Cortico-Spinal Repopulation from Precursors for Repair

描述（由申请人提供）：拟议实验的长期目标是通过i)诱导内源性前体（“干细胞”）产生新的皮质-脊髓运动神经元（CSMN）来修复受损或退化的皮质-脊髓电路；Ii)支持它们的存活，iii)与较低的运动神经元群体一起招募到功能性突触回路。最近，我们在成年小鼠中原位操纵内源性前体，使其在正常情况下不会发生的成年小鼠新皮层中进行神经发生（新神经元的诞生）。在合作工作中，我们用同源鸟类内源性前体诱导鸣禽的行为功能神经元原位替代。这些实验表明，即使在正常情况下不发生神经发生的成人新皮层中，也存在一个分子信号序列和组合，通过该序列和组合可以诱导新神经元的诞生。与这一建议更直接相关的是我们最近的发现：1)内源性前体也可以被特异性诱导原位分化为皮质-脊髓运动神经元（CSMN），并投射到脊髓；2)我们可以分离，FACS纯化（纯度达到99.5%），并在不同的发育阶段培养CSMN，以分析对存活和分化的特异性控制，特别是轴突伸长的速率/程度。我们假设，我们可以通过提高新生神经元的存活率和/或轴突生长到支持脊髓目标的速度和程度来大幅增加新招募的CSMN的数量。我们还假设新生神经元可以精确地分化成新的功能性CSMN，接受传入突触，并成为突触整合。我们的五个目标将在体外和体内直接测试这些和相关的假设。该研究将：目标1)对成人CSMN诱导的谱系特异性分化进行更完整的分析，并对新生儿CSMN诱导神经发生、神经元分化、连通性和长期存活的时间过程进行更详细的描述；目的2,3)在体外研究一组候选生长和神经营养因子，通过在不同发育阶段对CSMN进行FACS纯化，以及整体与局部因子应用，研究这些候选生长和神经营养因子对CSMN的生存和/或生长和电路连接的潜在特异性增强作用；目的4)通过体外实验中高度选择的候选生长因子在脑室内灌注来操纵和增加诱导的成年CSMN神经发生和脊髓投射。目的5)通过ICC和逆转录病毒gfp -大麦凝集素的表达来研究CSMN分化和潜在传入和输出突触形成的准确性。总之，这项工作的最终目标是通过原位操作内源性神经前体来修复皮质-脊髓运动神经元回路，而无需移植。

DESCRIPTION (provided by applicant): The long-term goal of the proposed experiments is the repair of damaged or degenerated cortico-spinal circuitry via i) induction of neurogenesis of new cortico-spinal motor neurons (CSMN) from endogenous precursors ("stem cells"); ii) support of their survival, and iii) recruitment into functional synaptic circuitry with the lower motor neuron population. Recently, we have manipulated endogenous precursors in situ in the adult mouse to undergo neurogenesis (birth of new neurons) de novo in adult mouse neocortex, where it does not normally occur. In collaborative work, we induced behaviorally functional neuronal replacement in situ in songbirds by homologous avian endogenous precursors. These experiments demonstrated that there exists a sequence and combination of molecular signals by which the birth of new neurons can be induced, even in the adult neocortex where neurogenesis does not normally occur. Even more directly relevant to this proposal are our recent findings that 1) endogenous precursors can also be specifically induced to differentiate in situ into cortico-spinal motor neurons (CSMN) with projections to the spinal cord; and 2) that we can isolate, FACS purify (to >99.5% purity), and culture CSMN at distinct developmental stages for analysis of lineage-specific controls over survival and differentiation, particularly rate / extent of axon elongation. We hypothesize that we can substantially increase the number of newly recruited CSMN by enhancing the survival of the newborn neurons and/or their rate and extent of axonal outgrowth to supportive spinal cord targets. We also hypothesize that newborn neurons can differentiate precisely into new functional CSMN, receive afferent synapses, and become synaptically integrated. Our five Aims will test these and related hypotheses directly, in vitro and in vivo. The proposed research will: Aim 1) undertake a more complete analysis of lineage-specific differentiation of the induced adult-born CSMN, and a more detailed characterization of the time course of induced neurogenesis, neuronal differentiation, connectivity, and long-term survival of newborn CSMN); Aims 2,3) investigate in vitro a select set of candidate growth and neurotrophic factors enabling potentially stage-specific enhanced survival and/or enhanced outgrowth and circuit connectivity of CSMN, employing FACS purification of CSMN at distinct developmental stages, and bath vs. localized factor application; Aim 4) manipulate and increase the induced adult CSMN neurogenesis and spinal projections by intraventricular infusion of highly selected candidate growth factors from the in vitro experiments and Aim 5) investigate the precision of CSMN differentiation and potential afferent and efferent synapse formation using ICC and retroviral GFP-barley lectin expression. Together, this work aims toward the ultimate goal of repair of cortico-spinal motor neuron circuitry by manipulation of endogenous neural precursors in situ, without transplantation.