabbit retina. We examined these cells' membrane potential and axon terminal calcium current differences when subjected to electrical stimulation. In addition, we investigated how differently healthy and degenerated cells behave with respect to various stimulation parameters, including pulse duration and cells' distance from the stimulating electrode. The results suggested that regardless of the position of the OFF CBCs in the retina model, there is not a significant difference between the membrane potential of healthy and degenerate cells when electrically stimulated. However, the healthy ON CBC axon terminal membrane potential rising time-constant is shorter (0.29 ± 0.03 ms) than the degenerated cells (0.8 ± 0.07 ms). Moreover, the ionic calcium channels at the axon terminals of the cells have a higher concentration and higher current in degenerated cells (32.24 ± 6.12 pA) than the healthy cells (13.64 ± 2.88 pA) independently of the cell's position.

Characterisation of functional deficits induced by AAV overexpression of alpha-synuclein in rats.

In the last decades different preclinical animal models of Parkinson's disease (PD) have been generated, aiming to mimic the progressive neuronal loss of midbrain dopaminergic (DA) cells as well as motor and non-motor impairment. Among all the available models, AAV-based models of human alpha-synuclein (h-aSYN) overexpression are promising tools for investigation of disease progression and therapeutic interventions. The goal with this work was to characterise the impairment in motor and non-motor domains following nigrostriatal overexpression of h-aSYN and correlate the behavioural deficits with histological assessment of associated pathology. Intranigral injection of an AAV9 expressing h-aSYN was compared with untreated animals, 6-OHDA and AAV9 expressing either no transgene or GFP. The animals were assessed on a series of simple and complex behavioural tasks probing motor and non-motor domains. Post-mortem neuropathology was analysed using immunohistochemical methods. Overexpression of h-aSYN led to progressive degeneration of DA neurons of the SN and axonal terminals in the striatum (STR). We observed extensive nigral and striatal pathology, resembling that of human PD brain, as well as the development of stable progressive deficit in simple motor tasks and in non-motor domains such as deficits in motivation and lateralised neglect. In the present work we characterized a rat model of PD that closely resembles human PD pathology at the histological and behavioural level. The correlation of cell loss with behavioural performance enables the selection of rats which can be used in neuroprotective or neurorestorative therapies.

Viral Tracing Confirms Paranigral Ventral Tegmental Area Dopaminergic Inputs to the Interpeduncular Nucleus Where Dopamine Release Encodes Motivated Exploration.

Midbrain dopaminergic (DAergic) neurons of the ventral tegmental area (VTA) are engaged by rewarding stimuli and encode reward prediction error to update goal-directed learning. However, recent data indicate that VTA DAergic neurons are functionally heterogeneous with emerging roles in aversive signaling, salience, and novelty, based in part on anatomic location and projection, highlighting a need to functionally characterize the repertoire of VTA DAergic efferents in motivated behavior. Previous work identifying a mesointerpeduncular circuit consisting of VTA DAergic neurons projecting to the interpeduncular nucleus (IPN), a midbrain area implicated in aversion, anxiety-like behavior, and familiarity, has recently come into question. To verify the existence of this circuit, we combined presynaptic targeted and retrograde viral tracing in the dopamine transporter-Cre mouse line. Consistent with previous reports, synaptic tracing revealed that axon terminals from the VTA innervate the caudal IPN; whereas, retrograde tracing revealed DAergic VTA neurons, predominantly in the paranigral region, project to the nucleus accumbens shell, as well as the IPN. To test whether functional DAergic neurotransmission exists in the IPN, we expressed the genetically encoded DA sensor, dLight 1.2, in the IPN of C57BL/6J mice and measured IPN DA signals in vivo during social and anxiety-like behavior using fiber photometry. We observed an increase in IPN DA signal during social investigation of a novel but not familiar conspecific and during exploration of the anxiogenic open arms of the elevated plus maze. Together, these data confirm VTA DAergic neuron projections to the IPN and implicate this circuit in encoding motivated exploration.

Direction selectivity of the retinotectal system of fish: Findings based on microelectrode extracellular recordings of the tectum opticum

Vision in fish plays an important role in different forms of visually guided behavior. The visual system of fish is available for research by different methods; it is a convenient experimental model for studying and understanding the mechanisms of vision in general. Responses of retinal direction-selective (DS) ganglion cells (GCs) are recorded extracellularly from their axon terminals in the superficial layers of the tectum opticum (TO). They can be divided into three distinct groups according to the preferred directions of stimulus movement: caudorostral, dorsoventral and ventrodorsal. Each of these groups comprises both ON and OFF units in equal proportions. Relatively small receptive fields (3-8?) and fine spatial resolution characterize retinal DS units as local motion detectors. Conversely, the responses of direction-selective tectal neurons (DS TNs) are recorded at two different tectal levels, deeper than the zone of retinal DS afferents. They are characterized by large receptive fields (up to 60?) and are indifferent to any sign of contrast, i.e., they can be considered as ON-OFF-type units. Four types of ON-OFF DS TNs preferring different directions of motion have been recorded. The preferred directions of three types of DS TNs match the preferred directions of three types of DS GCs. Matching the three preferred directions of ON and OFF DS GCs and ON-OFF DS TNs has allowed us to hypothesize that the GCs with caudorostral, ventrodorsal and dorsoventral preferences are input neurons for the corresponding types of DS TNs. On the other hand, the rostrocaudal preference in the fourth type of DS TNs, recorded exclusively in the deep tectal zone, is an emergent property of the TO. In this review, our findings are compared with the results of other authors examining direction selectivity in the fish retinotectal system.

Potential physiological and pathological roles for axonal ryanodine receptors.

Clinical disability following trauma or disease to the spinal cord often involves the loss of vital white matter elements including axons and glia. Although excessive Ca2+ is an established driver of axonal degeneration, therapeutically targeting externally sourced Ca2+ to date has had limited success in both basic and clinical studies. Contributing factors that may underlie this limited success include the complexity of the many potential sources of Ca2+ entry and the discovery that axons also contain substantial amounts of stored Ca2+ that if inappropriately released could contribute to axonal demise. Axonal Ca2+ storage is largely accomplished by the axoplasmic reticulum that is part of a continuous network of the endoplasmic reticulum that provides a major sink and source of intracellular Ca2+ from the tips of dendrites to axonal terminals. This "neuron-within-a-neuron" is positioned to rapidly respond to diverse external and internal stimuli by amplifying cytosolic Ca2+ levels and generating short and long distance regenerative Ca2+ waves through Ca2+ induced Ca2+ release. This review provides a glimpse into the molecular machinery that has been implicated in regulating ryanodine receptor mediated Ca2+ release in axons and how dysregulation and/or overstimulation of these internodal axonal signaling nanocomplexes may directly contribute to Ca2+-dependent axonal demise. Neuronal ryanodine receptors expressed in dendrites, soma, and axonal terminals have been implicated in synaptic transmission and synaptic plasticity, but a physiological role for internodal localized ryanodine receptors remains largely obscure. Plausible physiological roles for internodal ryanodine receptors and such an elaborate internodal binary membrane signaling network in axons will also be discussed.

Reconstitution of Membrane-tethered Postsynaptic Density Condensates Using Supported Lipid Bilayer.

Eukaryotic cells utilize sub-cellular compartmentalization to restrict reaction components within a defined localization to perform specified biological functions. One way to achieve this is via membrane enclosure; however, many compartments are not bounded with lipid membrane bilayers. In the past few years, it has been increasingly recognized that molecular components in non- or semi-membrane-bound compartments might form biological condensates autonomously (i.e., without requirement of energy input) once threshold concentrations are reached, via a physical chemistry process known as liquid-liquid phase separation. Molecular components within these compartments are stably maintained at high concentrations and separated from the surrounding diluted solution without the need for a physical barrier. Biochemical reconstitution using recombinantly purified proteins has served as an important tool for understanding organizational principles behind these biological condensates. Common techniques include turbidity measurement, fluorescence imaging of 3D droplets, and atomic force microscopy measurements of condensate droplets. Nevertheless, many molecular compartments are semi-membrane-bound with one side attached to the plasma membrane and the other side exposed to the cytoplasm and/or attached to the cytoskeleton; therefore, reconstitution in 3D solution cannot fully recapture their physiological configuration. Here, we utilize a postsynaptic density minimal system to demonstrate that biochemical reconstitution can be applied on supported lipid bilayer (SLB); we have also incorporated actin cytoskeleton into the reconstitution system to mimic the molecular organization in postsynaptic termini. The same system could be adapted to study other membrane-proximal, cytoskeleton-supported condensations.

Assessing Rewiring of the Retinal Circuitry by Electroretinogram (ERG) After Inner Retinal Lesion in Adult Zebrafish.

Adult zebrafish respond to retinal injury with a regenerative response that replaces damaged neurons with Müller glia-derived regenerated neurons. The regenerated neurons are functional, appear to make appropriate synaptic connections, and support visually mediated reflexes and more complex behaviors. Curiously, the electrophysiology of damaged, regenerating, and regenerated zebrafish retina has only recently been examined. In our previous work, we demonstrated that electroretinogram (ERG) recordings of damaged zebrafish retina correlate with the extent of the inflicted damage and that the regenerated retina at 80days post-injury exhibited ERG waveforms consistent with functional visual processing. In this paper we describe the procedure for obtaining and analyzing ERG recordings from adult zebrafish previously subjected to widespread lesions that destroy inner retinal neurons and engage a regenerative response that restores retinal function, in particular the synaptic connections between photoreceptor axon terminals and the dendritic trees of retinal bipolar neurons.

Electrophysiology of Dendritic Spines: Information Processing, Dynamic Compartmentalization, and Synaptic Plasticity.

For many years, synaptic transmission was considered as information transfer betweenpresynaptic neuron and postsynaptic cell. At the synaptic level, it was thought that dendritic arbors were only receiving and integrating all information flow sent along to the soma, while axons were primarily responsible for point-to-point information transfer. However, it is important to highlight that dendritic spines play a crucial role as postsynaptic components in central nervous system (CNS) synapses, not only integrating and filtering signals to the soma but also facilitating diverse connections with axons from many different sources. The majority of excitatory connections from presynaptic axonal terminals occurs on postsynaptic spines, although a subset of GABAergic synapses also targets spine heads. Several studies have shown the vast heterogeneous morphological, biochemical, and functional features of dendritic spines related to synaptic processing. In this chapter (adding to the relevant data on the biophysics of spines described in Chap. 1 of this book), we address the up-to-date functional dendritic characteristics assessed through electrophysiological approaches, including backpropagating action potentials (bAPs) and synaptic potentials mediated in dendritic and spine compartmentalization, as well as describing the temporal and spatial dynamics of glutamate receptors in the spines related to synaptic plasticity.

EFFECT OF GLYCOLYSIS SUPRESSION ON ULTRASTRUCTURE OF THE HIPPOCAMPAL GIANT SYNAPSES

Suppression of glycolysis in the rat hippocampal formations was induced by daily intracerebroventricular injection of 2-deoxy-D-glucose for 10 days. Using electron microscopy, a disturbance of morphological interactions of axonal terminals of granular neurons with perisynaptic astrocytic processes was found. At the same time, both the determinant ultrastructural characteristics of giant synapses and two types of functional contacts (asymmetric chemical active zones with dendritic spines and symmetric adhesive junctions with dendrites) were retained. However, they showed morphological signs of a weakening of synaptic processes, which were expressed in a decrease in the number of intraterminal active zones, clustering of synaptic vesicles, and their removal from neurotransmitter release sites. Compared to the norm, an increase in the number of small mitochondria with an electron-dense matrix and the evidence of initiation of glycogen synthesis in the form of glycosomes were found in the giant terminals. The data obtained indicate that the giant synapses of the hippocampal mossy fibers are a plastic, self-correcting system that is able to function by adapting its own bioenergetic metabolism, when brain glycolysis is suppressed.

MRNA transport, translation, and decay in adult mammalian central nervous system axons

Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knockout mice validate FMRP's role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the adult axonal transcriptome is stably maintained by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function invivo.

Hydrogen peroxide induced by nerve injury promotes axon regeneration via connective tissue growth factor

Regeneration of the neuromuscular junction (NMJ) leverages on extensive exchange of factors released from motor axon terminals (MATs), muscle fibers and perisynaptic Schwann cells (PSCs), among which hydrogen peroxide (H2O2) is a major pro-regenerative signal. To identify critical determinants of NMJ remodeling in response to injury, we performed temporal transcriptional profiling of NMJs from 2 month-old mice during MAT degeneration/regeneration, and cross-referenced the differentially expressed genes with those elicited by H2O2 in SCs. We identified an enrichment in extracellular matrix (ECM) transcripts, including Connective Tissue Growth Factor (Ctgf), which is usually expressed during development. We discovered that Ctgf levels are increased in a Yes-associated protein (YAP)-dependent fashion in response to rapid, local H2O2 signaling generated by stressed mitochondria in the injured sciatic nerve, a finding highlighting the importance of signals triggered by mechanical force to motor nerve repair. Through sequestration of Ctgf or inactivation of H2O2, we delayed the recovery of neuromuscular function by impairing SC migration and, in turn, axon-oriented re-growth. These data indicate that H2O2 and its downstream effector Ctgf are pro-regenerative factors that enable axonal growth, and reveal a striking ECM remodeling process during nerve regeneration upon local H2O2 signaling. Our study identifies key transcriptomic changes at the regenerating NMJ, providing a rich source of pro-regenerative factors with potential for alleviating the consequences of peripheral nerve injuries.