Looking for answers far away from the soma-the (un)known axonal functions of TDP-43, and their contribution to early NMJ disruption in ALS.

Axon degeneration and Neuromuscular Junction (NMJ) disruption are key pathologies in the fatal neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Despite accumulating evidence that axons and NMJs are impacted at a very early stage of the disease, current knowledge about the mechanisms leading to their degeneration remains elusive. Cytoplasmic mislocalization and accumulation of the protein TDP-43 are considered key pathological hallmarks of ALS, as they occur in ~ 97% of ALS patients, both sporadic and familial. Recent studies have identified pathological accumulation of TDP-43 in intramuscular nerves of muscle biopsies collected from pre-diagnosed, early symptomatic ALS patients. These findings suggest a gain of function for TDP-43 in axons, which might facilitate early NMJ disruption. In this review, we dissect the process leading to axonal TDP-43 accumulation and phosphorylation, discuss the known and hypothesized roles TDP-43 plays in healthy axons, and review possible mechanisms that connect TDP-43 pathology to the axon and NMJ degeneration in ALS.

Multiple Copies of microRNA Binding Sites in Long 3'UTR Variants Regulate Axonal Translation.

Rapid responses to changes within subcellular compartments of highly polarized cells, such as neuron axons, depend on local translation and post-transcriptional regulation. The mechanism by which microRNAs (miRNAs) regulate this process is not fully understood. Here, using live cell imaging and RNA sequencing analysis, we demonstrated how miRNAs can differentially control hundreds of transcripts at the subcellular level. We demonstrated that the seed match length of the miRNA target-sequence regulates both mRNA stability and protein translation rates. While longer seed matches have an increased inhibitory effect, transcriptome analysis did not reveal differences in seed match length between axonal and somata mRNAs of motor neurons. However, mRNA variants with longer 3'UTR are enriched in axons and contain multiple repeats of specific miRNA target sequences. Finally, we demonstrated that the long 3'UTR mRNA variant of the motor protein Kif5b is enriched explicitly in motor neuron axons and contains multiple sequence repeats for binding miR-129-5p. This subsequently results in the differential post-transcriptional regulation of kif5b and its synthesis in axons. Thus, we suggest that the number of miRNA binding sites at the 3'UTR of the mRNA, rather than the miRNA seed match length, regulates the axonal transcriptome.

MCP-1/CCR2 axis inhibition sensitizes the brain microenvironment against melanoma brain metastasis progression.

Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.

Microfluidic Neuromuscular Co-culture System for Tracking Cell-to-Cell Transfer and Axonal Transport of Labeled Proteins.

The molecular communication mechanisms within the Motor Neurons (MN) distant axon and its soma, as well as between MN and their neighboring cells and extracellular environment are of keen interest for our understanding of neurodevelopment and neurodegenerative diseases. One tool that has significantly improved our ability to study such processes with high spatiotemporal resolution is microfluidic devices. Here we describe a step-by-step guide to the neuromuscular co-culturing procedure and demonstrate how to track trophic factors transmission from muscle-to-neuron and their transport along the axons.

Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins.

Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.

A CRMP4-dependent retrograde axon-to-soma death signal in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal non-cell-autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1G93A -ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72-mutant patients, and the SOD1G93A -ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS-affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4-dynein interaction reduces MN loss in human-derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4-dependent retrograde death signal that underlies MN loss in ALS.

Multimodal single-molecule microscopy with continuously controlled spectral resolution.

Color is a fundamental contrast mechanism in fluorescence microscopy, providing the basis for numerous imaging and spectroscopy techniques. Building on spectral imaging schemes that encode color into a fixed spatial intensity distribution, here, we introduce continuously controlled spectral-resolution (CoCoS) microscopy, which allows the spectral resolution of the system to be adjusted in real-time. By optimizing the spectral resolution for each experiment, we achieve maximal sensitivity and throughput, allowing for single-frame acquisition of multiple color channels with single-molecule sensitivity and 140-fold larger fields of view compared with previous super-resolution spectral imaging techniques. Here, we demonstrate the utility of CoCoS in three experimental formats, single-molecule spectroscopy, single-molecule Förster resonance energy transfer, and multicolor single-particle tracking in live neurons, using a range of samples and 12 distinct fluorescent markers. A simple add-on allows CoCoS to be integrated into existing fluorescence microscopes, rendering spectral imaging accessible to the wider scientific community.

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Efficient gene transfer into primary muscle cells to analyze nerve-independent postsynaptic organization in vitro.

Acetylcholine receptor (AChR) clustering on the surface of muscle cells is a hallmark of postsynaptic differentiation at the vertebrate neuromuscular junction (NMJ). Even though the assembly of complex postsynaptic apparatuses is known to rely on both, pre- and postsynaptic signals, the identity of muscle-derived proteins modulating postsynaptic assembly and maintenance is still to be fully elucidated. Efficient gene transfer into muscle cells represents a powerful tool to analyze the contribution of muscle proteins on postsynaptic assembly and maintenance. Here, we describe a protocol that combines efficient electroporation of primary muscle satellite cells with the formation of aneural complex postsynaptic structures on the surface of myotubes. In vitro formed postsynaptic structures share various similarities with in vivo postsynaptic NMJ domains. While primary myotubes express increasing amounts of the ε AChR subunit, associated with NMJ maturation, surface AChR aggregates lack this AChR subunit. Our results also validate the functional expression of a luciferase reporter gene, as well as the response of complex postsynaptic structures to pharmacological treatment. Together, these methods in primary muscle cells are a valuable tool to perform a detailed and accurate analysis of the potential role of muscle-derived proteins on the maintenance of complex postsynaptic structures and to identify nerve-derived signals regulating functional NMJ maturation.

Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting both the upper and lower motor neurons (MNs), with no effective treatment currently available. Early pathological events in ALS include perturbations in axonal transport (AT), formation of toxic protein aggregates and Neuromuscular Junction (NMJ) disruption, which all lead to axonal degeneration and motor neuron death. Pridopidine is a small molecule that has been clinically developed for Huntington disease. Here we tested the efficacy of pridopidine for ALS using in vitro and in vivo models. Pridopidine beneficially modulates AT deficits and diminishes NMJ disruption, as well as motor neuron death in SOD1G93A MNs and in neuromuscular co-cultures. Furthermore, we demonstrate that pridopidine activates the ERK pathway and mediates its beneficial effects through the sigma-1 receptor (S1R). Strikingly, in vivo evaluation of pridopidine in SOD1G93A mice reveals a profound reduction in mutant SOD1 aggregation in the spinal cord, and attenuation of NMJ disruption, as well as subsequent muscle wasting. Taken together, we demonstrate for the first time that pridopidine improves several cellular and histological hallmark pathologies of ALS through the S1R.

Localization of RNAi Machinery to Axonal Branch Points and Growth Cones Is Facilitated by Mitochondria and Is Disrupted in ALS.

Local protein synthesis in neuronal axons plays an important role in essential spatiotemporal signaling processes; however, the molecular basis for the post-transcriptional regulation controlling this process in axons is still not fully understood. Here we studied the axonal mechanisms underlying the transport and localization of microRNA (miRNA) and the RNAi machinery along the axon. We first identified miRNAs, Dicer, and Argonaute-2 (Ago2) in motor neuron (MN) axons. We then studied the localization of RNAi machinery and demonstrated that mitochondria associate with miR-124 and RNAi proteins in axons. Importantly, this co-localization occurs primarily at axonal branch points and growth cones. Moreover, using live cell imaging of a functional Cy3-tagged miR-124, we revealed that this miRNA is actively transported with acidic compartments in axons, and associates with stalled mitochondria at growth cones and axonal branch points. Finally, we observed enhanced retrograde transport of miR-124-Cy3, and a reduction in its localization to static mitochondria in MNs expressing the ALS causative gene hSOD1G93A. Taken together, our data suggest that mitochondria participate in the axonal localization and transport of RNAi machinery, and further imply that alterations in this mechanism may be associated with neurodegeneration in ALS.

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS.

Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease's etiology is not fully understood, it is thought to involve a non-cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalized in vitro cocultures, we demonstrated that myocytes expressing diverse ALS-causing mutations promote axon degeneration and NMJ dysfunction, which were inhibited by applying Neuropilin1 blocking antibody. Finally, overexpressing miR126-5p is sufficient to transiently rescue axon degeneration and NMJ disruption both in vitro and in vivo Thus, we demonstrate a novel mechanism underlying ALS pathology, in which alterations in miR126-5p facilitate a non-cell-autonomous mechanism of motor neuron degeneration in ALS.SIGNIFICANCE STATEMENT Despite some progress, currently no effective treatment is available for amyotrophic lateral sclerosis (ALS). We suggest a novel regulatory role for miR126-5p in ALS and demonstrate, for the first time, a mechanism by which alterations in miR126-5p contribute to axon degeneration and NMJ disruption observed in ALS. We show that miR126-5p is altered in ALS models and that it can modulate Sema3 and NRP protein expression. Furthermore, NRP1 elevations in motor neurons and muscle secretion of Sema3A contribute to axon degeneration and NMJ disruption in ALS. Finally, overexpressing miR126-5p is sufficient to transiently rescue NMJ disruption and axon degeneration both in vitro and in vivo.

ALS Along the Axons - Expression of Coding and Noncoding RNA Differs in Axons of ALS models.

Amyotrophic lateral sclerosis (ALS) is a multifactorial lethal motor neuron disease with no known treatment. Although the basic mechanism of its degenerative pathogenesis remains poorly understood, a subcellular spatial alteration in RNA metabolism is thought to play a key role. The nature of these RNAs remains elusive, and a comprehensive characterization of the axonal RNAs involved in maintaining neuronal health has yet to be described. Here, using cultured spinal cord (SC) neurons grown using a compartmented platform followed by next-generation sequencing (NGS) technology, we find that RNA expression differs between the somatic and axonal compartments of the neuron, for both mRNA and microRNA (miRNA). Further, the introduction of SOD1G93A and TDP43A315T, established ALS-related mutations, changed the subcellular expression and localization of RNAs within the neurons, showing a spatial specificity to either the soma or the axon. Altogether, we provide here the first combined inclusive profile of mRNA and miRNA expression in two ALS models at the subcellular level. These data provide an important resource for studies on the roles of local protein synthesis and axon degeneration in ALS and can serve as a possible target pool for ALS treatment.

Tracking Quantum-Dot labeled neurotropic factors transport along primary neuronal axons in compartmental microfluidic chambers.

Neurons are highly polarized cells, with very long axons. Neurotrophic factors like the neuronal growth factor (NGF) are secreted from neuronal targets to promote neuron survival and proper function. These neurotrophic factors must undergo retrograde axonal transport towards the cell body, wherein they initiate signaling pathways important for neurons' various functions and overall health. This process of long-distance axonal signaling is conducted by the dynein motor protein, which transmits signaling endosomes of ligand-receptor complexes retrogradely along microtubule tracks. Here we describe step by step the use of polydimethylsiloxane (PDMS) compartmentalized microfluidic chambers for tracking axonal transport of trophic factors, with a focus on labeled NGF. We describe in detail how to fabricate the molds, assemble the PDMS platform, plate neurons and image, as well as analyze NGF transport along the axon. This method is useful for studying molecular communication mechanisms within the neuron's different compartments as well as between the neuron and its diverse microenvironments, both in health and under pathological conditions.

Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance.

Molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. Disruption in the structure and function of NMJs is a hallmark of various neurodegenerative processes during both development and pathological events. Still due to the complexity of this process, it is very difficult to elucidate the cellular mechanisms underlying it, generating a keen interest for developing better tools for investigating it. Here we describe a simplified method to study mechanisms of NMJs formation, maintenance and disruption. A spinal cord explant from mice expressing the Hb9::GFP motoneuron marker is plated on one side of a compartmental chamber, and myotubes derived from muscle satellite progenitor cells are plated on the other. The GFP labeled motoneurons extend their axons via microgrooves in the chamber to innervate the muscle cells and to form functional in-vitro NMJs. Next we provide procedures to measure axon growth and to reliably quantify NMJ activity using imaging of both muscle contractions and fast intracellular calcium changes. This platform allows precise control, monitoring and manipulation of subcellular microenvironments. Specifically, it enables to distinguish local from retrograde signaling mechanisms and allows restricted experimental intervention in local compartments along the muscle-neuron route.

A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.

Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF--facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.