Mechanisms of retinal degeneration in neuroinflammatory demyelinating diseases

多发性硬化症(MS)领域的一个重要问题是，白质中的髓鞘/轴突损伤是否有助于 灰质中的神经元胞体丢失，或由两个独立的突起引起。神经退行性变是其基础 无法补救的MS进行性下降，突出了阐明其机制的重要性。至 为了解决这个问题，我们利用了视觉系统，其中视神经中的所有有髓轴突都来自视网膜神经节 细胞(RGC)。在MS患者中，RGC丢失和视网膜变薄与全脑和灰质萎缩相关。 因此，视觉评估可能是神经退行性变的敏感生物标志物。此外，视觉系统正在被 用于确定治疗多发性硬化症的新临床试验药物的疗效本提案中提供的初步数据 证明阻断成熟少突胶质细胞的AMPA型谷氨酸受体(AMPAR)信号 预防实验性自身免疫性视神经轴突损伤和抑制视网膜神经节细胞丢失 脑脊髓炎(EAE)。谷氨酸失衡与多发性硬化症(MS)有关，但兴奋性毒性 OL特有的促进髓鞘降解和冲击性轴突损伤的机制尚未被探索。高架 谷氨酸水平在新的T2可见白质病变出现之前检测到，这表明谷氨酸是一种 MS患者中重要的兴奋性毒素我们之前报道过MOG反应性T细胞刺激小胶质细胞释放 来自系统XC转运体的谷氨酸，促进自身免疫性脱髓鞘过程中髓鞘的降解。在这里，我们展示 在成熟的OL上选择性地缺失AMPAR功能可以减轻EAE的临床症状，减少髓鞘 和视神经轴突丢失，并抑制RGC丢失。因此，我们假设减少AMPAR信号 在OL中，防止脱髓鞘和/或促进再髓鞘形成，从而保护潜在的轴突和 它的神经元胞体。这些研究将在慢性和复发缓解中利用可诱导的OL特异性AMPAR缺失 探讨自身免疫性炎症(EAE)对轴突完整性的影响，最终影响RGC的健康。删除的效果 AMPAR对离子型谷氨酸受体功能的影响将通过钙离子成像和细胞死亡机制来确定 以及增殖和分化的变化，这可能会促进重新髓鞘形成。轴突活性将通过以下方式进行评估 共聚焦3D重建、电子显微镜以及使用转基因小鼠的线粒体运输和形态 表达荧光线粒体。将用遗传荧光跟踪OL数量和成熟状态的变化 记者tdTomato，以确定新增殖的OL祖细胞与先前存在的髓鞘分开的命运。 此外，视网膜的病理评估将根据纵向功能视觉评估进行评估 (视觉诱发电位和视网膜电信号)和纵向结构视觉成像(光学相干断层扫描) 探讨OLS患者AMPAR功能的可诱导性缺失对视网膜功能的调节作用。 脱髓鞘疾病。

An important question in the field of multiple sclerosis (MS) is whether myelin/axonal damage in the white matter facilitates neuronal cell body loss in the gray matter, or results from two independent processes. Neurodegeneration underlies progressive decline in MS for which there is no remedy, highlighting the significance of elucidating its mechanisms. To address this question, we utilized the visual system, where all myelinated axons in the optic nerve arise from retinal ganglion cells (RGCs). In patients with MS, RGC loss and retinal thinning correlate with whole-brain and gray matter atrophy. Therefore, visual assessments may be a sensitive biomarker for neurodegeneration. Furthermore, the visual system is being used to determine the efficacy of new clinical trial drugs to treat MS. Preliminary data presented in this proposal demonstrate that blocking AMPA-type glutamate receptor (AMPAR) signaling in mature oligodendrocytes (OLs) prevents axonal damage in the optic nerve and inhibits retinal ganglion cell loss in experimental autoimmune encephalomyelitis (EAE). Glutamate dysregulation is implicated in multiple sclerosis (MS), but whether excitotoxic mechanisms specific to OLs contribute to myelin degradation and impact axonal injury has not been explored. Elevated levels of glutamate are detected prior to the appearance of new T2-visible white matter lesions implicating glutamate as an important excitotoxin in patients with MS. We previously reported that MOG-reactive T cells provoke microglia to release glutamate from the system xc- transporter, promoting myelin degradation during autoimmune demyelination. Here, we show that selective deletion of AMPAR function on mature OLs diminished the clinical symptoms of EAE, attenuated myelin and axonal loss in the optic nerve, and inhibited RGC loss. Therefore, we hypothesize that reducing AMPAR-signaling in OLs prevents demyelination and/or promotes remyelination, resulting in preservation of the underlying axon and its neuronal soma. These studies will utilize inducible OL-specific deletion of AMPARs in chronic and relapsing-remitting autoimmune inflammatory (EAE) to explore the impact on axon integrity and ultimately RGC health. Effects of deleting AMPARs on ionotropic glutamate receptor function will be determined by Ca2+ imaging and mechanisms of cell death as well as changes in proliferation and differentiation that may promote remyelination. Axon viability will be assessed by confocal 3D reconstruction, electron microscopy, and mitochondrial trafficking and morphology using transgenic mice that express fluorescent mitochondria. Changes in OL number and maturation state will be tracked with the genetic fluorescent reporter tdTomato, to determine the fate of newly proliferated OL progenitor cells separately from pre-existing myelin. Furthermore, pathological assessments of the retina will be evaluated in light of longitudinal functional visual assessments (visual evoked potentials and electroretinograms) and longitudinal structural visual imaging (optical coherence tomography) to investigate how inducible deletion of AMPAR function in OLs modulates retinal function at key time points in demyelinating diseases.