Utilizing microRNA in cerebrospinal fluid to evaluate the effects of tyrosine kinase inhibition via Nilotinib on gene regulation in individuals with Parkinson's disease or Alzheimer's disease

项目摘要/摘要： 据估计，美国40%和1%的人患有阿尔茨海默病(AD)和帕金森氏病(PD) 分别表示85岁以上的人群，使他们成为最常见的两种神经退行性变 疾病[1-6]。确定一个可靠的生物标志物来应对AD和PD的干预将有很大的意义 协助开发成功的疾病修正疗法。我们强有力的临床前证据 证明尼洛替尼是一种多激酶酪氨酸激酶抑制剂，可增加神经毒素的自噬清除 蛋白质和改善其他神经退行性病变。在人类身上，我们已经证明了尼洛替尼 穿透大脑，抑制脑脊液(CSF)Abelson酪氨酸激酶活性，减少脑脊液总量和 磷酸化tau，稳定脑脊液α-突触核蛋白水平，并增加脑脊液高香草酸，结束 多巴胺代谢的副产品。最近，microRNAs(MiRNAs)已经成为有吸引力的候选分子 神经退行性疾病中的生物标志物，因为它们在包括脑脊液和血浆在内的生物液中的稳定性。 MiRNA是一种小的非编码RNA，其作用是使基因靶标的表达在翻译后沉默。飞行员 研究发现PD和路易体痴呆(DBL)患者脑脊液中47miRNA的差异 与基线相比，尼洛替尼治疗6个月后表达。目标分析表明，许多 下调的miRNA靶基因与自噬和泛素化有关，这与许多 我们的临床前工作证明了自噬和泛素化导致蛋白分解在帕金森病和帕金森病中受到抑制 尼洛替尼治疗后失禁。在此，我建议通过尼洛替尼进一步评估TKI对基因的影响 NCT02954978收治的50例患者脑脊液miRNA测序的研究 尼洛替尼对帕金森病的影响和37例患者纳入NCT02954978以评估尼洛替尼对AD的影响。下一步- 世代测序将确定在尼洛替尼治疗的个体中差异表达的miRNAs 与基线相比。下一代测序将使用TaqMan实时聚合酶链式反应分析进行验证。基因 将进行靶向分析，以揭示哪些基因和途径与表达的miRNAs相关。 帕金森病进展的替代蛋白生物标记物将在脑脊液中使用酶联法进行测量 免疫吸附试验(ELISA)。总而言之，这些收集到的丰富数据将使我能够将miRNA 更改临床终点测量，包括蛋白质生物标记物和临床评定量表分数以进行验证 MiRNAs可作为帕金森病和阿尔茨海默病患者尼洛替尼对TKI反应的潜在可靠生物标志物。

Project summary/Abstract: Alzheimer’s disease (AD) and Parkinson’s disease (PD), are estimated to affect 40% and 1% of the United Stated population over 85 years of age, respectively, making them the two most common neurodegenerative diseases [1-6]. The identification of a reliable biomarker for response to interventions in AD and PD will greatly assist in the development of successful disease modifying therapies. Our strong preclinical evidence demonstrates that Nilotinib, a multi-kinase tyrosine kinase inhibitor, increases autophagic clearance of neurotoxic proteins and ameliorates other neurodegenerative pathologies. In humans we have shown that Nilotinib penetrates the brain, inhibits cerebrospinal fluid (CSF) Abelson tyrosine kinase activity, reduces CSF total and phosphorylated tau, stabilizes CSF alpha-synuclein levels, and increases CSF Homovanillic acid, an end byproduct of Dopamine metabolism. MicroRNAs (miRNAs) have recently emerged as attractive candidates as biomarkers in neurodegenerative diseases due to their stability in biological fluids including CSF and plasma. MiRNA are small non-coding RNA whose role is to post-translationally silence expression of gene targets. A pilot study identified 47 miRNA in the CSF of PD and Dementia with Lewy Bodies (DBL) patients differentially expressed after 6 months Nilotinib treatment compared to baseline. Target analysis indicates that many of the down regulated miRNA target genes are associated with autophagy and ubiquitination, agreeing with much of our preclinical work that demonstrates autophagy and ubiquitination leading to proteolysis is inhibited in PD and disinhibited after Nilotinib treatment. Here, I propose to further evaluate the effects of TKI via Nilotinib on gene regulation by sequencing the miRNA in CSF collected from 50 patients enrolled in NCT02954978 to evaluate Nilotinib’s impact in PD and 37 patients enrolled in NCT02954978 to evaluate Nilotinib’s impact in AD. Next- generation sequencing will identify miRNAs that were differentially expressed in Nilotinib treated individuals compared to baseline. Next-generation sequencing will be validated using TaqMan real-time PCR assays. Gene target analysis will be performed to reveal what genes and pathways are associated with the expressed miRNAs. Surrogate protein biomarkers for PD progression will be measured in the CSF using enzyme-linked immunosorbent assays (ELISAs). Together, this wealth of collected data will enable me to correlate miRNA changes to clinical endpoint measures including protein biomarkers and clinical rating scale scores to validate miRNAs as potential reliable biomarkers for the response to TKI via Nilotinib in PD and AD.