mGluR5 inhibits microglial activation and neuronal cell death after TBI

描述（由申请人提供）：创伤性脑损伤（TBI）导致神经元细胞死亡，并伴有星形胶质细胞增殖和与小胶质细胞激活相关的炎症，导致不可逆的组织损伤。这种继发性伤害发生在侮辱后的几秒钟到几分钟内，可能持续数天、数周，甚至可能持续数月到数年。我们实验室最近的证据显示，中枢神经系统的慢性炎症在损伤后至少持续6个月。炎症可能导致创伤后的慢性神经退行性变，以及阿尔茨海默病（AD）或帕金森综合征等疾病。我们实验室的初步研究表明，炎症可以通过激活小胶质细胞中的mGluR5受体来抑制。此外，我们的数据表明，mGluR5的作用可能是通过对NADPH氧化酶的作用介导的，NADPH氧化酶的作用是小胶质细胞产生活性氧（ROS），并可能在损伤后小胶质细胞的持续激活中发挥重要作用。提出的研究旨在解决以下假设：(1)NADPH氧化酶抑制减少脑外伤后小胶质细胞的激活，促炎因子的产生和相关的神经功能障碍；(2) mGluR5刺激可减弱脑外伤后小胶质细胞的活化和继发性神经元细胞死亡，改善脑外伤后的功能结局；(3) mGluR5，而不是mGluR1，在原代小胶质细胞培养物、小胶质细胞系和小胶质/神经元共培养物中调节小胶质细胞的激活和神经毒性，部分通过抑制NADPH氧化酶；(4) g1q蛋白信号转导通路是导致小胶质细胞NADPH氧化酶抑制和小胶质细胞活化抑制的mGluR5信号转导事件的关键组分。具体目的是证明：(1)在脑外伤后慢性神经元细胞丢失和相关神经功能障碍中，NADPH氧化酶在小胶质细胞激活和相关促炎因子产生中的重要性；(2)与mGluR5全基因敲除动物相比，mGluR5刺激会减弱小胶质细胞的激活，减少神经元细胞的死亡，改善脑外伤后的功能结果，而mGluR5全基因敲除动物表现出更大的小胶质细胞激活和神经元细胞死亡。我们还将使用条件/诱导敲除小鼠来区分小胶质细胞与神经元mGluR5的相对作用；(3)在小胶质细胞刺激的多细胞培养模型中，mGluR5而不是mGluR1调节小胶质细胞的激活和神经毒性；(4)在体外小胶质细胞模型中，g1q蛋白信号转导途径是由mGluR5刺激启动的，通过阻止NADPH氧化酶复合物的激活，对小胶质细胞活性的衰减至关重要。全国每年有100万例创伤性脑损伤（TBI）病例。创伤性脑损伤患者表现出持续至少3周的小胶质细胞激活的证据。我们将研究小胶质细胞和神经元mGluR5的神经保护作用，为TBI的新治疗方法提供初步研究。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) causes neuronal cell death, combined with astroglial proliferation and inflammation associated with the activation of microglia, which contributes to irreversible tissue damage. Such secondary injury begins within seconds to minutes after the insult and may continue for days, weeks and potentially even months to years. Recent evidence from our laboratory shows chronic inflammation in the CNS lasting at least 6 months after injury. Inflammation may contribute to chronic neurodegeneration after trauma as well as to disorders such as Alzheimer's disease (AD) or Parkinson's syndrome. Pilot studies in our laboratory have indicated that inflammation may be inhibited by activation of mGluR5 receptors in microglia. Furthermore, our data suggest that mGluR5 effects may be mediated by actions on the NADPH oxidase enzyme, which functions to produce reactive oxygen species (ROS) by microglia and may play a significant role in persistent activation of microglia following injury. The proposed studies are intended to address the following hypotheses: (1) NADPH oxidase inhibition reduces microglial activation, production of pro-inflammatory factors and associated neurological dysfunction after TBI; (2) mGluR5 stimulation attenuates microglia activation and secondary neuronal cell death, and improves functional outcomes after TBI; (3) mGluR5, but not mGluR1, modulates microglial activation and neurotoxicity in primary microglia cultures, a microglial cell line, and microglia/neuronal co-cultures, in part through inhibition of NADPH oxidase; and (4) the G1q-protein signal transduction pathway is the critical component of the mGluR5 signal transduction events leading to inhibition of microglial NADPH oxidase and suppression of microglial activation. Specific aims are to demonstrate: (1) the importance of NADPH oxidase in microglial activation and correlated production of pro-inflammatory factors in the chronic neuronal cell loss and associated neurological dysfunction after TBI; (2) that mGluR5 stimulation attenuates microglial activation, decreases neuronal cell death and improves functional outcomes after TBI in contrast to mGluR5 global knockout animals that show greater microglial activation and neuronal cell death. We will also distinguish the relative roles of microglial versus neuronal mGluR5 using conditional/inducible knockout mice; (3) that mGluR5, but not mGluR1, modulates microglial activation and neurotoxicity in multiple cell culture models of microglial stimulation; and (4) that the G1q-protein signal transduction pathway is initiated by mGluR5 stimulation and is critical for attenuation of microglial activity by preventing activation of the NADPH oxidase enzyme complex, using in vitro microglial cell models. PUBLIC HEALTH RELEVANCE There are 1 million incidences of traumatic brain injury (TBI) per year nationally. Patients with TBI demonstrate evidence of prolonged microglial activation that lasts at least 3 weeks post-injury. The proposed work, in which we will investigate the neuroprotective effects of microglial and neuronal mGluR5, will provide initial investigation to a novel therapeutic approach for TBI.