Axonal alterations in demyelinating diseases

描述(申请人提供)：我们假设在正常的有髓PNS轴突中，Kv1.1、Kv1.2、KCNQ2和KCNQ3的组合是复极化所必需的，并且Kv3.1b的错误表达对脱/重新髓鞘轴突的轴突传导具有有害影响。在未发表的工作中，我们发现Na，K-ATPase的α-亚型是唯一一个明确定位于轴突的亚型，它(令人惊讶地)被排除在节点之外，并且似乎因脱髓鞘而局灶性减少。因此，我们还假设，alphas的错误表达可能有助于去髓鞘/再髓鞘轴突的去极化。拟议的实验建立在这些发现的基础上，中心主题是阐明K+稳态是如何在正常和脱髓鞘/再髓鞘轴突中发挥作用的。 目的#1：Kv3.1b通道是否与脱髓鞘疾病的传导失败有关？我们将通过比较DTX-I和4-AP对KvS.lb缺失(KcnCL-/-)和KCNC1+/+背景下TremblerJ小鼠轴突传导的影响来研究KvS.lb是否是4-AP敏感通道。 目的#2：KCNQ2在有髓轴突中的作用是什么？由于KCNQ2基因缺失的小鼠在出生时死亡，在髓鞘形成和结节形成之前，我们将产生一只KCNQ2基因缺失的小鼠，并分析有髓轴突的结构和功能，包括KCNQ3的表达。 目的#3：有髓轴突和脱髓鞘轴突表达哪些Na，K-ATPase亚型？我们将通过免疫电子显微镜将Alpha1-3定位于CNS和PNS有髓轴突及其β亚基。

DESCRIPTION (provided by applicant): We hypothesize that in normal myelinated PNS axons, the combination of Kv1.1, Kv1.2, KCNQ2, and KCNQ3 is necessary for repolarization, and that the misexpression of Kv3.1 b has a deleterious effect on axonal conduction of de/remyelinated axons. In unpublished work, we have found the alphas isoform of Na,K-ATPase is the only one that is clearly localized to axons that it is (surprisingly) excluded from nodes, and appears to be focally diminished by demyelination. Thus, we also hypothesize that the misexpression of alphas may contribute to depolarization of de/remyelinated axons. The proposed experiments build on these findings, with the central theme of illuminating how K+ homeostasis works in normal and de/remyelinated axons. Aim #1: Do Kv3.1b channels contribute to conduction failure in demyelinating diseases? We will investigate whether KvS.lb is the 4-AP-sensitive channel by comparing the effects of DTX-I and 4- AP on axonal conduction in TremblerJ mice on a KvS.lb -null (Kcncl-/-) versus Kcnc1+/+ background. Aim #2: What is the role of KCNQ2 in myelinated axons? Because Kcnq2-null mice die at birth, before myelination and the formation of nodes, we will generate a conditional Kcnq2-null mouse and analyze the structure and function of myelinated axons, including the expression of KCNQ3. Aim #3: What Na,K-ATPase isoforms are expressed by myelinated axons and demyelinated axons? we will localize alpha1-3 by immunoelectron microscopy in CNS and PNS myelinated axons, along with their beta subunits.