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/β-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/β-catenin pathway is decreased, a finding possibly associated to the cytosolic aggregation of β-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/β-catenin and the BMP-dependent pathways could play relevant roles in the neurodegeneration of motor neurons in the context of ALS.
J. Neurochem. (2012) 122, 594–604. Abstract Bone morphogenetic proteins (BMPs) regulate several aspects of neuronal behavior. For instance, BMP‐2 has the ability to modulate, either positively or negatively, the outgrowth of neuronal processes in diverse cell types. In Drosophila motor neurons, the BMP type II receptor (BMPRII) homolog wishful thinking plays crucial roles on neuromuscular synaptogenesis signaling through Smad‐dependent and Smad‐independent pathways. However, a role for BMP signaling at the vertebrate neuromuscular junction has not been addressed. Herein, we have analyzed the expression of BMPRII and the effect of BMP‐2 during the morphological differentiation of motor neuron‐like NSC‐34 cells. Our data indicate that BMPRII is up‐regulated and becomes accumulated in somas and growth cones upon motor neuronal differentiation. BMP‐2 inhibits the differentiation of NSC‐34 cells, an effect that correlates with activation of a Smad‐dependent pathway, induction of the inhibitory Id1 transcription factor, and down‐regulation of the neurogenic factor Mash1. BMP‐2 also activates effectors of Smad‐independent pathways. Remarkably, BMP‐2 treatment significantly increases the expression of BMPRII. Our findings provide the first evidence to suggest a role for BMP pathways on the differentiation of motor neurons leading to successful assembly and/or regeneration of the vertebrate neuromuscular synapse.
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 β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-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, β-catenin-dependent signaling on the assembly of the vertebrate NMJ.
Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that affects motoneurons. Mutations in superoxide dismutase 1 (SOD1) have been described as a causative genetic factor for ALS. Mice overexpressing ALS-linked mutant SOD1 develop ALS symptoms accompanied by histopathological alterations and protein aggregation. The protein disulfide isomerase family member ERp57 is one of the main up-regulated proteins in tissue of ALS patients and mutant SOD1 mice, whereas point mutations in ERp57 were described as possible risk factors to develop the disease. ERp57 catalyzes disulfide bond formation and isomerization in the endoplasmic reticulum (ER), constituting a central component of protein quality control mechanisms. However, the actual contribution of ERp57 to ALS pathogenesis remained to be defined. Here, we studied the consequences of overexpressing ERp57 in experimental ALS using mutant SOD1 mice. Double transgenic SOD1G93A/ERp57WT animals presented delayed deterioration of electrophysiological activity and maintained muscle innervation compared to single transgenic SOD1G93A littermates at early-symptomatic stage, along with improved motor performance without affecting survival. The overexpression of ERp57 reduced mutant SOD1 aggregation, but only at disease end-stage, dissociating its role as an anti-aggregation factor from the protection of neuromuscular junctions. Instead, proteomic analysis revealed that the neuroprotective effects of ERp57 overexpression correlated with increased levels of synaptic and actin cytoskeleton proteins in the spinal cord. Taken together, our results suggest that ERp57 operates as a disease modifier at early stages by maintaining motoneuron connectivity.
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