| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In ALS G1uR2 underediting occurs in a disease specific and region selective manner. G1uR2 modification by RNA editing is a biologically crucial event for neuronal survival, and its deficiency is a direct cause of neuronal death. AMPA receptors containing the unedited form of GluR2Q have high Ca^<2+> permeability in contrast to the low Ca^<2+> conductance of those containing the edited form of G1uR2R. The role of Ca^<2+> -permeable AMPA receptors, particularly G1uR2 Q/R site RNA editing status, in neuronal death has been clearly demonstrated. Therefore, marked reduction of RNA editing in ALS motor neurons may be a direct cause of the selective motor neuron death seen in ALS. GluR2 RNA editing efficacy at Q/R site depends mainly on activity of adenosine deaminases acting on RNA (ADAR)2. Human ADAR2 gene has four alternative splicing isoforms at C terminal (2L-1, 2L-2, 2L-3, 2S). 2L-2 is active form. 2L-3 and 2S are inactive forms. Increase of ADAR2 activity may open the novel strategy fo
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r specific ALS therapy.
Transfection of active form of ADAR2 could be one of the strategies to increase ADAR2 activity. Another method is to find a drug which increases ADAR2 activity.
Over expression of four alternative splicing isoforms of ADAR2 gene
Alternative splicing isoforms at C terminal of ADAR2 gene (2L-2, 2L-3, 2S) were successfully transfected to COS 7 cells with Lipofectamines (Invitrogen). However, transfection efficacy was not sufficient enough to alter ADAR2 activity. We are under constructing adenovirus which contains each alternative splicing isoform.
Establishment of the methods to find a drug which could increase ADAR2 activity
Measurement of RNA editing efficiency: Culture cells were harvested, RNA was extracted and cRNA was obtained. The RT-PCR products derived from edited GluR2 mRNA were digested with BbvI, and two bands (66 and 116 bp) are detected with gel electrophoresis. ADAR2 activity was calculated. SH-SY5Y cells have 33-66% of GluR2 RNA editing at Q/R site. Then we used SH-SY5Y cells to measure RNA editing efficiency of the drugs. Drugs were loaded on SH-SY5Y cells for 24 hours. We found two drugs which increased ADAR2 activity. These drugs have potentials to treat ALS effectively. Less
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