6-hydroxydopamine (6-OHDA) Oxidative Stress Assay for Observing Dopaminergic Neuron Loss in Caenorhabditis elegans

Abstract

The nematode Caenorhabditis elegans is a powerful genetic model that can be used to investigate neuronal death. Research using C. elegans has been crucial to characterize cell death programmes that are conserved in mammals. Many neuronal signaling components, such as those mediating dopaminergic neurotransmission, are preserved as well. Dopaminergic neurons are progressively lost in Parkinson’s disease and an important risk factor to develop this disease appears to be oxidative stress, the increased occurrence of highly reactive oxygen species. Oxidative stress-induced dopaminergic neurodegeneration is mimicked in animal models by treatment with 6-hydroxydopamine (6-OHDA), a dopamine analog, which is specifically taken up into dopaminergic neurons. After exposing C. elegans to 6-OHDA, the loss of fluorescently labeled dopaminergic neurons can be easily monitored. An organisms’ sensitivity to oxidative stress is thought to be influenced by basal levels of intrinsic oxidative stress and the ability to counteract oxidative stress and oxidative stress-induced damage. The C. elegans ‘6-OHDA model’ led to the discovery of novel genes that are required to protect dopaminergic neurons and it has helped to determine the effects of conserved cell death and cell engulfment pathways in dopaminergic neurodegeneration. Here, we describe a simple protocol that allows for the easy detection of dopaminergic neuron loss after 6-OHDA treatment in C. elegans.

A functionalized hydroxydopamine quinone links thiol modification to neuronal cell death

“…Most literature regarding 6-OHDA suggests that the generation of reactive oxygen species (ROS) is the driving force of the molecule's toxicity [34,40,43,71,84,85]. In contrast, only few studies addressed protein modification by dopamine quinones [19,20,[44], [45], [46], [47], [48],86].…”

“…One notable oxidation product of dopamine is 6-hydroxydopamine (6-OHDA), which forms in the presence of iron via hydroxylation of dopamine [12,[34], [35], [36], [37], [38]]. Furthermore, 6-OHDA has been widely used in preclinical models to investigate PD in the animal model [[39], [40], [41]]. 6-OHDA undergoes a very similar series of reactions like dopamine, giving rise to 6-OHDA p -quinone, and eventually resulting in neuromelanin as well (Fig.…”

An unbiased, automated platform for scoring dopaminergic neurodegeneration in C. elegans

“…For instance, the high dose rotenone worms showing lower intensity features was a metric that would not have been able to be scored by the human eye. Likewise, while 6-OHDA has been shown to induce dopaminergic deterioration in worms [13,14,16], the studies have been limited by discrete, manual scoring systems. While this method of scoring is adequate for obtaining data like the percent of population experiencing deterioration, it provides less information about the exact degree of damage shown by each individual worm.…”

“…C. elegans is a well-studied model for examining factors that can lead to dopaminergic neurodegeneration [9][10][11][12][13][14]. This small nematode with a rapid life cycle and short life span makes examination of aging and aging-related pathologies such as neurodegeneration simple, and its transparent body makes fluorescence imaging in living animals feasible.…”

“…C. elegans possesses a fully characterized nervous system with eight dopaminergic neurons, of which the four cephalic (CEP) neurons have a particularly well-studied pattern of damage characterized by specific degenerative phenotypes including blebbing and breakage [9,15]. C. elegans demonstrate well conserved aging and metabolic pathways, both critical to the pathology of PD, and the CEP neurons have replicated degenerative effects induced by common PD models, such as rotenone and 6-OHDA treatment [13,15,16].…”