Building, Breaking, and Repairing Neuromuscular Synapses.

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.

Astragaloside IV protects against autoimmune myasthenia gravis in rats via regulation of mitophagy and apoptosis.

Astragaloside IV (AS­IV) has various pharmacological effects, including antioxidant and immunoregulatory properties, which can improve myasthenia gravis (MG) symptoms. However, the potential mechanism underlying the effects of AS­IV on MG remains to be elucidated. The present study aimed to investigate whether AS­IV has a therapeutic effect on MG and its potential mechanism of action. By subcutaneously immunizing rats with R97­116 peptide, an experimental autoimmune (EA) MG rat model was established. AS­IV (40 or 80 mg/kg/day) treatment was then applied for 28 days after modeling. The results demonstrated that AS­IV significantly ameliorated the weight loss, Lennon score and pathological changes in the gastrocnemius muscle of EAMG rats compared with the model group. Additionally, the levels of acetylcholine receptor antibody (AChR­Ab) were significantly decreased, whereas mitochondrial function [ATPase and cytochrome c (Cyt­C) oxidase activities] and ultrastructure were improved in the AS­IV treated rats. Moreover, the mRNA and protein expression levels of phosphatase and tensin homolog­induced putative kinase 1, Parkin, LC3II and Bcl­2, key signaling molecules for mitophagy and apoptosis, were upregulated, whereas the mRNA and protein expression levels of p62, Cyt­C, Bax, caspase 3 and caspase 9 were downregulated following AS­IV intervention. In conclusion, AS­IV may protect against EAMG in a rat model by modulating mitophagy and apoptosis. These findings indicated the potential mechanism underlying the effects of AS­IV on MG and provided novel insights into treatment strategies for MG.

CaMKIIß deregulation contributes to neuromuscular junction destabilization in Myotonic Dystrophy type I.

BACKGROUND: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. METHODS: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. RESULTS: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIß/ßM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. CONCLUSIONS: Our results indicate that CaMKIIß-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

Mapping seasonal migration in a songbird hybrid zone -- heritability, genetic correlations, and genomic patterns linked to speciation.

Seasonal migration is a widespread behavior relevant for adaptation and speciation, yet knowledge of its genetic basis is limited. We leveraged advances in tracking and sequencing technologies to bridge this gap in a well-characterized hybrid zone between songbirds that differ in migratory behavior. Migration requires the coordinated action of many traits, including orientation, timing, and wing morphology. We used genetic mapping to show these traits are highly heritable and genetically correlated, explaining how migration has evolved so rapidly in the past and suggesting future responses to climate change may be possible. Many of these traits mapped to the same genomic regions and small structural variants indicating the same, or tightly linked, genes underlie them. Analyses integrating transcriptomic data indicate cholinergic receptors could control multiple traits. Furthermore, analyses integrating genomic differentiation further suggested genes underlying migratory traits help maintain reproductive isolation in this hybrid zone.

Effects of DDT and DDE on placental cholinergic receptors.

The placental cholinergic system; known as an important factor in intracellular metabolic activities, regulation of placental vascular tone, placental development, and neurotransmission; can be affected by persistent organic pesticides, particularly organochlorine pesticides(OCPs), which can influence various epigenetic regulations and molecular pathways. Although OCPs are legally prohibited, trace amounts of the persistent dichlorodiphenyltrichloroethane(DDT) are still found in the environment, making prenatal exposure inevitable. In this study, the effects of 2,4'-DDT and 4,4'-DDT; and its breakdown product 4,4'-DDE in the environment on placental cholinergic system were evaluated with regards to cholinergic genes. 40 human placentas were screened, where 42,50% (17 samples) were found to be positive for the tested compounds. Average concentrations were 10.44 µg/kg; 15.07 µg/kg and 189,42 µg/kg for 4,4'-DDE; 2,4'-DDT and 4,4'-DDT respectively. RNA-Seq results revealed 2396 differentially expressed genes in positive samples; while an increase in CHRM1,CHRNA1,CHRNG and CHRNA2 genes at 1.28, 1.49, 1.59 and 0.4 fold change were found(p<0028). The increase for CHRM1 was also confirmed in tissue samples with immunohistochemistry. In vitro assays using HTR8/SVneo cells; revealed an increase in mRNA expression of CHRM1, CHRM3 and CHRN1 in DDT and DDE treated groups; which was also confirmed through western blot assays. An increase in the expression of CHRM1,CHRNA1, CHRNG(p<0001) and CHRNA2(p<0,05) were found from the OCPs exposed and non exposed groups.The present study reveals that intrauterine exposure to DDT affects the placental cholinergic system mainly through increased expression of muscarinic receptors. This increase in receptor expression is expected to enhance the sensitivity of the placental cholinergic system to acetylcholine.

Multiple cholinergic receptor subtypes coordinate dual modulation of acetylcholine on anterior and posterior paraventricular thalamic neurons.

Paraventricular thalamus (PVT) plays important roles in the regulation of emotion and motivation through connecting many brain structures including the midbrain and the limbic system. Although acetylcholine (ACh) neurons of the midbrain were reported to send projections to PVT, little is known about how cholinergic signaling regulates PVT neurons. Here, we used both RNAscope and slice patch-clamp recordings to characterize cholinergic receptor expression and ACh modulation of PVT neurons in mice. We found ACh excited a majority of anterior PVT (aPVT) neurons but predominantly inhibited posterior PVT (pPVT) neurons. Compared to pPVT with more inhibitory M2 receptors, aPVT expressed higher levels of all excitatory receptor subtypes including nicotinic α4, α7, and muscarinic M1 and M3. The ACh-induced excitation was mimicked by nicotine and antagonized by selective blockers for α4ß2 and α7 nicotinic ACh receptor (nAChR) subtypes as well as selective antagonists for M1 and M3 muscarinic ACh receptors (mAChR). The ACh-induced inhibition was attenuated by selective M2 and M4 mAChR receptor antagonists. Furthermore, we found ACh increased the frequency of excitatory postsynaptic currents (EPSCs) on a majority of aPVT neurons but decreased EPSC frequency on a larger number of pPVT neurons. In addition, ACh caused an acute increase followed by a lasting reduction in inhibitory postsynaptic currents (IPSCs) on PVT neurons of both subregions. Together, these data suggest that multiple AChR subtypes coordinate a differential modulation of ACh on aPVT and pPVT neurons.

Vagus nerve stimulation modulates distinct acetylcholine receptors on B cells and limits the germinal center response.

Acetylcholine is produced in the spleen in response to vagus nerve activation; however, the effects on antibody production have been largely unexplored. Here, we use a chronic vagus nerve stimulation (VNS) mouse model to study the effect of VNS on T-dependent B cell responses. We observed lower titers of high-affinity IgG and fewer antigen-specific germinal center (GC) B cells. GC B cells from chronic VNS mice exhibited altered mRNA and protein expression suggesting increased apoptosis and impaired plasma cell differentiation. Follicular dendritic cell (FDC) cluster dispersal and altered gene expression suggested poor function. The absence of acetylcholine-producing CD4+ T cells diminished these alterations. In vitro studies revealed that α7 and α9 nicotinic acetylcholine receptors (nAChRs) directly regulated B cell production of TNF, a cytokine crucial to FDC clustering. α4 nAChR inhibited coligation of CD19 to the B cell receptor, presumably decreasing B cell survival. Thus, VNS-induced GC impairment can be attributed to distinct effects of nAChRs on B cells.

Gut microbiota-derived butyrate restores impaired regulatory T cells in patients with AChR myasthenia gravis via mTOR-mediated autophagy.

More than 80% of patients with myasthenia gravis (MG) are positive for anti-acetylcholine receptor (AChR) antibodies. Regulatory T cells (Tregs) suppress overproduction of these antibodies, and patients with AChR antibody-positive MG (AChR MG) exhibit impaired Treg function and reduced Treg numbers. The gut microbiota and their metabolites play a crucial role in maintaining Treg differentiation and function. However, whether impaired Tregs correlate with gut microbiota activity in patients with AChR MG remains unknown. Here, we demonstrate that butyric acid-producing gut bacteria and serum butyric acid level are reduced in patients with AChR MG. Butyrate supplementation effectively enhanced Treg differentiation and their suppressive function of AChR MG. Mechanistically, butyrate activates autophagy of Treg cells by inhibiting the mammalian target of rapamycin. Activation of autophagy increased oxidative phosphorylation and surface expression of cytotoxic T-lymphocyte-associated protein 4 on Treg cells, thereby promoting Treg differentiation and their suppressive function in AChR MG. This observed effect of butyrate was blocked using chloroquine, an autophagy inhibitor, suggesting the vital role of butyrate-activated autophagy in Tregs of patients with AChR MG. We propose that gut bacteria derived butyrate has potential therapeutic efficacy against AChR MG by restoring impaired Tregs.

Postsynaptic receptors regulate presynaptic transmitter stability through transsynaptic bridges.

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABAA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABAA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

Rare disease research workflow using multilayer networks elucidates the molecular determinants of severity in Congenital Myasthenic Syndromes.

Exploring the molecular basis of disease severity in rare disease scenarios is a challenging task provided the limitations on data availability. Causative genes have been described for Congenital Myasthenic Syndromes (CMS), a group of diverse minority neuromuscular junction (NMJ) disorders; yet a molecular explanation for the phenotypic severity differences remains unclear. Here, we present a workflow to explore the functional relationships between CMS causal genes and altered genes from each patient, based on multilayer network community detection analysis of complementary biomedical information provided by relevant data sources, namely protein-protein interactions, pathways and metabolomics. Our results show that CMS severity can be ascribed to the personalized impairment of extracellular matrix components and postsynaptic modulators of acetylcholine receptor (AChR) clustering. This work showcases how coupling multilayer network analysis with personalized -omics information provides molecular explanations to the varying severity of rare diseases; paving the way for sorting out similar cases in other rare diseases.

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor.

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit ß1-ß2 and M2-M3 loops, with agonist binding re-positioning a key ß1-ß2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9' hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.

Calcitriol ameliorates motor deficits and prolongs survival of Chrne-deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2.

Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 â€‹kbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.

Myasthenia gravis: the changing treatment landscape in the era of molecular therapies.

Myasthenia gravis (MG) is an autoimmune disorder that affects the neuromuscular junction, leading to muscle weakness and fatigue. MG is caused by antibodies against the acetylcholine receptor (AChR), the muscle-specific kinase (MuSK) or other AChR-related proteins that are expressed in the postsynaptic muscle membrane. The standard therapeutic approach for MG has relied on acetylcholinesterase inhibitors, corticosteroids and immunosuppressants, which have shown good efficacy in improving MG-related symptoms in most people with the disease; however, these therapies can carry a considerable burden of long-term adverse effects. Moreover, up to 15% of individuals with MG exhibit limited or no response to these standard therapies. The emergence of molecular therapies, including monoclonal antibodies, B cell-depleting agents and chimeric antigen receptor T cell-based therapies, has the potential to revolutionize the MG treatment landscape. This Review provides a comprehensive overview of the progress achieved in molecular therapies for MG associated with AChR antibodies and MuSK antibodies, elucidating both the challenges and the opportunities these therapies present to the field. The latest developments in MG treatment are described, exploring the potential for personalized medicine approaches.

The YAP1/TAZ-TEAD transcriptional network regulates gene expression at neuromuscular junctions in skeletal muscle fibers.

We examined YAP1/TAZ-TEAD signaling pathway activity at neuromuscular junctions (NMJs) of skeletal muscle fibers in adult mice. Our investigations revealed that muscle-specific knockouts of Yap1 or Taz, or both, demonstrate that these transcriptional coactivators regulate synaptic gene expression, the number and morphology of NMJs, and synaptic nuclei. Yap1 or Taz single knockout mice display reduced grip strength, fragmentation of NMJs, and accumulation of synaptic nuclei. Yap1/Taz muscle-specific double knockout mice do not survive beyond birth and possess almost no NMJs, the few detectable show severely impaired morphology and are organized in widened endplate bands; and with motor nerve endings being mostly absent. Myogenic gene expression is significantly impaired in the denervated muscles of knockout mice. We found that Tead1 and Tead4 transcription rates were increased upon incubation of control primary myotubes with AGRN-conditioned medium. Reduced AGRN-dependent acetylcholine receptor clustering and synaptic gene transcription were observed in differentiated primary Tead1 and Tead4 knockout myotubes. In silico analysis of previously reported genomic occupancy sites of TEAD1/4 revealed evolutionary conserved regions of potential TEAD binding motifs in key synaptic genes, the relevance of which was functionally confirmed by reporter assays. Collectively, our data suggest a role for YAP1/TAZ-TEAD1/TEAD4 signaling, particularly through TAZ-TEAD4, in regulating synaptic gene expression and acetylcholine receptor clustering at NMJs.

Cholinergic sensing of allergen exposure by airway epithelium promotes type 2 immunity in the lungs.

BACKGROUND: Nonneuronal cells, including epithelial cells, can produce acetylcholine (ACh). Muscarinic ACh receptor antagonists are used clinically to treat asthma and other medical conditions; however, knowledge regarding the roles of ACh in type 2 immunity is limited. OBJECTIVE: Our aim was to investigate the roles of epithelial ACh in allergic immune responses. METHODS: Human bronchial epithelial (HBE) cells were cultured with allergen extracts, and their ACh production and IL-33 secretion were studied in vitro. To investigate immune responses in vivo, naive BALB/c mice were treated intranasally with different muscarinic ACh receptor antagonists and then exposed intranasally to allergens. RESULTS: At steady state, HBE cells expressed cellular components necessary for ACh production, including choline acetyltransferase and organic cation transporters. Exposure to allergens caused HBE cells to rapidly release ACh into the extracellular medium. Pharmacologic or small-interfering RNA-based blocking of ACh production or autocrine action through the M3 muscarinic ACh receptors in HBE cells suppressed allergen-induced ATP release, calcium mobilization, and extracellular secretion of IL-33. When naive mice were exposed to allergens, ACh was quickly released into the airway lumen. A series of clinical M3 muscarinic ACh receptor antagonists inhibited allergen-induced IL-33 secretion and innate type 2 immune response in the mouse airways. In a preclinical murine model of asthma, an ACh receptor antagonist suppressed allergen-induced airway inflammation and airway hyperreactivity. CONCLUSIONS: ACh is released quickly by airway epithelial cells on allergen exposure, and it plays an important role in type 2 immunity. The epithelial ACh system can be considered a therapeutic target in allergic airway diseases.

Transcriptome profile of subsynaptic myonuclei at the neuromuscular junction in embryogenesis.

Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre- and post-synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ-constituting genes, Dok7, Chrna1, and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (Dok7, Chrna1, and Chrnd) together with four representative ß-catenin regulators (Amotl2, Ptprk, Fam53b, and Tcf7l2). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where ß-catenin serves as a structural protein to organize the membrane-anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where ß-catenin serves as a transcriptional coactivator in Wnt/ß-catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2, but not of Amotl2 or Ptprk, impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA-seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/ß-catenin signaling that regulate the formation of the embryonic NMJ.

Exercise Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating Endoplasmic Reticulum Stress and Mitophagy Through M2 Acetylcholine Receptor.

Aims: Adaptive changes in the heart by exercise have been shown to reduce the risk of cardiovascular disease, and M2 Acetylcholine receptor (M2AChR), a receptor abundantly present on cardiac parasympathetic nerves, is closely associated with the development of cardiovascular disease. The present study intends to investigate whether exercise can regulate endoplasmic reticulum stress (ERS) and mitophagy through M2AChR to resist myocardial ischemia-reperfusion (I/R) injury and to elucidate its mechanism of action. Results: Exercise enhanced parasympathetic nerve function and increased myocardial M2AChR protein expression in I/R rats. In addition, it promoted the protein expression of MFN2 and inhibited the expression of Drp1, Chop, PINK1/Parkin, and PERK/eIF2α/ATF4 signaling pathways, effectively reducing mitophagy, ERS, and apoptosis. At the cellular level, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) reduced hypoxia/reoxygenation (H/R)-induced ERS through the downregulated expression of PERK/eIF2α/ATF4 pathway proteins in H9C2 cardiomyocytes. When intervened with M2AChR inhibitors, the levels of ERS and phosphorylation levels of the PERK/eIF2α/ATF4 pathway were increased in H/R cells. Innovation and Conclusion: Exercise intervention activated the parasympathetic state in rats. It inhibited myocardial mitophagy and ERS levels, and reduced myocardial apoptosis through M2AChR, thereby resisting I/R-induced myocardial injury and improving cardiac function. Antioxid. Redox Signal. 40, 209-221.

Molecular Analysis of a Congenital Myasthenic Syndrome Due to a Pathogenic Variant Affecting the C-Terminus of ColQ.

Congenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors.

The homeodomain transcriptional regulator DVE-1 directs a program for synapse elimination during circuit remodeling.

The elimination of synapses during circuit remodeling is critical for brain maturation; however, the molecular mechanisms directing synapse elimination and its timing remain elusive. We show that the transcriptional regulator DVE-1, which shares homology with special AT-rich sequence-binding (SATB) family members previously implicated in human neurodevelopmental disorders, directs the elimination of juvenile synaptic inputs onto remodeling C. elegans GABAergic neurons. Juvenile acetylcholine receptor clusters and apposing presynaptic sites are eliminated during the maturation of wild-type GABAergic neurons but persist into adulthood in dve-1 mutants, producing heightened motor connectivity. DVE-1 localization to GABAergic nuclei is required for synapse elimination, consistent with DVE-1 regulation of transcription. Pathway analysis of putative DVE-1 target genes, proteasome inhibitor, and genetic experiments implicate the ubiquitin-proteasome system in synapse elimination. Together, our findings define a previously unappreciated role for a SATB family member in directing synapse elimination during circuit remodeling, likely through transcriptional regulation of protein degradation processes.