Spinal motor neuron-enriched secreted protein R-spondin 2 induces Wnt-mediated neuromuscular junction formation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE51122 | GEO | 2016/09/11
SECONDARY ACCESSION(S): PRJNA221232
REPOSITORIES: GEO
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Project description:Spinal muscular atrophy (SMA), a recessive genetic disease, affects lower motoneurons leading to denervation, atrophy, paralysis and in severe cases death. Reduced levels of survival motor neuron (SMN) protein cause SMA. As a first step towards generating a genetic model of SMA in zebrafish, we identified three smn mutations. Two of these alleles, smnY262stop and smnL265stop, were stop mutations that resulted in exon 7 truncation, whereas the third, smnG264D, was a missense mutation corresponding to an amino acid altered in human SMA patients. Smn protein levels were low/undetectable in homozygous mutants consistent with unstable protein products. Homozygous mutants from all three alleles were smaller and survived on the basis of maternal Smn dying during the second week of larval development. Analysis of the neuromuscular system in these mutants revealed a decrease in the synaptic vesicle protein, SV2. However, two other synaptic vesicle proteins, synaptotagmin and synaptophysin were unaffected. To address whether the SV2 decrease was due specifically to Smn in motoneurons, we tested whether expressing human SMN protein exclusively in motoneurons in smn mutants could rescue the phenotype. For this, we generated a transgenic zebrafish line with human SMN driven by the motoneuron-specific zebrafish hb9 promoter and then generated smn mutant lines carrying this transgene. We found that introducing human SMN specifically into motoneurons rescued the SV2 decrease observed in smn mutants. Our analysis indicates the requirement for Smn in motoneurons to maintain SV2 in presynaptic terminals indicating that Smn, either directly or indirectly, plays a role in presynaptic integrity.
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Project description:Spinal Muscular Atrophy (SMA) is a childhood motor neuron disease caused by mutations or deletions within the SMN1 gene. At endstages of disease there is profound loss of motor neurons, loss of axons within ventral roots and defects at the neuromuscular junctions (NMJ), as evidenced by pathological features such as pre-synaptic loss and swelling and post-synaptic shrinkage. Although these motor unit defects have been widely described, the time course and interdependancy of these aspects of motor unit degeneration are unclear. Recent reports have also revealed an early upregulation of transcripts associated with the P53 signalling pathway. The relationship between the upregulation of these transcripts and pathology within the motor unit is also unclear. In this study, we exploit the prolonged disease timecourse and defined pre-symptomatic period in the Smn2B/- mouse model to perform a temporal analysis of the different elements of motor unit pathology. We demonstrate that NMJ loss occurs prior to cell body loss, and coincides with the onset of symptoms. The onset of NMJ pathology also coincides with an increase in P53-related transcripts at the cell body. Finally, using a tamoxifen inducible P53 knockout, we demonstrate that post-natal reduction in P53 levels can reduce NMJ loss, but does not affect other aspects of NMJ pathology, motor neuron loss or the phenotype of the Smn2B/- mouse model. Together this work provides a detailed temporal description of pathology within motor units of an SMA mouse model, and demonstrates that NMJ loss is a P53-dependant process. This work supports the role for P53 as an effector of synaptic and axonal degeneration in a die-back neuropathy.
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Project description:We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2-/-) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2-/- mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.
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Project description:Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found that early developmental spinal cord had higher levels of ECMproteins associated with neural development and axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin (TNC) inDNSCMwere identified as contributors tothese abilities. Furthermore,DNSCMdemonstrated superior performance as a delivery vehicle forNPCs and organoids in spinal cord injury (SCI)models. This suggests that ECMcues from early development stages might significantly contribute to the prominent regeneration ability in spinal cord.
2024-03-01 | GSE255188 | GEO
Project description:OBJECTIVE:We examined whether skeletal muscle overexpression of PGC-1α1 or PGC-1α4 affected myokine secretion and neuromuscular junction (NMJ) formation. METHODS:A microfluidic device was used to model endocrine signaling and NMJ formation between primary mouse myoblast-derived myotubes and embryonic stem cell-derived motor neurons. Differences in hydrostatic pressure allowed for fluidic isolation of either cell type or unidirectional signaling in the fluid phase. Myotubes were transduced to overexpress PGC-1α1 or PGC-1α4, and myokine secretion was quantified using a proximity extension assay. Morphological and functional changes in NMJs were measured by fluorescent microscopy and by monitoring muscle contraction upon motor neuron stimulation. RESULTS:Skeletal muscle transduction with PGC-1α1, but not PGC-1α4, increased NMJ formation and size. PGC-1α1 increased muscle secretion of neurturin, which was sufficient and necessary for the effects of muscle PGC-1α1 on NMJ formation. CONCLUSIONS:Our findings indicate that neurturin is a mediator of PGC-1α1-dependent retrograde signaling from muscle to motor neurons.
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Project description:At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ.
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Project description:Dominant mutations in GARS, encoding the essential enzyme glycyl-tRNA synthetase (GlyRS), result in a form of Charcot-Marie-Tooth disease, type 2D (CMT2D), predominantly characterized by lower motor nerve degeneration. GlyRS charges the amino acid glycine with its cognate tRNA and is therefore essential for protein translation. However, the underlying mechanisms linking toxic gain-of-function GARS mutations to lower motor neuron degeneration remain unidentified. The neuromuscular junction (NMJ) appears to be an early target for pathology in a number of peripheral nerve diseases and becomes denervated at later stages in two mouse models of CMT2D. We therefore performed a detailed longitudinal examination of NMJs in the distal lumbrical muscles and the proximal transversus abdominis (TVA) muscles of wild-type and Gars mutant mice. We determined that mutant lumbrical NMJs display a persistent defect in maturation that precedes a progressive, age-dependent degeneration. Conversely, the TVA remains relatively unaffected, with only a subtle, short-lived impairment in pre- and post-synaptic development and no reduction in lower motor neuron connectivity to muscle. Together, these observations suggest that mutant Gars is associated with compromised development of the NMJ prior to synaptic degeneration and highlight the neuromuscular synapse as an important site of early, selective pathology in CMT2D mice.
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Project description:Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. We sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins.
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Project description:Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations of the survival of motor neuron 1 (SMN1) gene. In the pathogenesis of SMA, pathological changes of the neuromuscular junction (NMJ) precede the motor neuronal loss. Therefore, it is critical to evaluate the NMJ formed by SMA patientsM-bM-^@M-^Y motor neurons (MNs), and to identify drugs that can restore the normal condition. We generated NMJ-like structures using motor neurons (MNs) derived from SMA patient-specific induced pluripotent stem cells (iPSCs), and found that the clustering of the acetylcholine receptor (AChR) is significantly impaired. Valproic acid and antisense oligonucleotide treatment ameliorated the AChR clustering defects, leading to an increase in the level of full-length SMN transcripts. Thus, the current in vitro model of AChR clustering using SMA patient-derived iPSCs is useful to dissect the pathophysiological mechanisms underlying the development of SMA, and to evaluate the efficacy of new therapeutic approaches.M-bM-^@M-^C To compare the gene expression pattern between control and patient derived iPSCs
2015-01-31 | E-GEOD-65470 | biostudies-arrayexpress
Project description:Spinal and bulbar muscular atrophy (SBMA) is an X-linked, neuromuscular neurodegenerative disease for which there is no cure. The disease is characterized by a selective decrease in fast-muscle power (e.g., tongue pressure, grip strength) accompanied by a selective loss of fast-twitch muscle fibers. However, the relationship between neuromuscular junction (NMJ) pathology and fast-twitch motor unit vulnerability has yet to be explored. In this study, we used a cross-model comparison of two mouse models of SBMA to evaluate neuromuscular junction pathology, glycolytic-to-oxidative fiber-type switching, and cytoskeletal alterations in pre- and postsynaptic termini of tibialis anterior (TA), gastrocnemius, and soleus hindlimb muscles. We observed significantly increased NMJ and myofiber pathology in fast-twitch, glycolytic motor units of the TA and gastrocnemius compared to slow-twitch, oxidative motor units of the soleus, as seen by decreased pre- and post-synaptic membrane area, decreased pre- and post-synaptic membrane colocalization, increased acetylcholine receptor compactness, a decrease in endplate area and complexity, and deficits in neurofilament heavy chain. Our data also show evidence for metabolic dysregulation and myofiber atrophy that correlate with severity of NMJ pathology. We propose a model in which the dynamic communicative relationship between the motor neuron and muscle, along with the developmental subtype of the muscle, promotes motor unit subtype specific vulnerability, metabolic alterations, and NMJ pathology.
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