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Unveiling synapse pathology in spinal bulbar muscular atrophy by genome-wide transcriptome analysis of purified motor neurons derived from disease specific iPSCs.
通过对疾病特异性 iPSCs 来源的纯化运动神经元进行全基因组转录组分析,揭开脊髓延髓肌萎缩的突触病理学。
- 影响因子:4.19
- DOI:10.1186/s13041-020-0561-1
- 作者列表:"Onodera K","Shimojo D","Ishihara Y","Yano M","Miya F","Banno H","Kuzumaki N","Ito T","Okada R","de Araújo Herculano B","Ohyama M","Yoshida M","Tsunoda T","Katsuno M","Doyu M","Sobue G","Okano H","Okada Y
- 发表时间:2020-02-19
Abstract
:Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients' pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.
摘要
: 脊髓延髓肌萎缩 (SBMA) 是由雄激素受体 (AR) 基因异常 CAG 重复扩增引起的成人起病,缓慢进展的运动神经元疾病。虽然通过对转基因小鼠模型和体外细胞培养模型的分析,配体 (睾酮) 依赖性突变型 AR 聚集已被证明在运动神经元变性中发挥重要作用, 由于模型小鼠和 SBMA 患者之间的差异,潜在的疾病机制仍有待完全阐明。因此,更准确地概括 SBMA 患者病理的新型人类疾病模型是更精确的病理生理分析和新型治疗药物开发所必需的。在此,我们从 4 例 SBMA 患者中建立了疾病特异性 iPSCs,并将其分化为脊髓运动神经元。为了研究运动神经元特异性病理,我们使用流式细胞术和基于运动神经元特异性报告基因 HB9e438:: Venus 的细胞分选来纯化 iPSC 来源的运动神经元, 并通过 RNA 序列进行全基因组转录组分析。结果揭示了 SBMA 中与突触、表观遗传学和内质网 (ER) 相关的病理学的参与。值得注意的是,我们证明了神经肌肉突触通过突触蛋白、 R-Spondin2 (RSPO2) 和 WNT 配体的显著上调参与了来自 SBMA 患者的运动神经元。已知与神经肌肉接头 (NMJ) 形成和乙酰胆碱受体 (AChR) 聚集有关。这些神经肌肉突触中异常的基因表达可能代表 SBMA 的一个新的治疗靶点。
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METHODS:BACKGROUND:Neuroinflammation has been recognized as an important factor in the pathogenesis of Alzheimer's disease (AD). One of the most recognized pathways in mediating neuroinflammation is the prostaglandin E2-EP1 receptor pathway. OBJECTIVE:Here, we examined the efficacy of the selective EP1 antagonist ONO-8713 in limiting amyloid-β (Aβ), lesion volumes, and behavioral indexes in AD mouse models after ischemic stroke. METHODS:Transgenic APP/PS1, 3xTgAD, and wildtype (WT) mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO) and sham surgeries. Functional outcomes, memory, anatomical outcomes, and Aβ concentrations were assessed 14 days after surgery. RESULTS:pdMCAO resulted in significant deterioration in functional and anatomical outcomes in the transgenic mice compared with the WT mice. No relevant differences were observed in the behavioral tests when comparing the ONO-8713 and vehicle-treated groups. Significantly lower cavitation (p = 0.0373) and percent tissue loss (p = 0.0247) were observed in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 mice. However, the percent tissue injury was significantly higher in APP/PS1 + ONO-8713 mice compared with WT + ONO-8713 group (p = 0.0373). Percent tissue loss was also significantly lower in the 3xTgAD + ONO-8713 mice than in the WT + ONO-8713 mice (p = 0.0185). ONO-8713 treatment also attenuated cortical microgliosis in APP/PS1 mice as compared with the vehicle (p = 0.0079); however, no differences were observed in astrogliosis across the groups. Finally, APP/PS1 mice presented characteristic Aβ load in the cortex while 3xTgAD mice exhibited very low Aβ levels. CONCLUSION:In conclusion, under the experimental conditions, EP1 receptor antagonist ONO-8713 showed modest benefits on anatomical outcomes after stroke, mainly in APP/PS1 mice.