Efficient in vivo protection of nigral dopaminergic neurons by lentiviral gene transfer of a modified Neurturin construct.
Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member Neurturin (NTN) have demonstrated neuroprotective effects on dopaminergic (DA) neurons, which are selectively lost during Parkinson's disease (PD). However, unlike GDNF, NTN has not previously been applied in PD models using an in vivo gene therapy approach. Difficulties with lentiviral gene delivery of wild type (wt) NTN led us to examine the role of the pre-pro-sequence, and to evaluate different NTN constructs in order to optimize gene therapy with NTN. Results from transfected cultured cells showed that wt NTN was poorly processed, and secreted as a pro-form. A similarly poor processing was found with a chimeric protein consisting of the pre-pro-part from GDNF and mature NTN. Moreover, we found that the biological activity of pro-NTN differs from mature NTN, as pro-NTN did not form a signaling complex with the tyrosine kinase receptor Ret and GFRalpha2 or GFRalpha1. Deletion of the pro-region resulted in significantly higher secretion of active NTN, which was further increased when substituting the wt NTN signal peptide with the immunoglobulin heavy-chain signal peptide (IgSP). The enhanced secretion of active mature NTN using the IgSP-NTN construct was reproduced in vivo in lentiviral-transduced rat striatal cells and, unlike wt NTN, enabled efficient neuroprotection of lesioned nigral DA neurons, similar to GDNF. An in vivo gene therapy approach with a modified NTN construct is therefore a possible treatment option for Parkinson's disease that should be further explored.
Fjord-Larsen
Lone
L
NsGene A/S, Baltorpvej 154, 2750 Ballerup, Denmark.
Johansen
Jens Leander
JL
Kusk
Philip
P
Tornøe
Jens
J
Grønborg
Mette
M
Rosenblad
Carl
C
Wahlberg
Lars U
LU
D000661:Amphetamine / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002452:Cell Count / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D020916:Conotoxins; D004298:Dopamine / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000502:physiology; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016086:Lentivirus / Q000502:physiology; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000627:therapeutic use*; D009474:Neurons / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000627:therapeutic use*; D051101:Neurturin; D016627:Oxidopamine; D010302:Parkinson Disease, Secondary / Q000139:chemically induced / Q000517:prevention & control*; D010857:Pigment Epithelium of Eye / Q000166:cytology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D013378:Substantia Nigra / Q000378:metabolism / Q000473:pathology*; D014162:Transfection / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
C483491:AnIB protein, Conus anemone; D020916:Conotoxins; D018765:Dopamine Uptake Inhibitors; C494987:GFRA1 protein, human; C494992:Gfra2 protein, rat; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; C495053:NRTN protein, human; D009414:Nerve Growth Factors; D018696:Neuroprotective Agents; D051101:Neurturin; C495057:Nrtn protein, rat; D011518:Proto-Oncogene Proteins; D016627:Oxidopamine; D000661:Amphetamine; D014446:Tyrosine 3-Monooxygenase; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C495001:Ret protein, rat; D004298:Dopamine