RGD domains neuroprotect the immature brain by a glial-dependent mechanism.
OBJECTIVE
Integrin binding to extracellular matrix ligands, including those presenting RGD motifs, modulate diverse cellular processes. In the brain, many endogenous RGD-containing molecules are induced after damage. Previously, the gene therapy vector termed NLSCt, which displays an RGD motif, was shown to neuroprotect after immature brain excitotoxicity. We analyze whether neuroprotection is mediated by the RGD motif.


METHODS
RGD-containing synthetic peptide GPenGRGDSPCA (GPen) was injected 2 hours after N-methyl-D-aspartate-mediated excitotoxicity to the postnatal day 9 rat brain. Damage and glial/inflammatory response were evaluated 3 days later. In addition, the neuroprotective effect of GPen and NLSCt after N-methyl-D-aspartate-induced cell death was also analyzed in vitro using neuron-purified and mixed neuron-glia primary cultures. To further characterize whether the neuroprotective effect was mediated by glial-derived soluble factors, we also tested the protective ability of conditioned media from RGD-treated microglia, astrocyte, or mixed glia cultures.


RESULTS
Animals treated with GPen peptide showed functional improvement, a significant reduction in lesion volume up to 28%, and a decrease in the number of degenerating neurons. In addition, N-methyl-D-aspartate-injected animals treated with both RGD-containing molecules at the neuroprotective doses showed a significant increase in microglial reactivity and microglia/macrophage cell number, but no differences in neutrophil infiltration and the astroglial response. Finally, in vitro studies showed that the neuroprotective effect was observed in mixed neuron-glia, but not in neuron-purified cultures. Conditioned media from RGD-treated microglial, astroglial, and mixed-glial cultures were not protective.


CONCLUSIONS
These results suggest that RGD-containing molecules neuroprotect by a glial-dependent mechanism.
Peluffo
Hugo
H
Medical Histology, Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Autonomous University of Barcelona, Bellaterra, Spain. hugo.peluffo@uab.es
González
Pau
P
Arís
Anna
A
Acarin
Laia
L
Saura
Josep
J
Villaverde
Antoni
A
Castellano
Bernardo
B
González
Berta
B
D000818:Animals; D001253:Astrocytes / Q000187:drug effects; D001522:Behavior, Animal / Q000187:drug effects; D001921:Brain / Q000254:growth & development* / Q000502:physiology*; D001923:Brain Chemistry / Q000187:drug effects; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D003627:Data Interpretation, Statistical; D003720:Densitometry; D018846:Excitatory Amino Acids / Q000633:toxicity*; D005260:Female; D007150:Immunohistochemistry; D017628:Microglia / Q000187:drug effects; D016202:N-Methylaspartate / Q000633:toxicity*; D009410:Nerve Degeneration / Q000517:prevention & control; D009457:Neuroglia / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D018696:Neuroprotective Agents; D020258:Neurotoxicity Syndromes / Q000517:prevention & control* / Q000523:psychology; D020556:Neutrophil Infiltration / Q000187:drug effects; D009842:Oligopeptides / Q000627:therapeutic use*; D011247:Pregnancy; D051381:Rats; D020318:Rats, Long-Evans
D017077:Culture Media, Conditioned; D018846:Excitatory Amino Acids; C085636:GpenGRGDSPCA; D018696:Neuroprotective Agents; D009842:Oligopeptides; D016202:N-Methylaspartate