Fusion of the tetanus toxin C fragment binding domain and Bcl-xL for protection of peripheral nerve neurons.
OBJECTIVE
Apoptosis has been shown to play an important role in motor neuron (MN) degeneration in both neurodegenerative disease and peripheral neuropathy. Bcl-xL, an antiapoptotic protein, is down-regulated in these etiologies [corrected] The carboxyl-terminal domain of the tetanus toxin heavy chain (Hc) has high affinity for axon terminal binding and uptake into motor and dorsal root ganglion (DRG) neurons. We report the development of a fusion protein between Hc and Bcl-xL to enhance uptake of Bcl-xL by MNs as a strategy for inhibiting peripheral neuronal apoptosis.


METHODS
The genes for Hc, Bcl-xL, and green fluorescent protein were cloned into an Escherichia coli expression system in 2 different arrangements. Fusion proteins were purified through chromatography. Cultured E15 rat spinal cord MNs and DRG cells were used to demonstrate neuron-specific uptake and retrograde transport of the fusion proteins mediated by Hc. Finally, glutamate-induced apoptosis was used as an in vitro model to measure the antiapoptotic effects of the fusion proteins.


RESULTS
Bcl-xL fusion proteins were found to bind specifically and undergo uptake into cultured rat spinal MNs. The fusion proteins were also taken up by DRG axonal terminals and transported back to the cell bodies in Campenot compartmentalized chambers (Tyler Research Corp., Edmonton, Canada). Finally, fusion protein application improved cell survival and decreased apoptosis in glutamate-mediated excitotoxicity of the SH-SY5Y neuronal cells.


CONCLUSIONS
Hc can be applied as a universal carrier for therapeutic cargo delivery specifically to MNs or DRGs. The fusion proteins between Bcl-xL and Hc constructed in this study might bear applications to the treatment of MN disease, neuropathy, or nerve injury through nerve or intramuscular injection.
Carlton
Erin
E
Department of Cell Biology, Cleveland Clinic, Cleveland, Ohio, USA.
Teng
Qingshan
Q
Federici
Thais
T
Yang
Jun
J
Riley
Jonathan
J
Boulis
Nicholas M
NM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018696:Neuroprotective Agents / Q000008:administration & dosage; D010446:Peptide Fragments / Q000008:administration & dosage* / Q000235:genetics; D020671:Posterior Horn Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage; D013744:Tetanus Toxin / Q000008:administration & dosage* / Q000235:genetics; D051020:bcl-X Protein / Q000008:administration & dosage* / Q000235:genetics
C494811:BCL2L1 protein, human; D018696:Neuroprotective Agents; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D051020:bcl-X Protein; C040635:tetanus toxin fragment C