Assay for Dopamine Neuron Viability Using C. Elegans
The loss of DA neurons is a major feature of Parkinson's disease and other neuro-generative disorders. Vanderbilt researchers have established an in vivo screen for DA neuron protective agents and genes using the nematode C. elegans. Using green fluorescent protein (GFP) expression in C. elegans DA neurons, researchers have established that the nematode is sensitive to the mammalian neurotoxins that target DA neurons in mammalian models, consistent with an environmentally triggered loss of DA neurons. They also demonstrate that agents that block the nematode DA transporter or genetic ablation of the DA transporters protect these DA neurons. Thus, researchers have established lines and conditions that can allow for the facile screening, in a high throughput format, for agents or genes that may protect DA neurons from exogenous or endogenous neurotoxin-induced cell death. The system should also be useful for identifying novel pathway controlling presynaptic DA neuron function with potential applications to Schizophrenia, ADHD and addiction, where altered DA signaling has been proposed.
Researchers have developed a strain of the nematode C. elegans expressing green fluorescent protein (GFP) selectively in DA neurons in the worm. Using this strain, they have established conditions under which the mammalian DA neuron toxin 6-hydroxydopamine leads to a loss of GFP fluorescence consistent with the death of the DA neurons. This loss can be blocked by agents known from in vitro studies to block the C. elegans DA transporter (CeDAT). Furthermore, using lines genetically ablated for CeDAT, researchers show that 6-OHDA sensitivity is lost in these lines. These tests establish an assay for genetic and pharmacologic modifiers of DA neuron vulnerability using their C. elegans GFP strains tested in the presence of the toxin 6-OHDA.
Since DA neuron loss is a critical feature of Parkinson's disease, as well other neuro-generative disorders, use of the C. elegans system with its powerful genetics and ease of culture should afford a powerful system for in vivo screening for DA neuron protective agents and genes. The DA transporter is a major target for psychoactive stimulants, including cocaine and methylphenidate. These technologies will also permit a study of genes and drugs that modulate DA transporter function, and thus may also be of use in addition research.
The major impediment is the question of whether the death of C. elegans DA neurons is a good model for the loss of these neurons in humans. The system described is rapid and amenable to high throughput design but will require some facility with the C. elegans model, as well as a willingness to consider model system genetic approaches to identify important pathways in DA neuron degeneration and pharmaceutical development.
Intellectual Property Status
U.S. patent 6,596,512 was issued July 22, 2003
U.S. patent 6,894,205 was issued May 17, 2005
U.S. patent 7,531,713 was issued May 12, 2009!doctype>