Rahul Chaurasia, M. Ayajuddin, Girish Ratnaparkhi, Shashidhara S. Lingadahalli, S. Yenisetti
{"title":"用简单的免疫荧光方法鉴定帕金森病果蝇模型全脑的神经变性和酪氨酸羟化酶减少情况","authors":"Rahul Chaurasia, M. Ayajuddin, Girish Ratnaparkhi, Shashidhara S. Lingadahalli, S. Yenisetti","doi":"10.21769/bioprotoc.4937","DOIUrl":null,"url":null,"abstract":"Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson’s Disease\",\"authors\":\"Rahul Chaurasia, M. Ayajuddin, Girish Ratnaparkhi, Shashidhara S. Lingadahalli, S. Yenisetti\",\"doi\":\"10.21769/bioprotoc.4937\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.\",\"PeriodicalId\":8938,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-02-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21769/bioprotoc.4937\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/bioprotoc.4937","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
摘要
多巴胺能(DAergic)神经变性是帕金森病(PD)的病理特征。果蝇在暴露于模拟帕金森病的神经毒物时也会表现出移动性缺陷和脑多巴胺水平降低。我们的实验室在散发性帕金森病的果蝇模型中证实,多巴胺能神经元的数量并没有减少;相反,酪氨酸羟化酶(TH)的荧光强度(FI)却显著降低。在此,我们提出了一种基于二抗(ab)荧光强度定量的灵敏检测方法。由于 FI 与 TH 的合成量成正比,因此在帕金森病条件下,FI 的降低表示 TH 合成的减少,从而提示 DAergic 神经元功能障碍。因此,FI 定量是了解 DAergic 神经变性早期阶段的一种精细而灵敏的方法。FI定量使用ZEN 2012 SP2单用户软件进行;必须获得许可证才能使用成像系统交互式控制图像采集、图像处理和分析。这种方法对生物学家很有帮助,因为它只需稍加改动就可用于描述不同类型细胞的变性程度以及变性程度对药物反应的变化。与昂贵而繁琐的共聚焦显微镜不同,本方法对于资金有限的神经生物学实验室来说是一种经济实惠的选择。主要特点 - 即使在神经元细胞体没有缺失的情况下,也能描述初生 DA 能和其他儿茶酚胺能神经变性的特征。- 对于发展中国家资金有限的神经生物学实验室来说,利用这种方法研究不同类型的细胞及其对药物/保健品的反应是一个不错的选择。
A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson’s Disease
Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.