Abul Khair Salema B, Dhanushkodi Nisha R, Ardah Mustafa T, Chen Wenfeng, Yang Yufeng, Haque M Emdadul
Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
Institute of Life Sciences, Fuzhou University, Fuzhou, China.
Front Neurosci. 2018 Feb 16;12:81. doi: 10.3389/fnins.2018.00081. eCollection 2018.
Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. We developed with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using activity monitor (DAM) system. Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.
溶酶体酶葡萄糖脑苷脂酶（GBA）的突变是帕金森病（PD）最常见的遗传风险因素。我们研究了在PD果蝇模型中，GCase活性降低如何影响α-突触核蛋白（α-syn）及其突变体（A30P和A53T）的聚集、神经退行性变、睡眠和运动行为。我们构建了GBA基因敲低（RNAi）（GCase活性降低）且同时表达野生型（WT）或A30P或A53T等突变体α-syn的果蝇。通过蛋白质免疫印迹法和共聚焦显微镜来研究这些果蝇中α-syn的聚集和神经退行性变。我们还使用活动监测器（DAM）系统研究了这些果蝇的睡眠和运动活性。蛋白质免疫印迹分析表明，与未进行GBA RNAi的相应A53T果蝇（对照）相比，GBA RNAi A53Tα-syn果蝇（30日龄）中Triton不溶性突触核蛋白（对应于α-syn聚集体）水平升高，而α-syn的mRNA表达保持不变。对30日龄果蝇全脑染色的共聚焦成像显示，与相应对照相比，在存在GBA RNAi的情况下，表达α-syn WT、A30P和A53T的果蝇中PPL1簇的神经元数量有统计学意义的减少。用针对α-syn聚集体的构象特异性抗体染色显示，与对照A53T果蝇相比，GBA RNAi的A53T果蝇大脑中α-syn聚集体染色的神经元数量增加，从而证实了我们的蛋白质分析结果，即在GBA酶活性降低的情况下，突变体A53T比未进行GBA沉默的对照A53T聚集更多。睡眠分析显示，与A53T对照果蝇和未表达突触核蛋白的GBA RNAi果蝇相比，表达突变体A53T的GBA沉默果蝇的总活动减少。在GCase活性降低的A53T果蝇中，α-syn聚集增加且多巴胺（DA）神经元丢失。这项研究表明，在与PD相关的杂合GBA1突变背景下以及老年时，GCase活性降低均会导致突变体α-syn A53T聚集增加，并加重PD果蝇模型中的表型。
