Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2driven and idiopathic Parkinson’s disease

Activating LRRK2 mutations cause Parkinson’s disease. Previously, we showed that cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2 mutant mice. Single nucleus RNA sequencing shows that cilia loss in cholinergic interneurons correlates with higher LRRK2 expression and decreased glial derived neurotrophic factor transcription. Nevertheless, much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia in mice and humans. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. Human striatal tissue from LRRK2 pathway mutation carriers and idiopathic Parkinson’s disease show similar cilia loss in cholinergic interneurons and astrocytes and overall loss of such neurons. These data strongly suggest that loss of cilia in specific striatal cell types de..., Collection/generation of RNA sequencing data: Mice were PBS perfused and brains were removed by surgical dissection. Dorsal striatal tissue was isolated in ice cold PBS using a light dissection micrscope. Striatal tissues were placed in 1.5 mL eppendorf tubes and quickly frozen in a 50:50 slurry containing 200 proof ethanol and dry ice.  1 mL of cold homogenization buffer was added and the samples were transferred to dounce homogenizers. The samples were dounced 10 times with the loose pestle and 10 times with the tight pestle on ice.  Homogenates were transferred to a conical tube through 30um strainers to remove large debris. Tubes were spun for 10 minutes at 4 C, 900xg. Supernatant was removed with only ~50 µL of solution remaining.  Samples were resuspended in 450 µL of blocking buffer. 10 µL of each sample was combined with 10 µL 0.4% Trypan Blue to assess nuclei quality and sample yield using a hemocytometer. Concentrations were adjusted to 1000 nuclei/µL. The Stanford Genomics ce..., , # Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2driven and idiopathic Parkinson’s disease [https://doi.org/10.5061/dryad.pk0p2ngvp](https://doi.org/10.5061/dryad.pk0p2ngvp) **General Information** Title of Dataset: Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2driven and idiopathic Parkinson’s disease Author/Principal Investigator Information Name: Suzanne Pfeffer ORCID: 0000-0002-6462-984X Institution: Stanford University Address: Beckman Center Room B413 279 Campus Drive Stanford, California 94305-5307 Email: [[e-mail]](mailto:[e-mail]) Author/Associate or Co-investigator Information Name: Shahzad Khan ORCID:0000-0003-3962-0226 Present Institution: University of North Carolina at Chapel Hill Address: Beckman Center Room B453 279 Campus Drive Stanford, California 94305-5307 Email: [[e-mail]](mailto:[e-mail]) Date of data collection: 2022-01-12 Geographic location of data co...激活突变的富亮氨酸重复激酶2（LRRK2，Leucine-rich repeat kinase 2）可引发帕金森病。此前本团队已证实，LRRK2突变小鼠的背侧纹状体中，胆碱能中间神经元与星形胶质细胞会丢失初级纤毛（primary cilia），而中型多棘神经元则无此现象。单核RNA测序结果显示，胆碱能中间神经元的纤毛丢失与LRRK2高表达以及胶质细胞源性神经营养因子（GDNF，glial cell line-derived neurotrophic factor）转录水平降低相关。然而，在小鼠与人类中均观察到，纤毛表型正常的中型多棘神经元内LRRK2表达量显著更高。与纹状体多巴胺能神经突起密度降低相伴随的是，LRRK2 G2019S突变神经元中，自闭症相关的接触蛋白5（CNTN5，Contactin 5）黏附蛋白表达量升高；胶质细胞则出现铁蛋白重链的显著丢失。来自LRRK2通路突变携带者以及特发性帕金森病患者的人类纹状体组织，同样表现出胆碱能中间神经元与星形胶质细胞的纤毛丢失，以及这类细胞的整体数量减少。上述数据强烈提示，特定纹状体细胞类型的纤毛丢失会…… RNA测序数据的收集与制备流程如下：实验小鼠经磷酸盐缓冲液（PBS，Phosphate Buffered Saline）灌流后，通过手术取出脑组织。在冰浴的PBS中使用光学解剖显微镜分离背侧纹状体组织，将其置于1.5 mL Eppendorf离心管中，随后放入由200度无水乙醇与干冰按1:1配比制成的混合浴中快速冷冻。加入1 mL预冷的匀浆缓冲液，将样品转移至杜恩匀浆器（dounce homogenizer）中，在冰上先用松散研杵匀浆10次，再用紧密研杵匀浆10次。匀浆液通过30 μm细胞筛过滤至锥形管中以去除大体积碎屑，4℃、900×g离心10分钟。弃去上清液，仅保留约50 μL溶液。用450 μL封闭液重悬沉淀，取10 μL样品与10 μL 0.4%台盼蓝（Trypan Blue）混合，通过血细胞计数板评估细胞核质量与样品得率。将样品浓度调整至1000个细胞核/μL。斯坦福基因组学中心…… # 致病性LRRK2驱动型与特发性帕金森病中的初级纤毛丢失及多巴胺能神经保护 [https://doi.org/10.5061/dryad.pk0p2ngvp](https://doi.org/10.5061/dryad.pk0p2ngvp) **通用信息** 数据集名称：致病性LRRK2驱动型与特发性帕金森病中的初级纤毛丢失及多巴胺能神经保护 作者/首席研究员信息 姓名：Suzanne Pfeffer ORCID：0000-0002-6462-984X 所属机构：斯坦福大学 通讯地址：贝克曼中心B413室，校园大道279号，斯坦福，加利福尼亚州94305-5307 电子邮箱：[e-mail] 合作/联合研究员信息 姓名：Shahzad Khan ORCID：0000-0003-3962-0226 现所属机构：北卡罗来纳大学教堂山分校 原通讯地址：贝克曼中心B453室，校园大道279号，斯坦福，加利福尼亚州94305-5307 电子邮箱：[e-mail] 数据收集日期：2022-01-12 数据采集地理区域……