PDF(Pubmed)
Abstract：
BACKGROUND: Astrocytes respond to injury and disease through a process known as reactive astrogliosis, of which inflammatory signaling is one subset. This inflammatory response is heterogeneous with respect to the inductive stimuli and the afflicted central nervous system region. This is of plausible importance in e.g. traumatic axonal injury (TAI), where lesions in the brainstem carries a particularly poor prognosis. In fact, astrogliotic forebrain astrocytes were recently suggested to cause neuronal death following axotomy. We therefore sought to assess if ventral brainstem- or rostroventral spinal astrocytes exert similar effects on motor neurons in vitro.
METHODS: We derived brainstem/rostroventral spinal astrocyte-like cells (ES-astrocytes) and motor neurons using directed differentiation of mouse embryonic stem cells (ES). We activated the ES-astrocytes using the neurotoxicity-eliciting cytokines interleukin- (IL-) 1α and tumor necrosis factor-(TNF-)α and clinically relevant inflammatory mediators. In co-cultures with reactive ES-astrocytes and motor neurons, we assessed neurotoxic ES-astrocyte activity, similarly to what has previously been shown for other central nervous system (CNS) regions.
RESULTS: We confirmed the brainstem/rostroventral ES-astrocyte identity using RNA-sequencing, immunocytochemistry, and by comparison with primary subventricular zone-astrocytes. Following cytokine stimulation, the c-Jun N-terminal kinase pathway down-stream product phosphorylated c-Jun was increased, thus demonstrating ES-astrocyte reactivity. These reactive ES-astrocytes conferred a contact-dependent neurotoxic effect upon co-culture with motor neurons. When exposed to IL-1β and IL-6, two neuroinflammatory cytokines found in the cerebrospinal fluid and serum proteome following human severe traumatic brain injury (TBI), ES-astrocytes exerted similar effects on motor neurons. Activation of ES-astrocytes by these cytokines was associated with pathways relating to endoplasmic reticulum stress and altered regulation of MYC.
CONCLUSIONS: Ventral brainstem and rostroventral spinal cord astrocytes differentiated from mouse ES can exert neurotoxic effects in vitro. This highlights how neuroinflammation following CNS lesions can exert region- and cell-specific effects. Our in vitro model system, which uniquely portrays astrocytes and neurons from one niche, allows for a detailed and translationally relevant model system for future studies on how to improve neuronal survival in particularly vulnerable CNS regions following e.g. TAI.
METHODS: We derived brainstem/rostroventral spinal astrocyte-like cells (ES-astrocytes) and motor neurons using directed differentiation of mouse embryonic stem cells (ES). We activated the ES-astrocytes using the neurotoxicity-eliciting cytokines interleukin- (IL-) 1α and tumor necrosis factor-(TNF-)α and clinically relevant inflammatory mediators. In co-cultures with reactive ES-astrocytes and motor neurons, we assessed neurotoxic ES-astrocyte activity, similarly to what has previously been shown for other central nervous system (CNS) regions.
RESULTS: We confirmed the brainstem/rostroventral ES-astrocyte identity using RNA-sequencing, immunocytochemistry, and by comparison with primary subventricular zone-astrocytes. Following cytokine stimulation, the c-Jun N-terminal kinase pathway down-stream product phosphorylated c-Jun was increased, thus demonstrating ES-astrocyte reactivity. These reactive ES-astrocytes conferred a contact-dependent neurotoxic effect upon co-culture with motor neurons. When exposed to IL-1β and IL-6, two neuroinflammatory cytokines found in the cerebrospinal fluid and serum proteome following human severe traumatic brain injury (TBI), ES-astrocytes exerted similar effects on motor neurons. Activation of ES-astrocytes by these cytokines was associated with pathways relating to endoplasmic reticulum stress and altered regulation of MYC.
CONCLUSIONS: Ventral brainstem and rostroventral spinal cord astrocytes differentiated from mouse ES can exert neurotoxic effects in vitro. This highlights how neuroinflammation following CNS lesions can exert region- and cell-specific effects. Our in vitro model system, which uniquely portrays astrocytes and neurons from one niche, allows for a detailed and translationally relevant model system for future studies on how to improve neuronal survival in particularly vulnerable CNS regions following e.g. TAI.
摘要：
背景：星形胶质细胞通过称为反应性星形胶质细胞增生的过程对损伤和疾病作出反应，其中炎症信号是一个子集。这种炎症反应相对于诱导刺激和患病的中枢神经系统区域是异质的。这在例如创伤性轴索损伤(TAI)中似乎很重要，脑干的病变预后特别差。事实上,星形胶质细胞的前脑星形胶质细胞最近被认为会导致轴突切开术后神经元死亡。因此，我们试图评估腹侧脑干或腹侧脊髓星形胶质细胞是否在体外对运动神经元发挥类似的作用。
方法：我们使用小鼠胚胎干细胞（ES）的定向分化来衍生脑干/rostroventral脊髓星形胶质细胞样细胞（ES-星形胶质细胞）和运动神经元。我们使用引起神经毒性的细胞因子白细胞介素-（IL-）1α和肿瘤坏死因子-（TNF-）α以及临床相关的炎症介质激活了ES星形胶质细胞。在与反应性ES-星形胶质细胞和运动神经元的共培养中，我们评估了神经毒性的ES-星形胶质细胞活性，类似于先前显示的其他中枢神经系统（CNS）区域。
结果：我们使用RNA测序证实了脑干/rostroventralES-星形胶质细胞的身份，免疫细胞化学,并与原发性脑室下区星形胶质细胞进行比较。细胞因子刺激后，c-JunN末端激酶途径下游产物磷酸化c-Jun增加，从而证明ES-星形胶质细胞反应性。这些反应性ES-星形胶质细胞在与运动神经元共培养时赋予接触依赖性神经毒性作用。当暴露于IL-1β和IL-6时，在人类严重创伤性脑损伤（TBI）后的脑脊液和血清蛋白质组中发现的两种神经炎症细胞因子，ES-星形胶质细胞对运动神经元产生类似的作用。这些细胞因子对ES-星形胶质细胞的激活与内质网应激和MYC调节改变有关的途径有关。
结论：从小鼠ES中分化出的腹侧脑干和腹侧脊髓星形胶质细胞在体外具有神经毒性作用。这突出了中枢神经系统病变后的神经炎症如何发挥区域和细胞特异性作用。我们的体外模型系统，它独特地描绘了一个小生境中的星形胶质细胞和神经元，允许详细和翻译相关的模型系统,用于关于如何在例如TAI之后改善特别脆弱的CNS区域中的神经元存活的未来研究。
方法：我们使用小鼠胚胎干细胞（ES）的定向分化来衍生脑干/rostroventral脊髓星形胶质细胞样细胞（ES-星形胶质细胞）和运动神经元。我们使用引起神经毒性的细胞因子白细胞介素-（IL-）1α和肿瘤坏死因子-（TNF-）α以及临床相关的炎症介质激活了ES星形胶质细胞。在与反应性ES-星形胶质细胞和运动神经元的共培养中，我们评估了神经毒性的ES-星形胶质细胞活性，类似于先前显示的其他中枢神经系统（CNS）区域。
结果：我们使用RNA测序证实了脑干/rostroventralES-星形胶质细胞的身份，免疫细胞化学,并与原发性脑室下区星形胶质细胞进行比较。细胞因子刺激后，c-JunN末端激酶途径下游产物磷酸化c-Jun增加，从而证明ES-星形胶质细胞反应性。这些反应性ES-星形胶质细胞在与运动神经元共培养时赋予接触依赖性神经毒性作用。当暴露于IL-1β和IL-6时，在人类严重创伤性脑损伤（TBI）后的脑脊液和血清蛋白质组中发现的两种神经炎症细胞因子，ES-星形胶质细胞对运动神经元产生类似的作用。这些细胞因子对ES-星形胶质细胞的激活与内质网应激和MYC调节改变有关的途径有关。
结论：从小鼠ES中分化出的腹侧脑干和腹侧脊髓星形胶质细胞在体外具有神经毒性作用。这突出了中枢神经系统病变后的神经炎症如何发挥区域和细胞特异性作用。我们的体外模型系统，它独特地描绘了一个小生境中的星形胶质细胞和神经元，允许详细和翻译相关的模型系统,用于关于如何在例如TAI之后改善特别脆弱的CNS区域中的神经元存活的未来研究。
