Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse.

The coordinated movement of many organisms relies on efficient nerve-muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non-myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ regeneration.

ß-catenin aggregation in models of ALS motor neurons: GSK3ß inhibition effect and neuronal differentiation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. A 20% of familial ALS cases are associated with mutations in the gene coding for superoxide dismutase 1 (SOD1). The accumulation of abnormal aggregates of different proteins is a common feature in motor neurons of patients and transgenic ALS mice models, which are thought to contribute to disease pathogenesis. Developmental morphogens, such as the Wnt family, regulate numerous features of neuronal physiology in the adult brain and have been implicated in neurodegeneration. ß-catenin is a central mediator of both, Wnt signaling activity and cell-cell interactions. We previously reported that the expression of mutant SOD1 in the NSC34 motor neuron cell line decreases basal Wnt pathway activity, which correlates with cytosolic ß-catenin accumulation and impaired neuronal differentiation. In this work, we aimed a deeper characterization of ß-catenin distribution in models of ALS motor neurons. We observed extensive accumulation of ß-catenin supramolecular structures in motor neuron somas of pre-symptomatic mutant SOD1 mice. In cell-cell appositional zones of NSC34 cells expressing mutant SOD1, ß-catenin displays a reduced co-distribution with E-cadherin accompanied by an increased association with the gap junction protein Connexin-43; these findings correlate with impaired intercellular adhesion and exacerbated cell coupling. Remarkably, pharmacological inhibition of the glycogen synthase kinase-3ß (GSK3ß) in both NSC34 cell lines reverted both, ß-catenin aggregation and the adverse effects of mutant SOD1 expression on neuronal differentiation. Our findings suggest that early defects in ß-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in familial ALS.

Physical nanocomposite hydrogels filled with low concentrations of TiO2 nanoparticles: Swelling, networks parameters and cell retention studies.

Physical nanocomposite hydrogels composed of poly(2-hydroxyethylmethacrylate) and titanium oxide nanoparticles at low concentrations (<1.0 wt%) were synthesized. The effect of the nanoparticle content on the water swelling and mechanical properties of the hydrogels was investigated. Additionally, to study the influence of the polymer-nanoparticle interactions, a second type of nanocomposite was synthesized using surface functionalized nanoparticles with 3-methacryloxypropyltrimethoxysilane as the filler. The pristine nanoparticles increased the swelling capacity, especially at short time scales, and greater solvent diffusion coefficients and initial swelling rates were achieved. In contrast, the nanocomposite filled with functionalized nanoparticles exhibited a diminished swelling capacity, a constant diffusion coefficient and a significant decrease in the initial swelling rate. The mechanical properties were studied by dynamic mechanical analyses using stress-relaxation tests. Two Maxwell models in parallel agreed well with the curves of the relaxation modulus as a function of time and indicated that at short relaxation times, the nanoparticles did not cause an effect, but that at longer times, the nanoparticles decreased the relaxation time. Finally, hydrogel network parameters determined by swelling measurements and mechanical experiments indicated that the hydrogel with well distributed nanoparticles decreases the molar mass between crosslink point and the mesh size, while poorly distributed nanoparticles lead to larger mesh size. Our functional studies show that the addition of titanium oxide nanoparticles improves the ability of nanocomposite hydrogels to retain aggregates of skeletal muscle cells, revealing their potential use as suitable scaffolds for tissue repair strategies.

ALS-linked protein disulfide isomerase variants cause motor dysfunction.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.

Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells.

Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of ß-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and ß-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, ß-catenin-dependent signaling on the assembly of the vertebrate NMJ.

Wnt signaling in skeletal muscle dynamics: myogenesis, neuromuscular synapse and fibrosis.

The signaling pathways activated by Wnt ligands are related to a wide range of critical cell functions, such as cell division, migration, and synaptogenesis. Here, we summarize compelling evidence on the role of Wnt signaling on several features of skeletal muscle physiology. We briefly review the role of Wnt pathways on the formation of muscle fibers during prenatal and postnatal myogenesis, highlighting its role on the activation of stem cells of the adult muscles. We also discuss how Wnt signaling regulates the precise formation of neuromuscular synapses, by modulating the differentiation of presynaptic and postsynaptic components, particularly regarding the clustering of acetylcholine receptors on the muscle membrane. In addition, based on previous evidence showing that Wnt pathways are linked to several diseases, such as Alzheimer's and cancer, we address recent studies indicating that Wnt signaling plays a key role in skeletal muscle fibrosis, a disease characterized by an increase in the extracellular matrix components leading to failure in muscle regeneration, tissue disorganization and loss of muscle activity. In this context, we also discuss the possible cross-talk between the Wnt/ß-catenin pathway with two other critical profibrotic pathways, transforming growth factor ß and connective tissue growth factor, which are potent stimulators of the accumulation of connective tissue, an effect characteristic of the fibrotic condition. As it has emerged in other pathological conditions, we suggests that muscle fibrosis may be a consequence of alterations of Wnt signaling activity.

Bone morphogenetic protein signaling in vertebrate motor neurons and neuromuscular communication.

An accurate communication between motor neurons and skeletal muscle fibers is required for the proper assembly, growth and maintenance of neuromuscular junctions (NMJs). Several signaling and extracellular matrix molecules play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in Drosophila have revealed crucial functions for early morphogens, such as members of the Wnt and Bone Morphogenetic Proteins (BMP) signaling pathways, during the assembly and maturation of the NMJ. Here, we bring together recent findings that led us to propose that BMPs also work in vertebrate organisms as diffusible cues to communicate motor neurons and skeletal muscles.

Characterization of Wnt/ß-catenin and BMP/Smad signaling pathways in an in vitro model of amyotrophic lateral sclerosis.

Different pathways activated by morphogens of the early embryonic development, such as the Wnt and the Bone Morphogenetic Protein (BMP) ligands, are involved in diverse physiological and pathological conditions of the nervous system, including neurodegeneration. In this work, we have analyzed the endogenous activity of the canonical Wnt/ß-catenin and BMP/Smad-dependent pathways in an in vitro model of amyotrophic lateral sclerosis (ALS), given by motor neuron-like NSC34 cells stably expressing wild-type or G93A mutated forms of human Cu/Zn superoxide dismutase-1 (SOD1). As ALS-derived motor neurons, NSC34 cells expressing mutated hSOD1 show a decreased proliferation rate, are more susceptible to oxidation-induced cell death and display Golgi fragmentation. In addition, they display an impaired ability to induce the expression of the motor neuronal marker Hb9 and, consistently, to morphologically differentiate into a motor neuronal phenotype. Regarding signaling, our data show that the transcriptional activity associated to the Wnt/ß-catenin pathway is decreased, a finding possibly associated to the cytosolic aggregation of ß-catenin. In turn, the BMP-dependent phosphorylation of Smad1 and the transcriptional activation of the BMP/Smad pathway is increased in the pathologic model. Together, these findings suggest that Wnt/ß-catenin and the BMP-dependent pathways could play relevant roles in the neurodegeneration of motor neurons in the context of ALS.

The Wnt and BMP families of signaling morphogens at the vertebrate neuromuscular junction.

The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction.

Evolution of the interaction between Runx2 and VDR, two transcription factors involved in osteoblastogenesis.

BACKGROUND: The mineralized skeleton is a major evolutionary novelty that has contributed to the impressive morphological diversifications of the vertebrates. Essential to bone biology is the solidified extracellular matrix secreted by highly specialized cells, the osteoblasts. We now have a rather complete view of the events underlying osteogenesis, from a cellular, molecular, genetic, and epigenetic perspective. Because this knowledge is still largely restricted to mammals, it is difficult, if not impossible, to deduce the evolutionary history of the regulatory network involved in osteoblasts specification and differentiation. In this study, we focused on the transcriptional regulators Runx2 and VDR (the Vitamin D Receptor) that, in mammals, directly interact together and stabilize complexes of co-activators and chromatin remodellers, thereby allowing the transcriptional activation of target genes involved in extracellular matrix mineralization. Using a combination of functional, biochemical, and histological approaches, we have asked if the interaction observed between Runx2 and VDR represents a recent mammalian innovation, or if it results from more ancient changes that have occurred deep in the vertebrate lineage. RESULTS: Using immunohistochemistry and in situ hybridization in developing embryos of chick, frog and teleost fishes, we have revealed that the co-expression of Runx2 and VDR in skeletal elements has been particularly strengthened in the lineage leading to amniotes. We show that the teleost Runx2 orthologue as well as the three mammalian Runx1, Runx2 and Runx3 paralogues are able to co-immunoprecipitate with the VDR protein present in nuclear extracts of rat osteoblasts stimulated with 1alpha,25-dihydroxyvitamin D3. In addition, the teleost Runx2 can activate the transcription of the mammalian osteocalcin promoter in transfection experiments, and this response can be further enhanced by 1alpha,25-dihydroxyvitamin D3. Finally, using pull-down experiments between recombinant proteins, we show that the VDR homologue from teleosts, but not from ascidians, is able to directly interact with the mammalian Runx2 homologue. CONCLUSIONS: We propose an evolutionary scenario for the assembly of the molecular machinery involving Runx2 and VDR in vertebrates. In the last common ancestor of actinopterygians and sacropterygians, the three Runx paralogues possessed the potential to physically and functionally interact with the VDR protein. Therefore, 1alpha,25-dihydroxyvitamin D3 might have been able to modulate the transcriptional activity of Runx1, Runx2 or Runx3 in the tissues expressing VDR. After the split from amphibians, in the lineage leading to amniotes, Runx2 and VDR became robustly co-expressed in developing skeletal elements, and their regulatory interaction was incorporated in the genetic program involved in the specification and differentiation of osteoblasts.

Wnt signaling promotes AChR aggregation at the neuromuscular synapse in collaboration with agrin.

Wnt proteins regulate the formation of central synapses by stimulating synaptic assembly, but their role at the vertebrate neuromuscular junction (NMJ) is unclear. Wnt3 is expressed by lateral motoneurons of the spinal cord during the period of motoneuron-muscle innervation. Using gain- and loss-of-function studies in the chick wing, we demonstrate that Wnt signaling is necessary for the formation of acetylcholine receptor (AChR) clusters without affecting muscle growth. Similarly, diaphragms from Dishevelled-1 mutant mice with deficiency in Wnt signaling exhibit defects in cluster distribution. In cultured myotubes, Wnt3 increases the number and size of AChR clusters induced by agrin, a nerve-derived signal critical for NMJ development. Wnt3 does not signal through the canonical Wnt pathway to induce cluster formation. Instead, Wnt3 induces the rapid formation of unstable AChR micro-clusters through activation of Rac1, which aggregate into large clusters only in the presence of agrin. Our data reveal a role for Wnts in post-synaptic assembly at the vertebrate NMJ by enhancing agrin function through Rac1 activation.

Common genetic variation within the low-density lipoprotein receptor-related protein 6 and late-onset Alzheimer's disease.

Genome-wide linkage studies have defined a broad susceptibility region for late-onset Alzheimer's disease on chromosome 12, which contains the Low-Density Lipoprotein Receptor-Related Protein 6 (LRP6) gene, a coreceptor for Wnt signaling. Here, we report the association between common LRP6 variants and late-onset Alzheimer's disease in a multicenter case-control series as well as in a large family-based series ascertained by the National Institute of Mental Health-National Institute on Aging Genetics Initiative. As shown in the genome-wide linkage studies, our association depends mainly on apolipoprotein E-epsilon4 (APOE-epsilon4) carrier status. Haplotype tagging single-nucleotide polymorphisms (SNPs) with a set of seven allelic variants of LRP6 identified a putative risk haplotype, which includes a highly conserved coding sequence SNP: Ile-1062 --> Val. Functional analyses revealed that the associated allele Val-1062, an allele previously linked to low bone mass, has decreased beta-catenin signaling in HEK293T cells. Our study unveils a genetic relationship between LRP6 and APOE and supports the hypothesis that altered Wnt/beta-catenin signaling may be involved in this neurodegenerative disease.