In vivo L-DOPA production by genetically modified primary rat fibroblast or 9L gliosarcoma cell grafts via coexpression of GTPcyclohydrolase I with tyrosine hydroxylase

Exp Neurol. 1998 Jun;151(2):249-64. doi: 10.1006/exnr.1998.6803.

Abstract

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.

MeSH terms

  • 3T3 Cells
  • Animals
  • Antioxidants / metabolism
  • Antiparkinson Agents / pharmacology
  • Apomorphine / pharmacology
  • Behavior, Animal / drug effects
  • Biopterin / analogs & derivatives
  • Biopterin / metabolism
  • Corpus Striatum / chemistry
  • Corpus Striatum / enzymology
  • Corpus Striatum / pathology
  • Dihydroxyphenylalanine / metabolism
  • Disease Models, Animal
  • Fibroblasts / cytology
  • Fibroblasts / enzymology
  • Fibroblasts / transplantation
  • GTP Cyclohydrolase / metabolism*
  • Gene Expression Regulation, Enzymologic / physiology
  • Genetic Therapy*
  • Gliosarcoma
  • Humans
  • Levodopa / biosynthesis*
  • Male
  • Mice
  • Microdialysis
  • Parkinson Disease, Secondary / metabolism
  • Parkinson Disease, Secondary / surgery
  • Parkinson Disease, Secondary / therapy*
  • Rats
  • Rats, Inbred F344
  • Recombinant Fusion Proteins / physiology
  • Retroviridae / genetics
  • Transformation, Genetic
  • Transgenes / physiology
  • Tumor Cells, Cultured / metabolism
  • Tumor Cells, Cultured / transplantation
  • Tyrosine 3-Monooxygenase / metabolism*

Substances

  • Antioxidants
  • Antiparkinson Agents
  • Recombinant Fusion Proteins
  • Biopterin
  • Levodopa
  • Dihydroxyphenylalanine
  • Tyrosine 3-Monooxygenase
  • GTP Cyclohydrolase
  • sapropterin
  • Apomorphine