Age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission in the mouse diaphragm.
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ABSTRACT:
SUBMITTER: Willadt S
PROVIDER: S-EPMC4837408 | biostudies-other | 2016 Apr
REPOSITORIES: biostudies-other
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Project description:In aging Fischer 344 rats, phrenic motor neuron loss, neuromuscular junction abnormalities, and diaphragm muscle (DIAm) sarcopenia are present by 24 mo of age, with larger fast-twitch fatigue-intermediate (type FInt) and fast-twitch fatigable (type FF) motor units particularly vulnerable. We hypothesize that in old rats, DIAm neuromuscular transmission deficits are specific to type FInt and/or FF units. In phrenic nerve/DIAm preparations from rats at 6 and 24 mo of age, the phrenic nerve was supramaximally stimulated at 10, 40, or 75 Hz. Every 15 s, the DIAm was directly stimulated, and the difference in forces evoked by nerve and muscle stimulation was used to estimate neuromuscular transmission failure. Neuromuscular transmission failure in the DIAm was observed at each stimulation frequency. In the initial stimulus trains, the forces evoked by phrenic nerve stimulation at 40 and 75 Hz were significantly less than those evoked by direct muscle stimulation, and this difference was markedly greater in 24-mo-old rats. During repetitive nerve stimulation, neuromuscular transmission failure at 40 and 75 Hz worsened to a greater extent in 24-mo-old rats compared with younger animals. Because type IIx and/or IIb DIAm fibers (type FInt and/or FF motor units) display greater susceptibility to neuromuscular transmission failure at higher frequencies of stimulation, these data suggest that the age-related loss of larger phrenic motor neurons impacts nerve conduction to muscle at higher frequencies and may contribute to DIAm sarcopenia in old rats. NEW & NOTEWORTHY Diaphragm muscle (DIAm) sarcopenia, phrenic motor neuron loss, and perturbations of neuromuscular junctions (NMJs) are well described in aged rodents and selectively affect FInt and FF motor units. Less attention has been paid to the motor unit-specific aspects of nerve-muscle conduction. In old rats, increased neuromuscular transmission failure occurred at stimulation frequencies where FInt and FF motor units exhibit conduction failures, along with decreased apposition of pre- and postsynaptic domains of DIAm NMJs of these units.
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Project description:Fragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor neuron degeneration is not well understood.Here we use a SOD1-ALS mouse model (low-copy Gurney G93A-SOD1 mouse) to show that motor neurons with Golgi fragmentation are retrogradely labeled by intramuscularly injected CTB (beta subunit of cholera toxin), indicating that Golgi fragmentation precedes neuromuscular denervation and axon retraction. We further show that Golgi fragmentation may occur in the absence of and precede two other pathological markers, i.e. somatodendritic SOD1 inclusions, and the induction of ATF3 expression. In addition, we show that Golgi fragmentation is associated with an altered dendritic organization of the Golgi apparatus, does not depend on intact apoptotic machinery, and is facilitated in transgenic mice with impaired retrograde dynein-dependent transport (BICD2-N mice). A connection to altered dynein-dependent transport also is suggested by reduced expression of endosomal markers in neurons with Golgi fragmentation, which also occurs in neurons with impaired dynein function.Together the data indicate that Golgi fragmentation is a very early event in the pathological cascade in ALS that is associated with altered organization of intracellular trafficking.
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Project description:ImportanceCongenital myasthenic syndromes (CMS) are heterogeneous disorders. Defining the phenotypic features, genetic basis, and pathomechanisms of a CMS is relevant to prognosis, genetic counseling, and therapy.ObjectivesTo characterize clinical, structural, electrophysiologic, and genetic features of a CMS and to search for optimal therapy.Design, settings, and participantsTwo sisters with CMS affecting the limb-girdle muscles were investigated between 2012 and 2014 at an academic medical center by clinical observation, in vitro analysis of neuromuscular transmission, cytochemical and electron microscopy studies of the neuromuscular junction, exome sequencing, expression studies in HEK293 and COS7 cells, and for response to therapy, and they were compared with 15 historical control participants.Main outcomes and measuresWe identified the disease gene and mutation, confirmed pathogenicity of the mutation by expression studies, and instituted optimal pharmacotherapy.ResultsQuantitative analysis of single EP regions was done for all 15 control participants and microelectrode studies of neuromuscular transmission and α-bgt binding sites per EP was conducted for 13 control participants. Examination of the older sister's intercostal muscle end plates (EPs) showed them to be abnormally small, with attenuated reactivities for the acetylcholine receptor and acetylcholinesterase. Most EPs had poorly differentiated or degenerate junctional folds, and some appeared denuded of nerve terminals. The amplitude of the EP potential (EPP), the miniature EPP, and the quantal content of the EPP were all markedly reduced. Exome sequencing identified a novel homozygous p.Glu1233Ala mutation in low-density lipoprotein receptor-related protein 4 (LRP4), a coreceptor for agrin to activate muscle-specific tyrosine kinase (MuSK), which is required for EP development and maintenance. Expression studies indicate that the mutation compromises the ability of LRP4 to bind to, phosphorylate, and activate MuSK. Treatment with albuterol sulfate improved the patients' symptoms. A previously identified patient harboring 2 heterozygous mutations in LRP4 had structurally abnormal intercostal EPs but no identifiable defect of neuromuscular transmission at these EPs.Conclusions and relevanceWe identified a second CMS kinship harboring mutations in LRP4, identified the mechanisms that impair neuromuscular transmission, and mitigated the disease by appropriate therapy.
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Project description:Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia.We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development.We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype.Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.
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Project description:BackgroundThe functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown.MethodsHb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1G93A ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed.ResultsMice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P < 0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79 ± 5.74 vs. 79.39 ± 3.77% fully occupied neuromuscular junctions for f/f, P < 0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88 ± 0.13 vs. 1.43 ± 0.48 μm for f/f, P < 0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment.ConclusionsOur current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.
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2023-06-19 | GSE226117 | GEO