Assay for toxin induced neuronal degeneration and viability in
Provided are in vivo screening methods to detect and identify substances that affect neuronal viability, and/or prevent neurodegeneration, and/or confer neuroprotective effects The screening methods utilize recombinant C. elegans expressing a detectable marker in neuronal sub-groups and the use of neurotoxins specific to specific neuronal cells. Also provided are methods for identifying modulators of neurotransmitter transporters such as the dopamine transporter. Therefore, the invention provides methods for identifying substances that can be used in the prevention and therapy of neurodegenerative diseases.
1. A method of screening for substances that affect neuronal viability comprising:
a) providing a recombinant Caenorhabditis elegans , the genome of which comprises a transgene that encodes a detectable marker and is under the control of a promoter, wherein said detectable marker is expressed in a neuronal cell of said recombinant C. elegans;
b) exposing said C. elegans to a candidate substance; and
c) detecting a change in the expression of the detectable marker relative to the expression of the detectable marker before said exposing;
wherein a change in the expression of the detectable marker corresponds to a change in the viability of the neuron.
2. The method of claim 1 , further comprising detecting the expression of the detectable marker in the neuronal cell in the absence of said candidate substance.
3. The method of claim 1 , wherein said substance is a neurotoxic substance.
4. The method of claim 3 , wherein the neurotoxic substance is 6-hydroxydopamine, 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine, or 5,7, di-hydroxy tryptamine.
5. The method of claim 3 , wherein the neurotoxic substance is 6-hydroxydopamine.
6. The method of claim 3 , wherein the neurotoxic substance is a generator of free radical species.
7. The method of claim 1 , further comprising the step of exposing said C. elegans to a known neurotoxin prior to step b).
8. The method of claim 1 , further comprising the step of exposing said C. elegans to a known neurotoxin after step b).
9. The method of claim 1 , wherein the change in detectable marker expression can be an increase in the detectable marker.
10. The method of claim 1 , wherein the change in detectable marker expression can be a decrease in the detectable marker.
11. The method of claim 1 , wherein the detectable marker is further defined as a marker that can be visually detected.
12. The method of claim 1 , wherein the detectable marker is further defined as a marker that can be spectroscopically detected.
13. The method of claim 12 , wherein the detectable marker is a green fluorescent protein.
14. The method of claim 12 , wherein the detectable marker is a yellow fluorescent protein.
15. The method of claim 12 , wherein the detectable marker is a blue fluorescent protein.
16. The method of claim 12 , wherein the detectable marker is a red fluorescent protein.
17. The method of claim 1 , wherein the detectable marker is β-galactosidase.
18. The method of claim 1 , wherein the detectable marker is an antigenic polypeptide.
19. The method of claim 1 , wherein the neuronal cell comprises a dopaminergic neuron.
20. The method of claim 1 , wherein the neuronal cell comprises a cholinergic neuron.
21. The method of claim 1 , wherein the neuronal cell comprises a GABA-ergic neuron.
22. The method of claim 1 , wherein the neuronal cell comprises a glycinergic neuron.
23. The method of claim 1 , wherein the neuronal cell comprises a serotonergic neuron.
24. The method of claim 1 , wherein the neuronal cell comprises a glutamatergic neuron.
25. The method of claim 1 , wherein the neuronal cell comprises a peptidergic neuron.