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.

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.

Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia.

Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration.

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.

Rabies Virus Hijacks and accelerates the p75NTR retrograde axonal transport machinery.

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.

Dynein interacts with the neural cell adhesion molecule (NCAM180) to tether dynamic microtubules and maintain synaptic density in cortical neurons.

Cytoplasmic dynein is well characterized as an organelle motor, but dynein also acts to tether and stabilize dynamic microtubule plus-ends in vitro. Here we identify a novel and direct interaction between dynein and the 180-kDa isoform of the neural cell adhesion molecule (NCAM). Optical trapping experiments indicate that dynein bound to beads via the NCAM180 interaction domain can tether projecting microtubule plus-ends. Live cell assays indicate that the NCAM180-dependent recruitment of dynein to the cortex leads to the selective stabilization of microtubules projecting to NCAM180 patches at the cell periphery. The dynein-NCAM180 interaction also enhances cell-cell adhesion in heterologous cell assays. Dynein and NCAM180 co-precipitate from mouse brain extract and from synaptosomal fractions, consistent with an endogenous interaction in neurons. Thus, we examined microtubule dynamics and synaptic density in primary cortical neurons. We find that depletion of NCAM, inhibition of the dynein-NCAM180 interaction, or dampening of microtubule dynamics with low dose nocodazole all result in significantly decreased in synaptic density. Based on these observations, we propose a working model for the role of dynein at the synapse, in which the anchoring of the motor to the cortex via binding to an adhesion molecule mediates the tethering of dynamic microtubule plus-ends to potentiate synaptic stabilization.

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve.

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.