CLN3: Modulation of Apoptosis and Ceramide Levels

描述（由申请人提供）：长期目标是基于CLN 3对细胞凋亡和神经酰胺生成的影响的分子、亚细胞和生物化学决定因素的特异性，开发用于幼年型Batten病和癌症的合理疗法。Batten病或神经元蜡样脂褐质沉积症是一组以进行性认知和运动障碍、失明、顽固性癫痫发作以及加速的神经元和感光细胞损失为特征的致命性疾病。多种证据表明，导致JNCL的CLN 3缺陷导致神经元凋亡。这些是：存在通过EM的核染色质浓缩和通过JNCL神经元的TUNEL染色的DNA断裂的证据，在存活的非凋亡JNCL神经元中Bcl-2的上调，以及JNCL脑中神经酰胺水平的增加。在细胞水平上，CLN 3蛋白的过表达导致对由血清剥夺和化疗剂诱导的细胞凋亡的抗性、半胱天冬酶-3活化的抑制和神经酰胺水平的调节。阻断CLN 3在人有丝分裂后神经元中的表达导致自发性细胞凋亡，这被抗细胞凋亡药物氟吡汀阻止。重要的是，这些抗凋亡机制也在癌症中起作用。CLN 3在人和小鼠癌细胞系以及实体人结肠癌中存在显著的过表达。反义-CLN 3腺病毒阻断这些细胞系中的CLN 3表达，导致癌细胞生长的抑制、细胞凋亡增加以及跨线粒体膜电位的丧失和神经酰胺水平的增加。来源于JNCL患者的细胞是用于研究CLN 3对细胞凋亡和神经酰胺的影响的极好模型。 总体假设：CLN 3蛋白影响凋亡途径，在凋亡中具有负调节作用。具体假设：a）该膜蛋白的亚细胞定位影响其在凋亡中的作用; B）CLN 3内的基序有助于其对凋亡的调节作用; c）CLN 3调节神经酰胺合成清除途径中的一个步骤;和d）CLN 3内的基序和/或CLN 3亚细胞定位可能对神经酰胺水平有影响。具体目标包括：1）通过分析整体基因表达来确定CLN 3对凋亡途径的影响; 2）通过定义亚细胞定位和导致这种调节的CLN 3氨基酸和基序来确定CLN 3调节凋亡的决定因素;和3）通过仔细检查CLN 3缺陷细胞中的神经酰胺合成/清除酶和代谢来确定神经酰胺合成/清除中的步骤是否由CLN 3调节。

DESCRIPTION (provided by applicant): Long term goals are the development of rational therapies for the juvenile form of Batten disease and cancer based on the specifics of molecular, subcellular and biochemical determinants of the impact of CLN3 on apoptosis and ceramide generation. Batten disease or the Neuronal Ceroid Lipofuscinosis are a group of fatal disorders characterized by progressive cognitive and motor impairment, blindness, intractable seizures, and accelerated neuronal and photoreceptor loss. Multiple lines of evidence have converged to suggest that the defect in CLN3 causing JNCL results in apoptotic neuronal death. These are: presence of nuclear chromatin condensation by EM and evidence of DNA fragmentation by TUNEL staining of JNCL neurons, upregulation of Bcl-2 in surviving, nonapoptotic JNCL neurons, and increased ceramide levels in JNCL brain. At a cellular level, overexpression of CLN3 protein results in resistance to apoptosis induced by serum deprivation and chemotherapeutic agents, inhibition of caspase-3 activation, and a modulation of ceramide levels. Blocking CLN3 expression in human post-mitotic neurons results in spontaneous apoptosis, which was prevented by the antiapoptotic drug, flupirtine. Importantly, these antiapoptotic mechanisms also operate in cancer. There is significant over-expression of CLN3 in human and mouse cancer cell lines, and solid human colon cancer. An antisense- CLN3 adeno-virus blocks CLN3 expression in these lines, causes inhibition of cancer cell growth, increased apoptosis with loss of potential across the mitochondrial membrane, and an increase in ceramide levels. Cells derived from JNCL patients are an excellent model for the study of effects of CLN3 on apoptosis and ceramide. Overall hypothesis: the CLN3 protein impacts apoptotic pathways has a negative regulatory role in apoptosis. Specific hypotheses: a) subcellular localization(s) for this membrane protein impacts its role in apoptosis; b) motifs within CLN3 contribute to its regulatory effects on apoptosis; c) CLN3 modulates one of the steps in ceramide synthesis clearance pathways; and d) motifs within CLN3 and or CLN3 subcellular localization(s) may have an effect on ceramide levels. Specific aims include: 1) to establish the impact of CLN3 on apoptotic pathways by analyzing global gene expression; 2) to determine the determinants of CLN3 for regulating apoptosis by defining subcellular localization and CLN3 amino acids and motifs resulting in this regulation; and 3) To determine whether a step in ceramide synthesis/clearance is modulated by CLN3 by scrutinizing ceramide synthesis/clearing enzymes and metabolism in CLN3-deficient cells.