Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson’s disease

Frontiers in Neuroscience

Neuroscience

Sarat Chandra Yenisetti¹  Priyanka Modi¹  Rahul Chaurasia¹  Abhik Das¹  Mohamad Ayajuddin¹  Zevelou Koza²  Limamanen Phom³ 

[1] Neurobiology Laboratory, Department of Zoology, Nagaland University (Central), Lumami, Nagaland, India;Neurobiology Laboratory, Department of Zoology, Nagaland University (Central), Lumami, Nagaland, India;Patkai Christian College (Autonomous), Dimapur, Nagaland, India;Neurobiology Laboratory, Department of Zoology, Nagaland University (Central), Lumami, Nagaland, India;Sao Chang Government College, Tuensang, Nagaland, India;

关键词: dopamine;    neurodegeneration;    fluorescence intensity;    tyrosine hydroxylase;    Drosophila;   

DOI  :  10.3389/fnins.2023.1158858

 received in 2023-02-04, accepted in 2023-05-03,  发布年份 2023

来源: Frontiers

【 摘 要 】

Death of dopaminergic (DAergic) neurons in the substantia nigra pars compacta of the human brain is the characteristic pathological feature of Parkinson’s disease (PD). On exposure to neurotoxicants, Drosophila too exhibits mobility defects and diminished levels of brain dopamine. In the fly model of sporadic PD, our laboratory has demonstrated that there is no loss of DAergic neuronal number, however, a significant reduction in fluorescence intensity (FI) of secondary antibodies that target the primary antibody-anti-tyrosine hydroxylase (TH). Here, we present a sensitive, economical, and repeatable assay to characterize neurodegeneration based on the quantification of FI of the secondary antibody. As the intensity of fluorescence correlates with the amount of TH synthesis, its reduction under PD conditions denotes the depletion in the TH synthesis, suggesting DAergic neuronal dysfunction. Reduction in TH protein synthesis is further confirmed through Bio-Rad Stain-Free Western Blotting. Quantification of brain DA and its metabolites (DOPAC and HVA) using HPLC-ECD further demonstrated the depleted DA level and altered DA metabolism as evident from enhanced DA turnover rate. Together all these PD marker studies suggest that FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using ZEN 2012 SP2, a licensed software from Carl Zeiss, Germany. This method will be of good use to biologists, as it with few modifications, can also be implemented to characterize the extent of degeneration of different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method using fluorescence microscopy will be a feasible option for fund-constrained neurobiology laboratories in developing countries.

【 授权许可】

Unknown   
Copyright © 2023 Ayajuddin, Chaurasia, Das, Modi, Phom, Koza and Yenisetti.