A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.
BACKGROUND
The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi).


RESULTS
In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi.


CONCLUSIONS
Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.
Desclaux
Mathieu
M
Biotechnology and Biotherapy, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, CNRS UMR 7225, INSERM UMRS 975, UPMC, Hôpital de la Pitié Salpêtrière, Paris, France.
Teigell
Marisa
M
Amar
Lahouari
L
Vogel
Roland
R
Gimenez Y Ribotta
Minerva
M
Privat
Alain
A
Mallet
Jacques
J
D000818:Animals; D001369:Axons; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics*; D002478:Cells, Cultured; D018920:Coculture Techniques; D017931:DNA Primers; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000166:cytology / Q000502:physiology*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D034622:RNA Interference; D014746:Vimentin / Q000235:genetics
D017931:DNA Primers; D005904:Glial Fibrillary Acidic Protein; D014746:Vimentin