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

描述（由申请人提供）：电压门控钠通道（VGSC）定位于轴突起始段、郎维尔结和神经肌肉接头，几乎专门负责动作电位的上升期。一个或两个β亚基可以与单个成孔α亚基相互作用，在细胞表面形成活性VGSC。VGSC的β亚基调节成年神经元中的钠通道密度和功能。我们最近鉴定了VGSC的β 2-亚基（β 2）作为参与阿尔茨海默病（AD）相关淀粉样前体蛋白加工的α-、β-和γ-分泌酶的底物。VGSC的α-和β-亚基的突变与癫痫症状有关，这是由VGSC活性降低和增加引起的，导致钠通道功能失衡。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，增加的BACE 1和PS1/γ-分泌酶介导的O2加工调节神经元细胞、BACE 1转基因小鼠和脑中BACE 1水平升高的AD患者中的VGSC α-亚基NaJ.1 mRNA和蛋白水平。升高的BACE 1活性抑制神经元细胞和成年小鼠海马神经元中的VGSC功能。在这里，我们提出了一个假设，即BACE 1和PS/V-分泌酶调节VGSC功能的释放P2-ICD和转录调节的α-亚基（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试验，然后进行MALDI-TOF MS分析，确定P2中确切的BACE 1和PS/γ-分泌酶切割位点。我们还将区分由PS/γ-分泌酶介导的β 2中潜在的γ-和e-裂解。将通过免疫沉淀和P2-ICD亲和层析鉴定P2-ICD的细胞质和细胞核结合伴侣。为了表征β-/γ-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P3中获得基因表达谱。|32-ICD转染的神经元细胞系。还将研究P2-ICD-DNA相关性。在具体目标2中，我们将确定β-/γ-分泌酶介导的β 2加工如何特异性调节VGSC α-亚基水平。Nav1.1启动子序列，介导P2反式激活，将使用荧光素酶报告基因测定和顺序Nav1.1启动子缺失进行鉴定。我们将确定在神经元细胞和原代神经元中增加的P-/γ-分泌酶介导的P2加工后VGSC α-亚基积累的亚细胞区室。将P2 +/-小鼠和BACE 1转基因小鼠杂交以测试体内确认P2错误加工诱导VGSC α-通道Nav1.1表达。VGSC α-亚基表达和成年海马神经元钠通道活性将在来自BACEHfZ ^动物的脑中评估。由于BACE 1活性和水平在AD脑中显著增加，因此VGSC活性的功能障碍可能有助于AD发病机制。此外，我们的初步数据表明，BACE 1和/或γ-分泌酶抑制剂可以影响VGSC功能，并有利于BACE 1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.