Exploring the Regenerative Capacity of Neurons in the Axolotl Brain.

描述（由申请人提供）：神经退行性疾病的特点是神经元亚型的选择性丧失，由于成人大脑不能产生新的神经元，这种丧失通常是不可逆的。迄今为止，有几种方法可以改善某些症状，但这些治疗方法都不能成功地取代人类失去的神经元。因此，大脑修复的新途径是必要的。一种通过从成体干细胞或现有神经元中产生新的神经元来补充丢失细胞的方法可能潜在地改善退行性表型。虽然大多数哺乳动物的神经元在没有外部操纵的情况下不容易再生或重新编程，但有几种蝾螈自然表现出这些能力。蝾螈就是这样一个拥有越来越多实验工具的物种。有趣的是，最近的研究表明，蝾螈具有再生潜力的各种器官——肢体、脊髓、心脏——都经历了自然的重编程，以取代失去的组织。虽然一般机制尚不清楚，但这种再生和重编程的耦合似乎是蝾螈各种器官再生潜力的必要条件。美西螈的大脑是另一个具有强大再生能力的器官。三分之一的端脑可以完全切除，大脑会在几个月内用新的神经元重新填充失去的组织。然而，在20世纪60年代分子和细胞生物学工具有限的情况下，对蝾螈大脑再生的研究才开始进行。总的来说，这项提议的目标是了解美西螈大脑的再生过程。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重建的保真度？2)新神经元从何而来？现有的神经元是否有可能去分化形成胚基，胚基随后分化成新的神经元？3)哪些分子变化与再生过程有关？回答这些问题不仅将揭示美西螈大脑再生的机制，而且还可能为在成人大脑中使用哪些分子来诱导神经发生和核重编程提供线索。Paola Arlotta博士的实验室与周边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。完成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。动物设施有能力在自由流动的水系统中容纳100多只蝾螈，并有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对美西螈大脑再生过程的理解，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手段。

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.