CLN3: Modulation of Apoptosis and Ceramide Levels

描述（由申请人提供）：长期目标是基于CLN3对细胞凋亡和神经酰胺生成影响的分子、亚细胞和生化决定因素的特点，开发合理的治疗幼年型巴滕病和癌症的方法。巴顿病或神经性Ceroid脂褐病是一组以进行性认知和运动障碍、失明、顽固性癫痫、加速神经元和光感受器丧失为特征的致命性疾病。多种证据表明，CLN3缺陷导致JNCL导致凋亡神经元死亡。这些是：EM显示的核染色质凝聚和TUNEL染色显示的JNCL神经元DNA断裂的证据，存活的、非凋亡的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，CLN3蛋白的过度表达导致对血清剥夺和化疗药物诱导的细胞凋亡的抵抗，抑制caspase-3的激活，以及神经酰胺水平的调节。阻断CLN3在人有丝分裂后神经元中的表达可导致细胞自发凋亡，而抗凋亡药物氟吡汀可阻止细胞自发凋亡。重要的是，这些抗凋亡机制也在癌症中起作用。CLN3在人和小鼠癌细胞系以及实体人结肠癌中均有显著过表达。一种反义的CLN3腺病毒阻断这些细胞系中CLN3的表达，导致癌细胞生长受到抑制，细胞凋亡增加，线粒体膜电位丧失，神经酰胺水平增加。来自JNCL患者的细胞是研究CLN3对细胞凋亡和神经酰胺影响的一个很好的模型。

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.