RESUMEN
In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue.
Asunto(s)
Diseño de Fármacos , Variación Genética/genética , Neurturina/química , Neurturina/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Anfetamina/farmacología , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Humanos , Macaca fascicularis , Masculino , Modelos Moleculares , Neurturina/genética , Oxidopamina/toxicidad , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/etiología , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Ratas , Ratas Wistar , Conducta Estereotipada/efectos de los fármacos , Simpaticolíticos/toxicidad , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
INTRODUCTION: Although glial cell line-derived neurotrophic factor (GDNF) has a strong clinical potential, little is known of how the posttranslational modifications of GDNF affect its biological activity and therapeutic potential. In mammalian cells GDNF is synthesized as a preproprotein. During secretion GDNF dimerizes, folds with -S-S- bonds, is modified by N-linked glycosylation, and undergoes proteolytic processing. After production in E. coli, unglycosylated GDNF is renaturated in vitro. Nevertheless, GDNF from E. coli was used in Parkinson's disease-related clinical trials. MATERIAL AND METHODS: Constructs encoding variants of human GDNF were generated and expressed in mammalian cells. The proteins were analysed by SDS-PAGE, Western blotting, RET-phosphorylation assays, and N-terminal sequencing. The stability of mammalian GDNF was compared to commercial GDNF produced in E. coli. RESULTS: Posttranslational processing of mammalian GDNF depends on the expression conditions. Two forms of GDNF with different N-termini are formed. GDNF without a prosequence is secreted and biologically active. GDNF is modified by N-linked glycosylation at one (Asn(49)) out of two consensus sites. N-linked glycosylation aids proteolytic processing of GDNF. Both glycosylated and unglycosylated GDNF from mammalian cells are more stable than GDNF from E. coli. DISCUSSION: Posttranslational modifications of GDNF influence its stability, which may be critical for its clinical use.