Cortico-Spinal Repopulation from Precursors for Repair

描述(申请人提供)：拟议实验的长期目标是通过以下方式修复受损或退化的皮质脊髓电路：i)从内源性前体(“干细胞”)诱导新的皮质脊髓运动神经元(CSMN)的神经发生；ii)支持其存活，以及iii)重新招募到与较低运动神经元群体的功能性突触电路中。最近，我们已经在成年小鼠的原位操纵内源性前体在成年小鼠的新皮质中进行神经再生(新神经元的诞生)，这在正常情况下是不会发生的。在合作工作中，我们通过同源鸟类内源性前体在原位诱导鸣禽的行为功能神经元替换。这些实验表明，存在一个分子信号序列和组合，可以通过这些信号诱导新神经元的诞生，即使在正常情况下不发生神经发生的成年新皮质中也是如此。与这一建议更直接相关的是我们最近的发现：1)内源性前体细胞也可以被特异性地诱导原位分化为皮质脊髓运动神经元(CSMN)，并向脊髓投射；以及2)我们可以分离、FACS纯化(纯度为99.5%)，并在不同的发育阶段培养CSMN，以分析谱系特异性控制存活和分化，特别是轴突延长的速度/程度。我们假设，我们可以通过提高新生神经元的存活率和/或其轴突向支持性脊髓靶点生长的速度和程度来显著增加新招募的CSMN的数量。我们还假设新生神经元可以精确地分化为新的功能CSMN，接收传入突触，并成为突触整合。我们的五个目标将直接在体外和体内检验这些和相关的假设。目的：1)对诱导的成体CSMN的谱系特异性分化进行更全面的分析，更详细地描述CSMN诱导神经发生、神经元分化、连接和新生CSMN长期存活的时间进程；2，3)体外研究一组候选生长和神经营养因子，使CSMN具有潜在的阶段特异性增强存活和/或增强突起和电路连接，采用FACS对不同发育阶段的CSMN进行纯化，并与局部因子应用比较；目的4)通过体外实验中精选生长因子的脑室注射，操纵和增加CSMN诱导的神经发生和脊髓投射；5)利用ICC和逆转录病毒GFP-大麦凝集素的表达，研究CSMN分化和潜在的传入和传出突触形成的精确度。总之，这项工作旨在通过原位操纵内源性神经前体来修复皮质-脊髓运动神经元回路的最终目标，而不是移植。

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.