Exploring the Regenerative Capacity of Neurons in the Axolotl Brain.

描述(申请人提供)：神经退行性疾病的特征是神经元亚型的选择性丧失，由于成人人脑无法产生新的神经元，这种亚型通常是不可逆的。到目前为止，已经有几种方法可以改善一些症状，但这些治疗方法都没有成功地取代人类丢失的神经元。因此，大脑修复的新途径是必要的。一种通过从成年干细胞或现有神经元中长出新神经元来补充丢失的细胞的方法可能会潜在地改善退化的表型。虽然大多数哺乳动物的神经元在没有外部操作的情况下不容易再生或重新编程，但几种火蜥蜴自然地表现出这些壮举。Axolotl就是这样一个物种，拥有越来越多的实验工具。有趣的是，最近的研究表明，各种具有再生潜力的Axolotl器官-肢体、脊髓、心脏-经历了自然的重新编程，以取代丢失的组织。虽然一般的机制尚不清楚，但这种再生和重编程的耦合似乎对Axolotl中各种器官的再生潜力是必要的。Axolotl脑是另一个具有很强再生能力的器官。三分之一的端脑可以被完全移除，大脑将在几个月内用新的神经元重新填充丢失的组织。然而，对紫杉醇脑再生的研究在20世纪60年代S之前才开始，当时分子和细胞生物学工具有限。总的来说，这项提议的目标是了解axolotl脑的再生过程。三个具体的问题推动了这一目标：1)特定的神经元亚型及其各自的电路在损伤后是否会逼真地重建？2)新的神经元从哪里来？有没有可能现有的神经元去分化形成胚泡，然后胚泡分化成新的神经元？3)再生过程中有什么分子变化？回答这些问题不仅可以揭示Axolotl中大脑再生的机制，还可以为哪些分子可能被用来诱导成年人脑中的神经发生和核重新编程提供线索。保拉·阿洛塔博士的实验室与周围的实验室和研究所的结合为回答这些问题提供了必要的工具。实现这一目标的方法包括免疫荧光、原位杂交、RNA-SEQ、逆行示踪、病毒注射和BrdU出生测定。该动物设施有能力在自由流动的水系统中容纳100多只Axolotl，并拥有一支专门的动物护理团队。这些50年前无法获得的资源和工具将极大地促进理解Axolotl中的大脑再生过程，以及将再生过程转化为哺乳动物系统并最终用于人类治疗的方法。

DESCRIPTION (provided by applicant): Neurodegenerative disorders are characterized by a selective loss of neuronal subtypes that is often irreversible due to the failure of the adult huma brain to generate new neurons. To date, several options to ameliorate some of the symptoms exist, but none of these treatments successfully replace the lost neurons in humans. Thus, new avenues for brain repair are necessary. A method to replenish lost cells by giving rise to new neurons from adult stem cells or from existing neurons could potentially ameliorate the degenerative phenotype. While most mammalian neurons do not readily regenerate or reprogram without external manipulation, several species of salamanders naturally exhibit these feats. The axolotl is one such species with an increasing array of experimental tools. Intriguingly, recent studies have demonstrated that the various axolotl organs with regenerative potential - limb, spinal cord, heart - undergo natural reprogramming in order to replace lost tissue. Though the general mechanism is still unclear, this coupling of regeneration and reprogramming seem to be necessary for the regenerative potential of various organs in the axolotl. The axolotl brain is another organ with great regenerative ability. One-third of the telencephalon can be completely removed and the brain will repopulate the lost tissue within a few months with new neurons. However, the research of axolotl brain regeneration has only been performed before the 1960's when molecular and cellular biological tools were limited. Broadly, the goal of this proposal is to understand the regenerative process of the axolotl brain. Three specific questions drive this goal: 1) Do the specific neuronal subtypes and their respective circuits reconstruct with fidelity upon injury? 2) Where do the new neurons come from? Is it possible that existing neurons dedifferentiate to form a blastema, which subsequently differentiates into new neurons? 3) What molecular changes are associated with the regenerative process? Answering these questions would not only reveal the mechanism of brain regeneration in the axolotl, but it also may give clues to what molecules may be used to induce neurogenesis and nuclear reprogramming in an adult human brain. The combination of the lab of Dr. Paola Arlotta and the surrounding labs and institutes provide the necessary tools to answer these questions. The methods of completing the goal include immunofluorescence, in situ hybridization, RNA-seq, retrograde tracing, viral injection, and BrdU birthdating. The animal facility has the capacity to hold over one hundred axolotls in a free-flowing water system and a dedicated animal care team. These resources and tools that were unavailable fifty years ago will greatly facilitate the understanding of the brain regeneration process in the axolotl as well as th means to translate the regenerative process to a mammalian system and eventually towards therapy in humans.