Jagtap Soham, Potdar Chandrakanta, Yadav Ravi, Pal Pramod Kumar, Datta Indrani
Department of Biophysics, National Institute of Mental Health and Neurosciences, Institute of National Importance, Bengaluru 560029, Karnataka, India.
Department of Neurology, National Institute of Mental Health and Neurosciences, Institute of National Importance, Bengaluru 560029, Karnataka, India.
ACS Chem Neurosci. 2022 Sep 7;13(17):2632-2645. doi: 10.1021/acschemneuro.2c00297. Epub 2022 Aug 25.
Being a large multidomain protein, LRRK2 has several confirmed pathological mutant variants for PD, and the incidence of these variants shows ethnicity biases. I1371V, a mutation in the GTPase domain, has been reported in East-Asian populations, but there are no studies reported on dopaminergic (DA) neurons differentiated from this variant. The aim here was to assess the yield, function, and α-synuclein pathology of DA neurons differentiated from LRRK2 I1371V iPSCs. FACS analysis of neural progenitors (NPs) showed a comparable immunopositive population of cells for neural and glial progenitor markers nestin and S100β; however, NPs from I1371V iPSCs showed lower clonogenic and proliferative capacities than healthy control NPs as determined by the neurosphere assay and Ki67 expression. Floor plate cells obtained from I1371V NPs primed with FGF8 showed distinctly lower immunopositivity for FOXA2 and CLIC5 than healthy control FPCs and similar DOC2B expression. On SHH addition, a similar mature neuronal population was obtained from both groups; however, the yield of TH-immunopositive cells was significantly lower in I1371V, with lower expression of mature DA neuronal markers En1, Nurr1, and DAT. Vesicular dopamine release and intracellular Ca response with KCl stimulation were lower in I1371V DA neurons, along with a significantly reduced expression of resting vesicle marker VMAT2. A concurrently lower expression of PSD95/Syn-I immunopositive puncta was observed in I1371V differentiated cells. Further, higher phosphorylation of α-synuclein and aggregation of oligomeric α-synuclein in I1371V DA neurons were observed. Our data demonstrated conclusively for the first time that mutations in the I1371V allele of LRRK2 showed developmental deficit from the FPC stage and generated a lower yield/number of TH-immunopositive neurons with impairment in their function and synapse density along with increased α-synuclein pathology.
作为一种大型多结构域蛋白，LRRK2有几种已被证实的帕金森病病理突变变体，这些变体的发病率存在种族差异。I1371V是GTPase结构域中的一种突变，在东亚人群中已有报道，但尚无关于由该变体分化而来的多巴胺能（DA）神经元的研究报道。本研究的目的是评估从LRRK2 I1371V诱导多能干细胞（iPSCs）分化而来的DA神经元的产量、功能和α-突触核蛋白病理学。对神经祖细胞（NPs）的荧光激活细胞分选（FACS）分析显示，神经和胶质祖细胞标志物巢蛋白和S100β的免疫阳性细胞群体相当；然而，通过神经球试验和Ki67表达确定，I1371V iPSCs来源的NPs的克隆形成能力和增殖能力低于健康对照NPs。用FGF8预处理的I1371V NPs获得的底板细胞对FOXA2和CLIC5的免疫阳性明显低于健康对照底板细胞，而DOC2B表达相似。添加音猬因子（SHH）后，两组获得了相似的成熟神经元群体；然而，I1371V组中酪氨酸羟化酶（TH）免疫阳性细胞的产量显著降低，成熟DA神经元标志物En1、Nurr1和多巴胺转运体（DAT）的表达也较低。I1371V DA神经元中，氯化钾刺激下的囊泡多巴胺释放和细胞内钙反应较低，静息囊泡标志物囊泡单胺转运体2（VMAT2）的表达也显著降低。在I1371V分化细胞中同时观察到突触后致密蛋白95/突触素-I（PSD95/Syn-I）免疫阳性斑点的表达较低。此外，在I1371V DA神经元中观察到α-突触核蛋白的磷酸化水平较高，以及寡聚α-突触核蛋白的聚集。我们的数据首次确凿地证明，LRRK2的I1371V等位基因突变从底板细胞阶段就表现出发育缺陷，产生的TH免疫阳性神经元产量/数量较低，其功能和突触密度受损，同时α-突触核蛋白病理学增加。
