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。
- 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
: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.
: 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。
METHODS::Identifying disease-causing pathways and drugs that target them in Parkinson's disease (PD) has remained challenging. We uncovered a PD-relevant pathway in which the stress-regulated heterodimeric transcription complex CHOP/ATF4 induces the neuron prodeath protein Trib3 that in turn depletes the neuronal survival protein Parkin. Here we sought to determine whether the drug adaptaquin, which inhibits ATF4-dependent transcription, could suppress Trib3 induction and neuronal death in cellular and animal models of PD. Neuronal PC12 cells and ventral midbrain dopaminergic neurons were assessed in vitro for survival, transcription factor levels and Trib3 or Parkin expression after exposure to 6-hydroxydopamine or 1-methyl-4-phenylpyridinium with or without adaptaquin co-treatment. 6-hydroxydopamine injection into the medial forebrain bundle was used to examine the effects of systemic adaptaquin on signaling, substantia nigra dopaminergic neuron survival and striatal projections as well as motor behavior. In both culture and animal models, adaptaquin suppressed elevation of ATF4 and/or CHOP and induction of Trib3 in response to 1-methyl-4-phenylpyridinium and/or 6-hydroxydopamine. In culture, adaptaquin preserved Parkin levels, provided neuroprotection and preserved morphology. In the mouse model, adaptaquin treatment enhanced survival of dopaminergic neurons and substantially protected their striatal projections. It also significantly enhanced retention of nigrostriatal function. These findings define a novel pharmacological approach involving the drug adaptaquin, a selective modulator of hypoxic adaptation, for suppressing Parkin loss and neurodegeneration in toxin models of PD. As adaptaquin possesses an oxyquinoline backbone with known safety in humans, these findings provide a firm rationale for advancing it towards clinical evaluation in PD.
METHODS::Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by brain atrophy particularly in the striatum that produces motor impairment, and cognitive and psychiatric disturbances. Multiple pathogenic mechanisms have been proposed including dysfunctions in neurotrophic support and calpain-overactivation, among others. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is an essential mediator of neurotrophin signaling. In adult brain, Kidins220 presents two main isoforms that differ in their carboxy-terminal length and critical protein-protein interaction domains. These variants are generated through alternative terminal exon splicing of the conventional exon 32 (Kidins220-C32) and the recently identified exon 33 (Kidins220-C33). The lack of domains encoded by exon 32 involved in key neuronal functions, including those controlling neurotrophin pathways, pointed to Kidins220-C33 as a form detrimental for neurons. However, the functional role of Kidins220-C33 in neurodegeneration or other pathologies, including HD, has not been explored. In the present work, we discover an unexpected selective downregulation of Kidins220-C33, in the striatum of HD patients, as well as in the R6/1 HD mouse model starting at early symptomatic stages. These changes are C33-specific as Kidins220-C32 variant remains unchanged. We also find the early decrease in Kidins220-C33 levels takes place in neurons, suggesting an unanticipated neuroprotective role for this isoform. Finally, using ex vivo assays and primary neurons, we demonstrate that Kidins220-C33 is downregulated by mechanisms that depend on the activation of the protease calpain. Altogether, these results strongly suggest that calpain-mediated Kidins220-C33 proteolysis modulates onset and/or progression of HD.
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.