in-vitro-neuromuscular-junction-induced-from-human-induced

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Kyoto University

Kyoto Prefectural University of Medicine

Borna disease virus (BDV) infection causes neurological disease in cats. Here, we report BDV infection in 199 hospitalized domestic cats in the Tokyo area. BDV infection was evaluated by detection of plasma antibodies against BDV-p24 or -p40. BDV-specific antibodies were detected in 54 cats (27.1%). Interestingly, the percentage of seropositive cats was not significantly different among the three clinical groups, i.e., healthy (29.8%), neurologically asymptomatic disease (22.2%) and neurological disease (33.3%). The specific antibodies were present even in cats aged below one year. The seropositive ratio was constant, irrespective of age and sampling season. The present study suggests that additional factors are required for onset of Borna disease in naturally infected cats and that BDV is transmitted through vertical routes in cats. 

A study on Borna disease virus infection in domestic cats in Japan.

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 patients' motor neurons (MNs), and to identify drugs that can restore the normal condition. We generated NMJ-like structures using 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. 

Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs.

Dravet syndrome (DS) is a severe childhood epilepsy typically caused by de novo dominant mutations in SCN1A. Although patients with DS frequently have neurocognitive abnormalities, the precise neural mechanisms responsible for their expression have not been elucidated. There are wide phenotypic differences among individuals with SCN1A mutations, suggesting that factors other than the SCN1A mutation modify the phenotype. Therefore, a well-controlled cellular model system is required to improve our understanding of the mechanisms underlying DS. Here we generated induced pluripotent stem cell (iPSC) lines from an individual with SCN1A mutation mosaicism, and separately cloned iPSC lines both with and without the SCN1A mutation. These clones theoretically have the same genetic backgrounds, except for the SCN1A gene, and should serve as an ideal pair for investigating the pathophysiology caused by SCN1A mutations. Quantitative reverse transcription-PCR and western blot analysis revealed higher tyrosine hydroxylase mRNA and protein expression levels in mutant neurons than in wild-type neurons. Moreover, dopamine concentrations in media collected from mutant neural cultures were higher than those from wild-type neural cultures. Our findings suggest that SCN1A mutation leads to changes in the dopamine system that may contribute to the behavioral abnormalities in DS. 

Establishment of isogenic iPSCs from an individual with SCN1A mutation mosaicism as a model for investigating neurocognitive impairment in Dravet syndrome.

Syntaxin-binding protein 1 (STXBP1) is essential for synaptic vesicle exocytosis. Mutations of its encoding gene, STXBP1, are among the most frequent genetic causes of epileptic encephalopathies. However, the precise pathophysiology of STXBP1 haploinsufficiency has not been elucidated. Using patient-derived induced pluripotent stem cells (iPSCs), we aimed to establish a neuronal model for STXBP1 haploinsufficiency and determine the pathophysiologic basis for STXBP1 encephalopathy. We generated iPSC lines from a patient with Ohtahara syndrome (OS) harboring a heterozygous nonsense mutation of STXBP1 (c.1099C>T; p.R367X) and performed neuronal differentiation. Both STXBP1 messenger RNA (mRNA) and STXBP1 protein expression levels of OS-derived neurons were approximately 50% lower than that of control-derived neurons, suggesting that OS-derived neurons are a suitable model for elucidating the pathophysiology of STXBP1 haploinsufficiency. Through Western blot and immunocytochemistry assays, we found that OS-derived neurons show reduced levels and mislocalization of syntaxin-1, a component of soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. In addition, OS-derived neurons have impaired neurite outgrowth. In conclusion, this model enables us to investigate the neurobiology of STXBP1 encephalopathy throughout the stages of neurodevelopment. Reduced expression of STXBP1 leads to changes in the expression and localization of syntaxin-1 that may contribute to the devastating phenotype of STXBP1 encephalopathy. 

Mislocalization of syntaxin-1 and impaired neurite growth observed in a human iPSC model for STXBP1-related epileptic encephalopathy.

The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.

iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.

The purpose of this study was to retrospectively investigate the effect of the severity of acute graft-versus-host disease (GVHD) on physical function after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Article	Year
A study on Borna disease virus infection in domestic cats in Japan. The Journal of veterinary medical science\| PubMed ID: 24805904	2014
Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs. Stem cell reports\| PubMed ID: 25801509	2015
Establishment of isogenic iPSCs from an individual with SCN1A mutation mosaicism as a model for investigating neurocognitive impairment in Dravet syndrome. Journal of human genetics\| PubMed ID: 26841829	2016
Mislocalization of syntaxin-1 and impaired neurite growth observed in a human iPSC model for STXBP1-related epileptic encephalopathy. Epilepsia\| PubMed ID: 26918652	2016
iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases. JCI insight\| PubMed ID: 31534050	2019
Effect of the severity of acute graft-versus-host disease on physical function after allogeneic hematopoietic stem cell transplantation. Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer\| PubMed ID: 31712955	2020

Michiko Yoshida

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