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

描述(申请人提供)：电压门控钠通道(VGSC)定位于轴突起始段、Ranvier结节和神经肌肉接头，几乎完全负责动作电位的上升阶段。一个或两个(3个)亚基可以与单个形成孔的α-亚基相互作用，在细胞表面形成活性的VGSC。VGSC的P亚基调节成年神经元的钠通道密度和功能。我们最近发现VGSC的32亚基(02)是参与阿尔茨海默病(AD)相关淀粉样前体蛋白加工的a-、P-和y-分泌酶的底物。VGSC a-和p-亚基的突变都与癫痫症状有关，这是由于VGSC活性降低和增加导致钠通道功能失衡所致。有趣的是，癫痫发作的增加与阿尔茨海默病有关。我们的初步数据表明，02的BACE1和PS1/y-分泌酶介导的处理增加调节了神经细胞、BACE1转基因小鼠和脑中BACE1水平升高的AD患者VGSC a亚单位NaJ.1的mRNA和蛋白水平。BACE1活性升高抑制神经细胞和成年小鼠海马神经元的VGSC功能。在这里，我们提出了BACE1和PS/v-分泌酶通过释放P2-ICD和转录调控a-亚基来调节VGSC功能的假说(S)。具体目标1将侧重于p-/v-分泌酶介导的p2加工的生物学功能。我们将使用体外和无细胞切割分析以及MALDI-TOF MS分析来确定P2中BACE1和PS/y-分泌酶的确切切割位点。我们还将区分(32)中潜在的y-和e-裂解，由PS/y-分泌酶介导。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。为了研究p-/y-分泌酶介导的P2对基因表达的影响，我们将从全长P2、32-CTF和|32-ICD转染神经细胞系中获得基因表达谱。P2-ICD-DNA相关性也将被调查。在具体目标2中，我们将确定p-/y-分泌酶介导的(32)处理如何具体调节VGSC a亚基水平。将使用荧光素酶报告分析和顺序的Nav1.1启动子缺失来鉴定介导P2反式激活的Nav1.1启动子序列。我们将确定在神经细胞和原代神经元中，随着p/y-分泌酶介导的P2处理的增加，VGSC a亚基积累的亚细胞室。P2“‘”小鼠和BACE1转基因小鼠将进行交叉试验，以在体内证实P2错误处理诱导了VGSC a通道Nav1.1的表达。VGSC a亚基的表达和成年海马神经元的钠通道活性将在BACEHfiZ？动物的大脑中进行评估。由于BACE1活性和水平在AD脑中显著升高，因此VGSC活性异常可能参与了AD的发病。此外，我们的初步数据表明，BACE1和/或γ-分泌酶抑制剂可以影响VGSC的功能，并有助于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.