Evolutionary Conserved Mechanisms that Control Central Nervous System Development Regeneration and Degeneration

脑功能的逐渐丧失和神经退行性变是整个不同门的衰老的共同特征。老年性痴呆，包括阿尔茨海默病（AD），可能涉及成体神经干细胞（NSC）数量、活力和/或功能的失败。我们的实验室研究NSC在中枢神经系统（CNS）发育、成人再生和发病性痴呆（如AD）中的作用。我们正在建立再生医学和衰老研究领域，作为第一个前瞻性地分离人类NSC并将其用于已发表的临床前和临床试验研究的实验室。在这一建议中，我们试图了解NSC参与的基本原理和进化上保守的因素在群体被囊动物Botryllus schlosseri的神经元再生，退化和衰老。Botryllus有两种繁殖模式：有性繁殖产生具有简单中枢神经系统（脊索动物大脑）的原始脊索动物，该脊索动物将蜕变为无性繁殖的无根无脊椎动物，通过出芽繁殖。我们已经发现，Botryllus芽含有自我更新的种系干细胞和体细胞干细胞，这些干细胞自组织形成由遗传相同的个体组成的集落。这一阶段表现出候选NSC每周可检测的中枢神经系统组织再生，并在整个成年期经历反复的神经变性，这一过程类似于脊椎动物的成年神经发生和神经变性。因此，通过观察年轻和年老菌落的每周再生周期（例如<3个月vs. 7年），Botryllus提供了一个独特的机会来研究中枢神经系统产生和退化的细胞和分子机制。我们的目标是识别在NSC中积累的突变和/或表观遗传变化，并通过自我更新在生物体的整个生命中保持存在。我们对老的和年轻的Botryllus菌落的中枢神经系统细胞进行了系统的分子（脑转录组学）分析，并对每个中枢神经系统谱系细胞进行了形态学和行为学表征。该分析揭示了93个与阿尔茨海默病相关的同源基因，包括APP、GRN、PSEN1、GLUD2和VPS35，这些基因在年轻和年老菌落之间存在差异表达。此外，老菌落的大脑含有较少的细胞数量，并且减少了神经元介导的对感觉刺激的反应。由于干细胞是唯一能够自我更新并在整个群体生命中维持的细胞，我们假设随着时间的推移，在NSC及其祖细胞中积累的基因突变或表观遗传变化是与年龄相关的神经退行性疾病的主要原因。为了验证这一假设，我们计划在脊索动物幼虫和幼、老菌落中表征Botryllus大脑的分子和细胞多样性，在单细胞水平上分离它们的NSCs，鉴定NSC DNA中积累的突变，并测试它们对大脑再生和功能的影响。Morpholino反义rna还将被用于操纵基因的活性，这些基因的遗传变异可以预测阿尔茨海默病的发病，并测试它们对大脑再生和退化能力的影响，还将尝试通过CRISPR引入它们的基因组的永久改变。

Gradual loss of brain function and neurodegeneration are common features of aging throughout diverse phyla. Senile dementias, including Alzheimer’s Disease (AD), likely involve failures of adult neural stem cell (NSC) number, viability, and/or functions. Our lab studies NSC’s role in central nervous system (CNS) development, adult regeneration, and in onset dementia such as AD. We are establishing a field of research in regenerative medicine and aging as the first lab to prospectively isolate human NSC and use them in published preclinical and clinical trial studies. In this proposal we seek to understand basic principles and evolutionarily conserved elements of NSC involvement in neuronal regeneration, degeneration, and aging in the colonial tunicate Botryllus schlosseri.  Botryllus has two reproductive modes: sexual reproduction which produces a primitive chordate with a simple CNS (the chordate brain), that will undergo metamorphosis into an asexually reproducing sessile invertebrate which propagates by budding. We have found that Botryllus buds contain self-renewing germline stem cells, and somatic stem cells which self-organize to form a colony composed of genetically identical individuals. This stage exhibits weekly assayable CNS tissue regeneration from candidate NSC and undergoes repeated neurodegeneration throughout its adult life, a process that resembles adult neurogenesis and neurodegeneration in vertebrates. Thus, Botryllus offers a unique opportunity to study the cellular and molecular mechanisms of CNS generation and degeneration through observing weekly regeneration cycles in young and old colonies (e.g. <3 months vs. >7 years). We aim to identify the mutations and/or epigenetic changes that accumulate in the NSC, and through self-renewal remain present throughout an organism's life. We have undertaken a systematic molecular (brain transcriptomic) analysis of CNS cells of old and young Botryllus colonies, paired with morphological and behavioral characterization of each of their CNS lineage cells. This analysis revealed 93 homologous genes that correlate with Alzheimer’s disease, including APP, GRN, PSEN1, GLUD2, and VPS35 that are differentially expressed between young and old colonies. Furthermore, the brains of old colonies contain a lower number of cells and have reduced neuron-mediated responses to sensory stimuli. Since stem cells are the only cells that self-renew and are maintained throughout the colony’s life, we hypothesize that genetic mutations or epigenetic changes that accumulate over time in NSC and their progenitors are the main cause of age-related neurodegenerative diseases. To test this hypothesis, we plan to characterize the molecular and cellular diversity of the Botryllus brain in chordate larvae and young and old colonies, isolate their NSCs at the single cell level, identify mutations that accumulate in NSC DNA, and test their effect on brain regeneration and function. Morpholino anti-sense RNAs will also be used to manipulate the activity of genes whose genetic variation predicts the onset of Alzheimer’s disease and test their effect on brain regeneration and degeneration capacity, and also attempt to introduce permanent alterations of their genomes by CRISPR.