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

大脑功能的逐渐丧失和神经退化是不同门类衰老的共同特征。老年性痴呆，包括阿尔茨海默病(AD)，可能涉及成人神经干细胞(NSC)数量、活性和/或功能的丧失。我们的实验室研究了NSC在中枢神经系统(CNS)发育、成人再生以及AD等痴呆症发病中的作用。我们正在建立一个再生医学和衰老领域的研究领域，作为第一个前瞻性分离人类神经干细胞并将其用于已发表的临床前和临床试验研究的实验室。在这项建议中，我们试图了解NSC参与殖民地被囊藻Botryllus schlosseri中神经元再生、退化和衰老的基本原理和进化保守元件。Botryllus有两种繁殖模式：有性繁殖产生具有简单CNS(脊索脑)的原始脊索，将经历蜕变为通过萌芽繁殖的无性繁殖无脊椎动物。我们发现Botryllus的芽含有自我更新的生殖系干细胞，以及自组织形成由基因相同的个体组成的群体的体细胞干细胞。这一阶段表现为候选NSC每周可分析的CNS组织再生，并在其成年一生中经历反复的神经退化，这一过程类似于脊椎动物的成年神经发生和神经退化。因此，Botryllus提供了一个独特的机会，通过观察年轻和老年群体的每周再生周期(例如&lt；3个月与&gt；7年)，研究CNS生成和退化的细胞和分子机制。我们的目标是确定在NSC中积累的突变和/或表观遗传变化，并通过自我更新在有机体的整个生命中保持存在。我们对老的和年轻的Botryllus群体的CNS细胞进行了系统的分子(脑转录)分析，并对它们的每个CNS谱系细胞进行了形态和行为特征的描述。这项分析揭示了93个与阿尔茨海默病相关的同源基因，包括APP、GRN、PSEN1、GLUD2和VPS35，这些基因在年轻和老年群体中差异表达。此外，老蜂群的大脑包含的细胞数量较少，神经元对感觉刺激的反应也较少。由于干细胞是唯一自我更新并在整个群体一生中保持的细胞，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，我们计划表征脊索幼虫和年轻和老年群体中Botryllus大脑的分子和细胞多样性，在单细胞水平分离他们的NSCs，识别NSC DNA中积累的突变，并测试它们对大脑再生和功能的影响。吗啡反义RNA还将被用来操纵那些基因变异预测阿尔茨海默病发病的基因的活性，并测试它们对大脑再生和退化能力的影响，还将试图通过CRISPR引入它们的基因组永久改变。

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.