Loss of mitochondrial protein CHCHD10 in skeletal muscle causes neuromuscular junction impairment.

The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Acetylcholine receptors (AChRs) are restricted at the synaptic region for proper neurotransmission. Mutations in the mitochondrial CHCHD10 protein have been identified in multiple neuromuscular disorders; however, the physiological roles of CHCHD10 at NMJs remain elusive. Here, we report that CHCHD10 is highly expressed at the postsynapse of NMJs in skeletal muscles. Muscle conditional knockout CHCHD10 mice showed motor defects, abnormal neuromuscular transmission and NMJ structure. Mechanistically, we found that mitochondrial CHCHD10 is required for ATP production, which facilitates AChR expression and promotes agrin-induced AChR clustering. Importantly, ATP could effectively rescue the reduction of AChR clusters in the CHCHD10-ablated muscles. Our study elucidates a novel physiological role of CHCHD10 at the peripheral synapse. It suggests that mitochondria dysfunction contributes to neuromuscular pathogenesis.

APC2CDH1 negatively regulates agrin signaling by promoting the ubiquitination and proteolytic degradation of DOK7.

Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.

Differences in Cadmium Uptake and Accumulation in Seedlings of Wheat Varieties with Low- and High-Grain Cadmium Accumulation under Different Drought Stresses.

Cadmium (Cd) and drought, as abiotic stresses, have long been significant challenges for crop growth and agricultural production. However, there have been relatively few studies conducted on the effects of drought stress on Cd uptake, especially regarding the differences in Cd uptake characterization in varieties with varying Cd accumulation under different drought stress. To investigate the effects of drought conditions on Cd uptake by wheat in different genotypes under specific background levels of Cd pollution, we validated the differences in root absorption characteristics of low- (YM) and high-grain Cd accumulating wheat genotypes (XM) using non-invasive micro-test technology, and we conducted a hydroponic experiment on the Cd addition and different drought levels in a climate-controlled chamber. The biomass, root morphology, Cd uptake, and accumulation were determined under Cd (100 µmol L-1) and different drought levels of 0% (0 MPa), 5% (-0.100 Mpa), 10% (-0.200 Mpa), and 15% (-0.388 Mpa) simulated by polyethylene glycol (PEG-6000). We found that the simultaneous exposure to Cd and drought had a suppressive effect on the total root lengths, root surface areas, and root volumes of XM and YM, albeit with distinct patterns of variation. As the concentration of PEG-6000 increased, the Cd concentrations and the amount of Cd accumulated in the roots and shoots of XM and YM decreased. Specifically, the Cd concentration in the roots exhibited a reduction ranging from 12.51% to 66.90%, while the Cd concentration in the shoots experienced an even greater decrease of 50.46% to 80.57%. The PEG-6000 concentration was significantly negatively correlated (p < 0.001) with Cd concentration of roots and shoots and Cd accumulation in roots, shoots, and the whole plants and significantly negatively correlated (p < 0.05) with the total length, surface area, and volume of roots. This study confirms that drought stress (5% PEG-6000) can decrease the uptake and accumulation of Cd in wheat seedlings without significant inhibition of biomass, and the change of root morphology (root length) and the decrease of Cd concentration in roots may be the main direct pathways for achieving these effects under drought stress. This research provides a new perspective and idea for water management in Cd-contaminated farmland.

Motoneurons innervation determines the distinct gene expressions in multinucleated myofibers.

BACKGROUND: Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal myofibers. At the postsynaptic side in myofibers, acetylcholine receptor (AChR) proteins are clustered by the neuronal agrin signal. Meanwhile, several nuclei in each myofiber are specially enriched around the NMJ for postsynaptic gene transcription. It remains mysterious that how gene expressions in these synaptic nuclei are systematically regulated, especially by motoneurons. RESULTS: We found that synaptic nuclei have a distinctive chromatin structure and gene expression profiling. Synaptic nuclei are formed during NMJ development and maintained by motoneuron innervation. Transcriptome analysis revealed that motoneuron innervation determines the distinct expression patterns in the synaptic region and non-synaptic region in each multinucleated myofiber, probably through epigenetic regulation. Myonuclei in synaptic and non-synaptic regions have different responses to denervation. Weighted gene co-expression network analysis revealed that the histone lysine demethylases Kdm1a is a negative regulator of synaptic gene expression. Inhibition of Kdm1a promotes AChR expression but impairs motor functions. CONCLUSION: These results demonstrate that motoneurons innervation determines the distinct gene expressions in multinucleated myofibers. Thus, dysregulation of nerve-controlled chromatin structure and muscle gene expression might cause muscle weakness and atrophy in motoneuron degenerative disorders.

UBQLN2-HSP70 axis reduces poly-Gly-Ala aggregates and alleviates behavioral defects in the C9ORF72 animal model.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in the intron of the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Transcripts carrying G4C2 repeat expansions generate neurotoxic dipeptide repeat (DPR) proteins, including poly-Gly-Ala (poly-GA), which tends to form protein aggregates. Here, we demonstrate that UBQLN2, another ALS/FTD risk factor, is recruited to reduce poly-GA aggregates and alleviate poly-GA-induced neurotoxicity. UBQLN2 could recognize HSP70 ubiquitination, which facilitates the UBQLN2-HSP70-GA complex formation and promotes poly-GA degradation. ALS/FTD-related UBQLN2 mutants fail to bind HSP70 and clear poly-GA aggregates. Disruption of the interaction between UBQLN2 and HSP70 inhibits poly-GA aggregation in C9-ALS/FTD iPSC-derived neurons. Finally, enhancing HSP70 by the chemical compound 17AAG at the adult stage mitigates behavioral defects in poly-GA animals. Our findings suggest a critical role of the UBQLN2-HSP70 axis in protein aggregate clearance in C9-ALS/FTD.

Differences in root surface adsorption, root uptake, subcellular distribution, and chemical forms of Cd between low- and high-Cd-accumulating wheat cultivars.

The differences in the mechanism of cadmium (Cd) accumulation in the grains of different wheat (Triticum aestivum L.) cultivars remain unclear. Thus, we conducted a hydroponic experiment in a greenhouse to compare root surface adsorption, root uptake, subcellular distribution, and chemical forms of Cd between low- and high-Cd-accumulating wheat cultivars at seedling stage, to improve our understanding of the differences between cultivars. The results showed that Cd adsorbed on the root surface was mainly in a complexed form, and the total amount of Cd on the Yaomai16 (YM, high-Cd-accumulating genotypes) root surface was higher (p < 0.05) than that on Xinmai9817 (XM, low-Cd-accumulating genotypes). A large amount of Cd ions adsorbed on root surface would cause plant damage and inhibit growth. Comparing the root-to-shoot translocation factors of Cd, the transfer coefficients of YM were 1.017, 1.446, 1.464, and 1.030 times higher than those of XM under 5, 10, 50, and 100 µmol L-1 Cd treatments, respectively. The subcellular distribution of Cd under Cd exposure is mainly in the cell wall and soluble fraction. The proportions of Cd in YM shoot soluble fraction were higher than those in XM, which was the main detoxification mechanism limiting the activity of Cd and may be responsible for low Cd accumulation in grains, while the effects of the chemical forms of Cd on migration and detoxification were not found to be related to Cd accumulation in the kernels.

Reducing water use by alternate-furrow irrigation with livestock wastewater reduces antibiotic resistance gene abundance in the rhizosphere but not in the non-rhizosphere.

Livestock wastewater is rich in nutrients but may contain antibiotics and antibiotic resistance genes (ARGs). Their discharge to watercourses or soil may result in proliferation of ARGs. Irrigation with wastewater appears to be the most feasible option of disposing of it. One efficient irrigation technology used in arid regions is alternate-furrow irrigation (AFI) by alternately drying part of the plant roots for a prolonged period to physiologically reduce transpiration without compromising yield. However, the extent to which AFI with wastewater influences the concentration of antibiotics and spread of ARGs in soil is poorly understood. The purpose of this paper is to investigate how AFI using swine wastewater alters antibiotic kinetics and ARGs abundance under different irrigation rates, using pepper as the model plant. We examined three AFI treatments using 50%, 65% and 80% of the amount of water employed in sufficient conventional furrow irrigation. Each treatment had a groundwater irrigation control. The results showed that antibiotic concentrations and relative ARGs abundance in the top 20 cm of soil did not increase with the irrigation amount, although they were higher than those in the groundwater-irrigated soils. The relative ARGs abundance in the soil was modulated by irrigation amount and reducing the irrigation amount in AFI reduced ARGs dispersion only in rhizosphere. When the soil moisture was close to field capacity, ARGs were more abundant in rhizosphere than in non-rhizosphere, possibly because the rhizosphere is rich in microbes and increasing antibiotic concentrations due to an increase in irrigation rate favors antibiotic-resistant microbiome in competing for substrates. These, however, were not mirrored in the relative ARGs abundance in the roots. These results have important implications as it revealed that reducing the input of antibiotics and ARGs into soil with AFI does not necessarily reduce ARGs proliferation.

Peanut-Shell Biochar and Biogas Slurry Improve Soil Properties in the North China Plain: A Four-Year Field Study.

Biochar and biogas slurry have been proved to improve the quality of some soil types, but the long-term effects on fluvo-aquic soil are not fully understood. This study aimed to compare the continuity effects of peanut-shell biochar and biogas slurry on the physicochemical properties, microbial population size, and enzyme activities of fluvo-aquic soil. We conducted a four-year field experiment of winter wheat-summer maize rotation in the North China Plain. Along with equal nitrogen inputs, three treatments were applied-conventional fertilizers, peanut-shell biochar, and hoggery biogas slurry-after which various soil quality indicators were compared. Compared with those of control, both biochar and biogas slurry increased the soil total nitrogen and organic matter content, and improved soil aggregation, microbial biomass, and actinomycetes. Biogas slurry decreased soil pH and improved urease and protease activities. With biochar and biogas slurry treatments, wheat yield increased by 8.46% and 23.47%, and maize yield by 18% and 15.46%, respectively. Biogas slurry increased the content of crude protein and starch in the grains. Both biogas slurry and peanut-shell biochar improved fluvo-aquic soil nutrient content, water-stable macroaggregates, and microbial population, which might be related to their high nutrient content, large specific surface area, adsorption capacity, and functional groups. Biogas slurry generally exhibited stronger effects than biochar probably because of its richness in nutrients and bioactive substances.

Introducing Autoimmunity at the Synapse by a Novel Animal Model of Experimental Autoimmune Myasthenia Gravis.

The neuromuscular junction (NMJ) is a peripheral synapse between motor neurons and skeletal muscle fibers that controls muscle contraction. The NMJ is the target of various disorders including myasthenia gravis (MG), an autoimmune disease in which auto-antibodies (auto-Abs) attack the synapse, and thus cause muscle weakness in patients. There are multiple auto-Abs in the MG patient sera, but not all the Abs are proven to be pathogenic, which increases the difficulties in clinical diagnoses and treatments. To establish the causative roles of auto-Abs in MG pathogenesis, the experimental autoimmune MG (EAMG) induced by the active immunization of auto-antigens (auto-Ags) or the passive transfer of auto-Abs is required. These models simulate many features of the human disease. To date, there are three kinds of EAMG models reported, of which AChR-EAMG and MuSK-EAMG are well characterized, while the recent LRP4-EAMG is much less studied. Here, we report a current summary of LRP4-EAMG and its pathogenic mechanisms. The features of LRP4-EAMG are more similar to those of AChR-EAMG, indicating a similar clinical treatment for LRP4- and AChR-positive MG patients, compared to MuSK-positive MG patients.

Motoneuron Wnts regulate neuromuscular junction development.

The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Unlike extensively investigated postsynaptic differentiation, less is known about mechanisms of presynaptic assembly. Genetic evidence of Wnt in mammalian NMJ development was missing due to the existence of multiple Wnts and their receptors. We show when Wnt secretion is abolished from motoneurons by mutating the Wnt ligand secretion mediator (Wls) gene, mutant mice showed muscle weakness and neurotransmission impairment. NMJs were unstable with reduced synaptic junctional folds and fragmented AChR clusters. Nerve terminals were swollen; synaptic vesicles were fewer and mislocated. The presynaptic deficits occurred earlier than postsynaptic deficits. Intriguingly, these phenotypes were not observed when deleting Wls in muscles or Schwann cells. We identified Wnt7A and Wnt7B as major Wnts for nerve terminal development in rescue experiments. These observations demonstrate a necessary role of motoneuron Wnts in NMJ development, in particular presynaptic differentiation.