The Effect of GTP Cyclohydrolase I on the Activity and Expression of Tyrosine Hydroxylase and Cell Growth

ABSTRACT

BACKGROUND: Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by resting tremor, rigidity, and bradykinesia. L-3,4-dihydroxyphenylalanine (L-dopa) has been used for over last 3 decades to treat this disorder, however, its usage is limited due to the reducing effectiveness on time and severe side effects. The best strategy for treating this disorder without serious side effects would be to keep a constant level of dopamine in the brain. This could be achieved by gene or cell therapy using gene(S) involved in dopamine biosynthesis or cells from other individual. For Parkinson's gene therapy, however, there still are controversies on which gene In what combination will yield the best result. In this report, we propose a biochemical background for using GTP cyc]ohydrolase I (GTPCH I) in addition to TH for higher and/or more stable expression of TH. METHODS: TH and GTPCH I cDNA were subcloned into retroviral vectos and resulting recombinant retrovirus packaged in BOSC 23 cells were used to infect NIH-3T3. Confirming successful infections by westers blot analysls, the new cell lines were used to examine steady state TH expression level and TH activity. Furthermore, the effect of ectopic expression of BH4 to the proliferation of these cells were studied. RESULTS: NIH-3T3 cells expressing both TH and GTPCH I showed approximately 10 fold higher expression of TH protein than the cells expressing TH alone. The activity of KNTH2GC6 was approximately 4-6 fold higher than that of striatal tissue and 60 fold higher than KNTH2. Furthermore, growth rate of KNTH2GC6 was strikingly reduced by inhibiting the biosynthesis of BH4. CONCLUSIONS: We showed that the use of GTPCH I in addition to TH not only increased the stability and/or expression of TH protein but also the activity of the enzyme. These improved characteristics of TH protein are very likely due to the expression of BH4 and should be very seriously considered for Parkinson's gene therapy.