Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice.
To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 +/- 8.5%; lumbar cord = 62 +/- 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 +/- 3.9%; lumbar cord = 27 +/-4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.
Benn
Susanna C
SC
Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
Ay
Ilknur
I
Bastia
Elena
E
Chian
Ru-Ju
RJ
Celia
Samuel A
SA
Pepinsky
R Blake
RB
Fishman
Paul S
PS
Brown
Robert H
RH
Francis
Jonathan W
JW
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002452:Cell Count / Q000379:methods; D002490:Central Nervous System / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007276:Injections, Intraventricular / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D010751:Phosphopyruvate Hydratase; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000134:cerebrospinal fluid*; D000072105:Superoxide Dismutase-1; D013744:Tetanus Toxin / Q000378:metabolism / Q000494:pharmacology*; D014018:Tissue Distribution / Q000187:drug effects
D010446:Peptide Fragments; D011994:Recombinant Proteins; C000606290:SOD1 protein, human; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D010751:Phosphopyruvate Hydratase