Double transduction with GTP cyclohydrolase I and tyrosine hydroxylase is necessary for spontaneous synthesis of L-DOPA by primary fibroblasts

Abstract

Gene transfer of tyrosine hydroxylase (TH) in animal models of Parkinson's disease (PD), using either genetically modified cells or recombinant virus vectors, has produced partial restoration of behavioral and biochemical deficits. The limited success of this approach may be related to the availability of the cofactor, tetrahydrobiopterin (BH4), because neither the dopamine-depleted striatum nor the cells used for gene transfer possess a sufficient amount of BH4 to support TH activity. To determine the role of BH4 in gene therapy, fibroblast cells transduced with the gene for TH were additionally modified with the gene for GTP cyclohydrolase l; an enzyme critical for BH4 synthesis. In contrast to cells transduced with only TH, doubly transduced fibroblasts spontaneously produced both BH4 and 3, 4-dihydroxy-L-phenylalanine. To examine further the importance of GTP cyclohydrolase I in gene therapy for PD, in vivo micro-dialysis was used to assess the biochemical changes in the dopamine-denervated striatum containing grafts of genetically modified fibroblasts. Only denervated striata grafted with fibro-blasts possessing both TH and GTP cyclohydrolase I genes displayed biochemical restoration. However, no significant differences from controls were observed in apomorphine-induced rotation. This is partly attributable to a limited duration of gene expression in vivo. These differences between fibroblasts transduced with TH alone and those additionally modified with the GTP cyclohydrolase I gene indicate that BH4 is critical for biochemical restoration in a rat model of PD and that GTP cyclohydrolase I is sufficient for production of BH4.

Fig. 1.

Schematic of the retroviral vector plasmids.A, pLNChTH contains the cDNA for human tyrosine hydroxylase type 2 (hTH-2) under the control of a cytomegalovirus promoter (CMV) and the selectable marker aminoglycoside phosphotransferase (neo) under the control of the retroviral long terminal repeat (LTR). B, pΔgHCGC contains the rat GTP cyclohydrolase I (GTPCH1) cDNA and the selectable marker hygromycin-B-phosphotransferase (HPH) within the same backbone as pLNChTH.

Fig. 2.

Immunostaining with polyclonal antibody against TH. A, Unmodified primary fibroblast cells (PF).B, Fibroblasts transduced with LNChTH (PFTH). C, Fibroblasts doubly transduced with LNChTH and ΔgHCGC (PFTHGC). Scale bar, 100 μm.

Fig. 3.

The effect of exogenous BH₄in genetically modified fibroblast cells. A, Uptake of BH₄ and production ofl-DOPA by primary fibroblasts transduced with hTH2 cDNA only (PFTH) incubated with exogenous BH₄. B, Production ofl-DOPA by primary fibroblasts doubly transduced with hTH2 and rat GTP cyclohydrolase I cDNAs (PFTHGC). Confluent cells were incubated for 4 hr at 37°C in DMEM with 10% fetal calf serum with various concentration of cofactor (0–200 μm). Data represent mean ± SEM (n = 3).

Fig. 4.

The effect of 2,4-diamino-6-hydroxy-pyrimidine (DAHP) on BH₄ production (A) and on l-DOPA production (B) in fibroblast cells cotransduced with TH and GTP cyclohydrolase I (PFTHGC). The cells were incubated for 1 hr at 37°C for the measurement. Data represent mean ± SEM (n = 3).

Fig. 5.

Apomorphine-induced contralateral rotation in unilateral 6-OHDA-lesioned rats with genetically modified grafts. The PF group had grafts of unmodified fibroblasts, PFTH had grafts of cells with the TH gene only, and PFTHGC had graft of cells with both TH and GTP cyclohydrolase I genes. Data represent mean ± SEM (n = 7).

Fig. 6.

Histology of genetically modified fibroblast grafts and schematic of graft and microdialysis probe locations.A, Nissl-stained section showing the location of a representative graft expressing both TH and GTP cyclohydrolase I (PFTHGC) within the striatum. Scale bar, 200 μm. B, Schematic sections showing the relative positions of the grafts (hatched) and microdialysis probe (solid) between the two grafts.

Fig. 7.

Microdialysis in striatum of freely moving rats with genetically modified fibroblast grafts.l-DOPA was measured by HPLC in the dialysates at 15 min intervals. Dialysates were monitored for 1 hr before and 4 hr after NSD 1015 (100 mg/kg, i.p.). Data represent mean ± SEM (n = 6) concentration of l-DOPA in 20 μl dialysate samples.

Fig. 8.

Histology and TH immunohistochemistry (B–F) of primary fibroblast grafts at 4 d.A, High-power (400×) view of Nissl staining showing the fibroblast morphology of PFTHGC cells shown in Figure 5. B, Fibroblast grafts modified with TH alone (PFTH). C, Fibroblasts doubly transduced with TH and GTP cyclohydrolase I (PFTHGC). B and C were taken at 100× magnification; scale bar, 100 μm. D, Unmodified fibroblast grafts at 200× magnification. Nomarski optics were used to demonstrate the morphology because the TH immunostaining was negative in this control PF graft. Scale bar, 50 μm. E, F, High-power (400×) views of the grafts in B andC. Scale bar, 50 μm.