Yeast-based methods of identifying nucleic acids and compounds for treating neurodegenerative diseases
The disclosure encompasses methods for the screening of small molecules or nucleic acids that may reverse the inhibition of growth of the unicellular yeast Schizosaccharomyces pombe by rotenone. The use of a yeast as the screening target allows for the high-throughput screening of small molecule and nucleic acid libraries for candidates that may then be screened in animal models as therapeutic agents for the treatment of neurodegenerative diseases. The plant-derived isoflavonoid, rotenone, while only moderately inhibitory to S. pombe cell growth on complex rich medium, is highly inhibitory to growth on synthetic minimal medium. S. pombe cells carrying a deletion in the gene pmk1 are hypersensitive to rotenone. S. pombe , therefore, provides a model for elucidating complex 1-independent targets of rotenone, and can serve as a screening tool for identifying compounds or oligonucleotides potentially able to reverse the effects of rotenone or Parkinson's disease in animal or human subjects.
1. A method of identifying a test compound characterized by reducing rotenone inhibition of proliferation of a strain of a yeast Schizosaccharomyces pombe , comprising:
(a) obtaining a culture of a strain of the yeast, wherein the proliferation of the yeast when in contact with rotenone is reduced compared to the proliferation of the yeast when not in contact with rotenone;
(b) contacting the yeast in contact with rotenone with a test compound; and
(c) determining whether the proliferation of the yeast in contact with rotenone and with the test compound increases compared to the proliferation of the yeast in contact with rotenone but not
with the test compound, thereby indicating whether the test compound reduces inhibition of yeast proliferation by rotenone, wherein the Schizosaccharomyces pombe is maintained under culture conditions comprising a limited nutrient minimal medium and/or comprises a mutation of at least one gene, said mutation conferring sensitivity of the yeast to proliferative inhibition by rotenone, wherein the at least one gene is aks1, pmk1, or psd1, psd2 and psd3.
2. The method of claim 1 , wherein the step of contacting the yeast in contact with rotenone with the test compound comprises delivering to the yeast at least one test heterologous nucleic acid, and wherein the method further comprises the steps:
(i) isolating the yeast receiving the at least one test heterologous nucleic acid and having increased proliferation in the presence of rotenone as compared to the proliferation of an untransformed yeast in contact with rotenone; and
(ii) identifying the at least one heterologous nucleic acid, wherein said heterologous nucleic acid confers resistance to rotenone on the unicellular fungus yeast.
3. The method of claim 1 , wherein the culture medium is rich complete medium and the yeast comprises the mutation of the at least one gene.
4. The method of claim 1 , wherein the at least one gene is aks1 or pmk1.
5. The method of claim 1 , wherein the at least one gene is psd1, psd2 and psd3.
6. The method of claim 1 , wherein the Schizosaccharomyces pombe is maintained under culture conditions comprising a limited nutrient minimal medium.