Glutamine Transporters SNAT1 and SNAT2 in Rett Syndrome Microglia

描述（由申请人提供）：Rett综合征（RTT）是一种毁灭性的神经发育障碍，由X连锁MECP 2基因的功能缺失突变引起。MECP 2编码甲基CpG结合蛋白2（MeCP 2），其是一种表观遗传调节剂，其结合靶基因中的甲基CpG二核苷酸以调节转录。MeCP 2缺乏如何导致神经功能缺损仍然知之甚少，但它显然与树突和突触异常有关。我们以前报道过，MeCP 2缺陷型小胶质细胞（MDM）通过组成性释放比野生型小胶质细胞多5倍的谷氨酸，从而破坏树突和突触，从而引起兴奋性毒性。我们随后发现MeCP 2是谷氨酰胺转运蛋白SNAT 1和SNAT 2的有效转录抑制因子。因此，MDM表现出SNAT 1和SNAT 2的过度表达，导致谷氨酰胺摄取增加，小胶质细胞谷氨酰胺稳态破坏，线粒体氧化应激和谷氨酸的过度产生。这种新的MeCP 2调节途径对于鉴定治疗靶点以阻断RTT中的小胶质细胞神经毒性具有高度意义。因为我们的研究 揭示了小胶质细胞谷氨酸的产生是由一个主要的表观遗传因子MeCP 2调节的，对这一通路的研究将推进我们对神经活动如何通过DNA甲基化的表观遗传界面调节异质性小胶质细胞反应模式和神经元-胶质细胞相互作用的认识。 因此，为了巩固对该机制的支持，本提案的目的是（1）牢固地建立MeCP 2调节的SNAT 1/SNAT 2表达与MDM异常之间的联系，以及（2）检查该途径与RTT的相关性。在目的1中，我们将确定SNAT 1/SNAT 2表达与MDM表型之间的因果关系，例如线粒体异常和谷氨酸过度产生。我们将确定野生型小胶质细胞中SNAT 1和/或SNAT 2的过表达是否诱导MDM表型。我们还将确定在MDM中敲低SNAT 1和/或SNAT 2表达是否会降低MDM表型。在目标2中，我们将使用从MECP 2敲除小鼠和对照野生型小鼠的脑中新鲜分离的小胶质细胞，确定不同疾病阶段小胶质细胞异常（SNAT 1/SNAT 2表达/活性和线粒体异常）的进展。我们最近已经建立了一系列的技术来研究新鲜分离的小胶质细胞从青少年和成熟的啮齿动物的大脑，这将最好地代表小胶质细胞在体内。我们将采用qRT-PCR、流式细胞术、膜片钳、电子显微镜和荧光成像来研究该制剂中的MDM表型。 这些研究的结果预计将具有高度意义：1）帮助我们了解MeCP 2缺陷如何通过小胶质细胞介导的机制导致RTT中的树突和突触异常; 2）探索谷氨酰胺转运蛋白在小胶质细胞病理学中的作用，目前对此知之甚少; 3）提供首批研究之一，以检查小胶质细胞功能的表观遗传控制。 公共卫生相关性：在Rett综合征中，小胶质细胞（脑免疫细胞）的异常可能导致神经网络中断。我们将研究谷氨酰胺转运蛋白SNAT 1和SNAT 2的异常水平是否会导致Rett综合征的小胶质细胞异常。

DESCRIPTION (provided by applicant): Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked MECP2 gene. MECP2 encodes methyl-CpG-binding protein 2 (MeCP2), an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. How MeCP2 deficiency causes neurological deficits remains poorly understood, but it is clearly related to dendritic and synaptic abnormalities. We previously reported that MeCP2-deficient microglia (MDM) cause excitotoxicity by constitutively releasing five times more glutamate than wild-type microglia, thus damaging dendrites and synapses. We subsequently found that MeCP2 is a potent transcriptional suppressor of the glutamine transporters SNAT1 and SNAT2. MDM consequently show over-expression of SNAT1 and SNAT2, resulting in increased glutamine uptake, disruption of microglial glutamine homeostasis, mitochondrial oxidative stress, and over-production of glutamate. This novel MeCP2-regulated pathway is highly significant for identification of therapeutic targets to block microglial neurotoxicity in RTT. Because our studies reveal that microglial glutamate production is regulated by a major epigenetic factor MeCP2, the research into this pathway will advance our knowledge about how neural activities regulate heterogeneous microglia response patterns and neuron-glia interactions through the epigenetic interface of DNA methylation. To consolidate the support for this mechanism, the purpose of this proposal, therefore, is (1) to firmly establish the link between MeCP2-regulated SNAT1/SNAT2 expression and MDM abnormalities and (2) to examine the relevance of this pathway to RTT. In Aim 1, we will determine the causal relationship between SNAT1/SNAT2 expression and the MDM phenotype, such as mitochondrial abnormalities and glutamate over- production. We will determine if over-expression of SNAT1 and/or SNAT2 in wild-type microglia induces the MDM phenotype. We will also determine if knock-down of SNAT1 and/or SNAT2 expression in MDM reduces the MDM phenotype. In Aim 2, we will determine the progression of microglial abnormalities (SNAT1/SNAT2 expression/activity and mitochondrial abnormalities) at different disease stages, using microglia freshly isolated from brains of MECP2 knockout mice and control wild-type mice. We recently have established a series of techniques to study microglia freshly isolated from juvenile and mature rodent brains, which would best represent microglia in vivo. We will employ qRT-PCR, flow cytometry, patch-clamp, electron microscopy, and fluorescent imaging to study the MDM phenotype in this preparation. The results of these studies are expected to be highly significant by 1) helping us to understand how MeCP2 deficiency causes dendritic and synaptic abnormalities in RTT through a microglia-mediated mechanism; 2) exploring the role of glutamine transporters in microglial pathology, which is presently poorly understood; and 3) providing one of the first few studies to examine epigenetic control of microglia function. PUBLIC HEALTH RELEVANCE: In Rett syndrome, abnormalities of microglia, the brain immune cells, may cause interruption of the neuronal network. We will investigate if abnormal levels of glutamine transporters SNAT1 and SNAT2 cause microglial abnormalities in Rett syndrome.