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

Niemann-Pick C型(NPC)是由NPC1(95%)和NPC2(5%)突变引起的致死性脂质沉积病 基因。鼻咽癌的特点是胆固醇和鞘脂蓄积，复杂进行性 神经退化，寿命不到20年。NPC1/2在晚期内小体/溶酶体中的作用 (LE/L)，与可溶性NPC2结合未酯化胆固醇并将其转移到完整的膜NPC1上，NPC1 促进其走出LE/L。最近发现，HDAC抑制可以在表观遗传上 将低活性NPC1mut蛋白的表达提高到可以纠正胆固醇缺陷的水平。 然而，目前正在尝试用于治疗鼻咽癌的HDAC抑制剂不能进入中枢系统，也不能治疗鼻咽癌 鼻咽癌的神经后遗症。然而，我发表的结果表明，FTY720/Fingolimod用于 治疗多发性硬化症，也是一种HDAC抑制剂，增加NPC1/2的表达并纠正 NPC1mut成纤维细胞中胆固醇和鞘磷脂储存缺陷。此外，我在小鼠身上的初步数据 表明FTY720确实在小脑中蓄积，增加NPC1/2在脑和肝脏中的表达， 并降低胆固醇水平。因此，在我的第一个目标中，我将把这种方法扩展到活体小鼠模型 NPC更准确地模拟了人类的疾病，监测大脑和肝脏NPC1/2的表达， 胆固醇和鞘脂的动态平衡，以及疾病的进展。而神经鞘脂类的积累 虽然与鼻咽癌相关神经缺陷的发生有关，但其机制尚不清楚。 最近发表的研究和我的初步数据支持这样一个假设，即鞘氨醇激酶1的缺陷 (SphK1)，它将鞘磷脂代谢物鞘氨醇转化为生物活性介体鞘氨醇-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集中在SphK1的作用上 通过详细研究其表达，定位， 以及对鼻咽癌突变成纤维细胞的调控。此外，通过使用一种新的SphK1激活剂，我们最近 发现，并且CRISPR Cas9介导了SphK1的表达和活性的调节，我将检查是否 鞘磷脂分解代谢增加会抑制鼻咽癌细胞的表型。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 鞘磷脂的代谢途径是相互依赖的，SphK1活性降低会导致两者的积累 鞘脂和胆固醇。因此，我将研究同时针对胆固醇和 在鼻咽癌小鼠模型中，神经鞘脂脂通过一种新的“双脂”减少策略积累。我希望 我的结果将验证使用FTY720/Fingolimod有效地恢复正常的NPC1/2活动水平， 回答长期以来关于神经鞘脂脂在鼻咽癌中积累机制的问题，并为 寻找一种针对鼻咽癌疾病两种病原性血脂的新治疗策略。

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.