Regeneration of retinal neurons from Müller glia

项目概要/摘要： 本申请中提出的项目的目标是替换因疾病或损伤而丢失的视网膜神经元 通过刺激视网膜的常驻神经胶质细胞Müller神经胶质（MG）， 神经发生青光眼、黄斑变性和视网膜色素变性等疾病， 视网膜中的各种神经元群体，并导致数百万人的视力受损或失明 世界各地视觉可以说是人类拥有的最重要的感觉，目前只有 一旦视网膜细胞退化，恢复视力的临床选择非常有限。令人惊讶的是，许多 非哺乳动物物种具有从MG再生其视网膜神经元并恢复视觉的能力 损坏后的功能。最近的发现阐明了这些物种修复其 视网膜导致我提出的项目，以重现MG介导的视网膜再生小鼠。生成 最近已经在年轻小鼠中使用转基因MG定向的方法获得了来自MG的未成熟视网膜神经元， 前神经转录因子Ascl 1的表达。在我的初步研究中，我发现 MG特异性Ascl 1表达和添加组蛋白脱乙酰酶抑制剂的组合使MG能够 在受损的成年老鼠视网膜上产生新的神经元。这一令人兴奋的发现现在为许多人开辟了道路 研究再生神经元的长期存活力和功能的额外研究。 以下三个提出的目标将1）确定MG衍生的神经元是否稳定更长时间 时间段，2）确定它们是否保留了它们的一些神经胶质特征，以及3）确定是否存在 更有效地使这些MG衍生神经元成熟的方法。使用最先进的成像技术， 例如超分辨率ZeissAiryscan共焦显微镜和连续块面扫描电子显微镜 显微镜下，我将解决在何种程度上MG衍生的神经元实现成熟的神经元形态 和连通性。使用全细胞电生理学并测量MG衍生神经元对光的反应 将解决神经元的功能和成熟。最后，使用单细胞mRNA测序对FACS纯化的 MG衍生的神经元将解决MG衍生的神经元发展基因表达模式的程度。 表达类似于已知的成熟视网膜细胞神经元类型，或者它们是否保留一定程度的神经胶质细胞， 基因表达。通过利用这些技术来表征细胞的神经元状态， 然后可以修改范例，如研究策略中所述，以确定MG衍生的神经元是否 可以被驱动到一个更成熟的状态。拟议的研究强调并扩展了唯一已知的 协议产生新的神经元在成年哺乳动物视网膜，并提出了一个独特的机会， 扩大再生医学领域，使其更接近潜在的临床治疗。

Project Summary/Abstract: The goal of the project proposed in this application is to replace retinal neurons that are lost to disease or injury with new neurons by stimulating the resident glial cells of the retina, the Müller glia (MG), to undergo neurogenesis. Diseases ranging from glaucoma, macular degeneration, and Retinitis Pigmentosa, destroy various neuronal populations in the retina and result in visual impairment or blindness for millions of people around the world. Sight is arguably the most important sense humans possess, and there are currently only very limited clinical options to restore vision once the cells in the retina have degenerated. Amazingly, many non-mammalian species have the ability to regenerate their retinal neurons from MG and restore visual function after damage. Recent discoveries elucidating the mechanisms by which these species repair their retinas have led to my proposed project to reproduce MG-mediated retinal regeneration in mice. Generating immature retinal neurons from MG has recently been achieved in young mice using transgenic MG directed expression of the proneural transcription factor Ascl1. In my preliminary studies, I have found that the combination of MG-specific Ascl1 expression and the addition of a histone deacetylase inhibitor enables MG to generate new neurons in a damaged adult mouse retina. This exciting finding now opens the way for many additional studies to investigate the long-term viability and functionality of the regenerated neurons. The following three proposed Aims will 1) determine whether the MG-derived neurons are stable for longer periods of time, 2) determine if they retain some of their glial characteristics, and 3) determine if there are methods for more effectively maturing these MG-derived neurons. Using state-of-the-art imaging techniques, such as super-resolution Zeiss Airyscan confocal microscopy, and serial block-face scanning electron microscopy I will address the extent to which the MG-derived neurons achieve mature neuronal morphology and connectivity. Using whole-cell electrophysiology and measuring the MG-derived neuron's response to light will address neuronal functionality and maturity. Lastly, using single-cell mRNA-sequencing on FACS-purified MG-derived neurons will address the extent to which the MG-derived neurons develop a pattern of gene expression similar to known mature retinal cell neuronal types, or whether they retain some degree of glial gene expression. By utilizing these techniques to characterize the neuronal state of the cells, the experimental paradigm can then be modified, as described in the Research Strategy, to determine if MG-derived neurons can be driven to a more mature state. The proposed study highlights and expands upon the only known protocol for generating new neurons in the adult mammalian retina, and presents a unique opportunity to expand the field of regenerative medicine and push it closer to a potential clinical therapy.