PS1 and BACE1 regulate processing and activity of voltage-gated sodium channels

描述（由申请人提供）：定位于轴突起始段、Ranvier节点和神经肌肉连接处的电压门控钠通道（VGSC）几乎完全负责动作电位的上升阶段。一个或两个（3个）亚基可以与单个成孔a亚基相互作用，在细胞表面形成活性VGSC。VGSC的p亚基调节成人神经元钠通道密度和功能。我们最近发现VGSC的32亚基（02）作为a-、P-和y-分泌酶的底物，参与阿尔茨海默病（AD）相关淀粉样前体蛋白的加工。VGSC的a-和p-亚基突变与癫痫症状有关，由VGSC活性的降低和增加引起钠通道功能的不平衡。有趣的是，癫痫发作的发病率增加与阿尔茨海默病有关。我们的初步数据表明，增加BACE1-和PS1/y-分泌酶介导的02加工调节VGSC a亚基NaJ。1神经元细胞、BACE1转基因小鼠和AD患者脑内BACE1水平升高的mRNA和蛋白水平。升高的BACE1活性抑制神经元细胞和成年小鼠海马神经元的VGSC功能。本研究提出BACE1和PS/v分泌酶通过释放P2-ICD和a-亚基的转录调节VGSC功能的假说。特异性目标1将重点关注p-/v-分泌酶介导的p2加工的生物学功能。我们将通过体外和无细胞裂解实验，以及MALDI-TOF质谱分析，确定P2中BACE1和PS/y-分泌酶的确切裂解位点。我们还将区分由PS/y分泌酶介导的（32）中潜在的y-和e-裂解。通过免疫沉淀和P2-ICD亲和层析鉴定P2-ICD的细胞质和核结合伙伴。为了表征p-/y-分泌酶介导的P2加工对基因表达的影响，将从全长P2、32-CTF和|32- icd转染的神经元细胞系中获得基因表达谱。P2- ICD-DNA的关联也将被研究。在Specific Aim 2中，我们将确定p-/y-分泌酶介导的（32）加工如何特异性调节VGSC a亚基水平。介导P2转激活的Nav1.1启动子序列将通过荧光素酶报告基因检测和Nav1.1启动子序列缺失进行鉴定。我们将确定在神经细胞和原代神经元中p-/y-分泌酶介导的P2加工增加后，VGSC a-亚单位积累的亚细胞区室。将P2“’”小鼠与BACE1转基因小鼠杂交，在体内验证P2错误加工诱导VGSC a通道Nav1.1的表达。将在BACEHfiZ^动物的大脑中评估VGSC a-亚基表达和成年海马神经元钠通道活性。由于BACE1的活性和水平在AD大脑中显著升高，因此VGSC活性的功能障碍可能有助于AD的发病。此外，我们的初步数据表明，BACE1和/或y分泌酶抑制剂可以影响VGSC功能，并有利于BACE1活性升高的AD患者的膜兴奋性正常化。

DESCRIPTION (provided by applicant): Localized to axonal initial segments, nodes of Ranvier, and neuromuscular junctions, voltage-gated sodium channels (VGSC) are almost exclusively responsible for the rising phase of action potentials. Either one or two (3 subunits can interact with a single pore-forming a-subunit to form the active VGSC on the cell surface. p subunits of the VGSC regulate both sodium channel density and function in adult neurons. We have recently identified the (32-subunit (02) of the VGSC as a substrate for a-, P-, and y-secretases involved in processing of the Alzheimer's disease (AD)-associated amyloid precursor protein. Mutations in both a- and p-subunits of the VGSCs have been linked to epileptic symptoms, induced by both decreased and increased VGSC activity resulting in an imbalance in sodium channel function. Interestingly, increased incidence of epileptic seizures has been associated with Alzheimer's disease. Our preliminary data indicate that increased BACE1- and PS1/y-secretase-mediated processing of 02 regulates VGSC a-subunit NaJ.1 mRNA and protein levels in neuronal cells, BACE1 transgenic mice, and in AD patients with elevated levels of BACE1 in brain. Elevated BACE1 activity inhibits VGSC function in neuronal cells and adult mouse hippocampal neurons. Here we propose to address the hypothesis that BACE1 and PS/v-secretase regulate VGSC function by release of P2-ICD and transcriptional regulation of a-subunit(s). Specific Aim 1 will focus on the biological function of p-/v-secretase-mediated processing of p2. We will determine the exact BACE1 and PS/y-secretase cleavage sites in P2 using in vitro and cell-free cleavage assays followed by MALDI-TOF MS analysis. We will also distinguish between potential y- and e-cleavages in (32, mediated by PS/y-secretase. Cytoplasmic and nuclear binding partners of P2-ICD will be identified by immunoprecipitation and P2-ICD affinity chromatography. To characterize effects of p-/y-secretase-mediated processing of P2 on gene expression, gene expression profiles will be obtained from full-length P2, 32-CTF, and |32-ICD-transfected neuronal cell lines. P2- ICD-DNA association will also be investigated. In Specific Aim 2, we will determine how p-/y-secretase-mediated processing of (32 specifically regulates VGSC a-subunit levels. Nav1.1 promoter sequences, which mediate P2 transactivation, will be identified using luciferase reporter assays and sequential Nav1.1 promoter deletions. We will determine the subcellular compartment where VGSC a-subunits accumulate following increased p-/y- secretase-mediated processing of P2 in neuronal cells and in primary neurons. P2"'" mice and BACE1 transgenic mice will be crossed to test for in vivo confirmation that P2 misprocessing induces VGSC a-channel Nav1.1 expression. VGSC a-subunit expression and adult hippocampal neuron sodium channel activity will be assessed in brains from BACEHfiZ^ animals. Since BACE1 activity and levels are significantly increased in AD brains, consequent dysfunction in VGSC activity may contribute to AD pathogenesis. Moreover, our preliminary data suggest that BACE1 and/or y-secretase inhibitors could affect VGSC function, and be beneficial in normalizing membrane excitability in AD patients with elevated BACE1 activity.