Purkinje cell survival in organotypic cultures: implication of Rho and its downstream effector ROCK.
Organotypic cultures of postnatal day 1 (P1) to P7 mouse cerebella are well-established models for studying cell survival. In the present work, we investigate the involvement of the Rho/ROCK intracellular pathway in Purkinje cell survival by using organotypic cultures of P3 Swiss mice. Specific inhibitors of Rho or ROCK were applied at different concentrations to the slice cultures, which were maintained for 5 days in vitro. We show that the bacterial exoenzyme C3 transferase, a specific inhibitor of the small GTPase Rho, increases Purkinje cell survival. There is a 4.5- and 2.5-fold increase in Purkinje cell survival when C3 intracellular uptake is promoted either by the PEP-1 peptide or by the C2IN carrier protein, respectively, and not with the commonly used TAT peptide. Moreover, treatment with Y27632 and H-1152, two specific inhibitors of the Rho kinase ROCK, also strongly reduces apoptotic cell death and results in 6.5- and 8.5-fold increases in cell survival, respectively. In immunohistochemical analysis, we also show that H-1152 did not change either glial fibrillary acidic protein or isolectin-B4 staining, indicating that this compound did not alter the cellular composition in our cultures. Thus, our data demonstrate that inhibition of Rho and its downstream effector ROCK may be used to enhance cell survival in neurodegenerative diseases.
Julien
Sylvie
S
Section for Experimental Vitreoretinal Surgery, University Eye Hospital, Tübingen, Germany. sylvie.julien@med.uni-tuebingen.de
Schnichels
Sven
S
Teng
Henry
H
Tassew
Nardos
N
Henke-Fahle
Sigrid
S
Mueller
Bernhard K
BK
Monnier
Philippe P
PP
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001905:Botulinum Toxins / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D051379:Mice; D018696:Neuroprotective Agents / Q000494:pharmacology; D009924:Organ Culture Techniques; D011689:Purkinje Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D013194:Staining and Labeling; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism*
C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000577:Amides; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D037102:Lectins; D018696:Neuroprotective Agents; D011725:Pyridines; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D054460:rho-Associated Kinases; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins