New approach for cholesterol and sphingolipid reduction in Niemann-Pick C1 disease

尼曼-匹克C型（NPC）是由NPC 1（95%）和NPC 2（5%）突变引起的致命性脂质储存病。 基因. NPC的特点是胆固醇和鞘脂蓄积，复杂进行性 神经退行性变，寿命不到20年。NPC 1/2在晚期内体/溶酶体中的功能 (LE/L），可溶性NPC 2结合未酯化的胆固醇并将其转移到整合膜NPC 1， 便于其移出LE/L。最近发现HDAC抑制可以表观遗传地 将低活性NPC 1 mut蛋白的表达增加至能够纠正胆固醇缺陷的水平。 然而，HDAC抑制剂目前正在尝试用于治疗NPC不进入中枢神经系统，不治疗 NPC的神经系统后遗症。然而，我发表的结果表明，FTY 720/芬戈莫德，用于 治疗多发性硬化症，也是一种HDAC抑制剂，增加NPC 1/2的表达，并纠正 NPC 1 mut成纤维细胞胆固醇和鞘脂储存缺陷。此外，我在老鼠身上的初步数据 显示FTY 720确实在小脑中积累，增加了脑和肝中的NPC 1/2表达， 降低胆固醇水平因此，在我的第一个目标中，我将把这种方法扩展到体内小鼠模型， 更准确地模拟人类疾病的NPC，监测大脑和肝脏NPC 1/2表达， 胆固醇和鞘脂稳态以及疾病进展。虽然鞘脂的积累 虽然这些基因与NPC相关神经缺陷的发展有关，但其机制仍不清楚。 最近发表的工作和我的初步数据支持鞘氨醇激酶1缺乏的前提， （SphK 1），其将鞘脂代谢物鞘氨醇转化为生物活性介质鞘氨醇-1-。 磷酸盐可能在鼻咽癌的发病机制中起重要作用。因此，我的目标2a侧重于SphK 1的作用 通过详细检查其表达、定位 和调控。此外，通过采用一种新的SphK 1激活剂，我们最近 发现，和CRISPR CAS9介导的SphK 1表达和活性的调节，我将研究是否 增加鞘脂催化剂抑制NPC细胞表型。最后，最有疗效的 方法的重点是减少胆固醇的积累，但我们的初步数据表明，胆固醇和 鞘脂代谢途径是相互依赖的，SphK 1活性降低导致两者的积累 鞘脂和胆固醇。因此，我将研究针对胆固醇和 在NPC小鼠模型中通过新的“双重脂质”减少策略的鞘脂积累。我预计 我的研究结果将验证FTY 720/Fingolimod的使用，以有效地恢复正常的NPC 1/2活性水平， 回答了长期以来关于NPC中鞘脂蓄积机制的问题， 针对NPC疾病中两种致病性脂质的新治疗策略。

Niemann-Pick Type C (NPC) is fatal lipid storage disease caused by mutations in NPC1 (95%) and NPC2 (5%) genes. NPC is characterized by cholesterol and sphingolipid accumulation, complex progressive neurodegeneration, and a lifespan of less than 20 years. NPC1/2 function in late endosomes/lysosomes (LE/L), with soluble NPC2 binding unesterified cholesterol and transferring it to integral membrane NPC1 that facilitates its movement out of the LE/L. Recently it was discovered that HDAC inhibition can epigenetically increase expression of the low activity NPC1mut protein to levels that can correct the cholesterol defect. However, HDAC inhibitors now being tried for treatment NPC do not get into the CNS and do not treat the neurological sequelea of NPC. My published results, however, suggest that FTY720/Fingolimod, used for treatment of multiple sclerosis, is also a HDAC inhibitor that increases expression of NPC1/2 and corrects cholesterol and sphingolipid storage defects in NPC1mut fibroblasts. Furthermore, my preliminary data in mice show that FTY720 does accumulate in the cerebellum, increases NPC1/2 expression in brain as well as liver, and reduces cholesterol levels. Hence, in my first Aim, I will extend this approach to an in vivo mouse model of NPC that more accurately mimics the human disorder, monitoring brain and liver NPC1/2 expression, cholesterol and sphingolipid homeostasis, and disease progression. While accumulation of sphingolipids have been implicated in development of NPC-associated neurological defects, the mechanisms remain unknown. Recent published work and my preliminary data support the premise that a deficiency of sphingosine kinase 1 (SphK1), which converts the sphingolipid metabolite sphingosine to the bioactive mediator sphingosine-1- phosphate, may be important in pathogenesis of NPC. Consequently, my Aim 2a focuses on the role of SphK1 in sphingolipid accumulation on a cellular level through a detailed examination of its expression, localization, and regulation in NPC mutant fibroblasts. Furthermore, by employing a novel SphK1 activator we recently discovered, and CRISPR CAS9 mediated modulation of SphK1 expression and activity, I will examine whether increasing sphingolipid catabolism suppresses the NPC cellular phenotype. Finally, most therapeutic approaches focus on reducing cholesterol accumulation, but our preliminary data suggest that cholesterol and sphingolipid metabolic pathways are interdependent, decreased SphK1 activity leads to accumulation of both sphingolipids and cholesterol. Therefore, I will examine the benefits of targeting both cholesterol and sphingolipid accumulation through a novel “dual lipid” reduction strategy in a mouse model of NPC. I expect that my results will validate the use of FTY720/Fingolimod to effectively restore normal NPC1/2 activity levels, answer longstanding questions about the mechanism of sphingolipid accumulation in NPC, and pave the way for a new therapeutic strategy targeting both pathogenic classes of lipids in NPC disease.