A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice.
Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinson's Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.
Larsen
Kristin E
KE
Columbia University, Department of Neurology, New York, NY 10032, and Cecil B. Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Charlestown 02129, USA.
Benn
Susanna C
SC
Ay
Ilknur
I
Chian
Ru-Ju
RJ
Celia
Samuel A
SA
Remington
Mary P
MP
Bejarano
Michelle
M
Liu
Meiqin
M
Ross
Joshua
J
Carmillo
Paul
P
Sah
Dinah
D
Phillips
Kester A
KA
Sulzer
David
D
Pepinsky
R Blake
RB
Fishman
Paul S
PS
Brown
Robert H
RH
Francis
Jonathan W
JW
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D019771:Axotomy / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D009046:Motor Neurons / Q000187:drug effects*; D009447:Neuroblastoma; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D013116:Spinal Cord / Q000166:cytology*; D013744:Tetanus Toxin / Q000737:chemistry / Q000494:pharmacology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D051100:Glial Cell Line-Derived Neurotrophic Factor; D018696:Neuroprotective Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine