Mechanisms of retrograde signaling between muscle and motor neurons

描述(申请人提供)：在运动神经元(MN)因轴突切断而失去与肌肉的功能连接后，MN的特性发生了各种变化，包括切换到再生模式。当运动轴突与肌肉重新建立突触联系时，这些变化恢复正常。这一证据表明，突触接触介导了肌肉和MN之间的重要相互作用，通常使正常MN特性得以表达并抑制再生，但其潜在机制尚不清楚。我们已经证明，阻断运动终板乙酰胆碱受体(ACHR)会使MN电流的棘波激活阈值(血流基电流)发生类似于切断轴突的变化，这是MN兴奋性的一个重要指标。我们获得的更多证据表明，肌肉纤维动作电位或机械活动并不参与这一信号传递。这些观察表明，肌肉和MNS之间的逆行信号可能是通过激活ACHR来完成的。一个重要的问题是，Achr介导的信号丢失是否会引发更广泛的轴突切断后影响。如果是这样，那么轴突切断的效果可能是基于Achr介导的肌肉逆行信号的丢失，而不是损伤本身。这一新的想法将在特定的目标1中进行测试。其他现有证据表明，终板ACHR的激活启动了显著的钙内流。这种电流可以用来激活肌肉中的下游机制，从而将ACHR的激活与最终产生逆行信号连接到MN。位于运动终板的几个钙敏感分子中包括神经元型一氧化氮合酶(NNOS)。CA2激活nNOS产生一氧化氮(NO)，它可能通过扩散到运动终末直接发出MNS信号，或者激活运动终板的进一步下游级联，最终提供逆行信号。在特定的目标2中，我们将通过确定外源性NO是否能阻止ACHR阻断后MN特性的切断样改变来测试nNOS在MNS逆行信号转导中的作用。这些研究的结果将为MNS中触发轴突反应的因素提供新的见解，并开始识别肌肉中的分子机制，这些机制是控制MN兴奋性最低的信号基础。 公共卫生相关性：这项工作的重点是了解肌肉和运动神经元之间逆行信号传递的潜在机制。根据我们收集的数据，我们提出了一个假设，即该信号是由运动终板乙酰胆碱受体激活启动的。本研究的目的是测试这一信号的丢失是否是运动神经元对轴突切断反应的基础，并开始鉴定肌肉中介导逆行信号的分子机制。

DESCRIPTION (provided by applicant): After motoneurons (MNs) lose functional connectivity with muscle by axotomy, a variety of changes occur in MN properties including a switch to a regenerative mode. These changes return to normal when motor axons re-establish synaptic contact with muscle. This evidence indicates that synaptic contact mediates important interactions between muscle and MNs that normally enable expression of normal MN properties and inhibit regeneration, but underlying mechanisms are poorly understood. We have shown that blockade of motor endplate acetylcholine receptors (ACHRs) produces axotomy-like changes in MN current threshold for spike activation (rheobase current), an important metric of MN excitability. Additional evidence we have obtained shows that muscle fiber action potential or mechanical activity is not involved in this signaling. These observations suggest that retrograde signaling between muscle and MNs may be accomplished via ACHR activation. An important question is whether loss of ACHR-mediated signaling can provoke a wider range of post-axotomy effects. If so, then the effects of axotomy may be based on the loss of ACHR-mediated retrograde signaling from muscle rather than injury itself. This novel idea will be tested in Specific Aim 1. Other available evidence indicates that a significant Ca2+ influx is initiated by activation of endplate ACHRs. Such currents could serve to activate downstream mechanisms within muscle and thus link ACHR activation to eventual production of retrograde signals to MNs. Included among several Ca2+-sensitive molecules located at the motor endplate is neuronal nitric oxide synthase (nNOS). Ca2+ activation of nNOS produces nitric oxide (NO) which may signal MNs directly via diffusion to motor terminals or activate further downstream cascades at the motor endplate that ultimately provide retrograde signaling. In Specific Aim 2, we will test the involvement of nNOS in retrograde signaling to MNs by determining whether exogenous NO can prevent axotomy-like changes in MN properties after ACHR blockade. The results of these studies will add new insight into factors that trigger the axotomy response in MNs and begin the identification of molecular mechanisms in muscle which underlie signaling that controls at a minimum MN excitability. PUBLIC HEALTH RELEVANCE: This work is focused on understanding mechanisms underlying retrograde signaling between muscle and motor neurons. Based on data we collected, we have developed the hypothesis that this signaling is initiated by motor endplate acetylcholine receptor activation. The purpose of the proposed studies is to test whether loss of this signaling underlies the motor neuron response to axotomy and to begin identification of molecular mechanisms in muscle that mediate retrograde signaling.