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 S ，Schnichels S ，Teng H ，Tassew N ，Henke-Fahle S ，Mueller BK ，Monnier PP ... -  《-》

Inhibition of Rho-ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase.

Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes Clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho-ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD-labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB-induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho-ROCK-flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes.

Krijnen PA ，Sipkens JA ，Molling JW ，Rauwerda JA ，Stehouwer CD ，Muller A ，Paulus WJ ，van Nieuw Amerongen GP ，Hack CE ，Verhoeven AJ ，van Hinsbergh VW ，Niessen HW ... -  《-》

Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina.

Tura A ，Schuettauf F ，Monnier PP ，Bartz-Schmidt KU ，Henke-Fahle S ... -  《-》

Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing.

Höltje M ，Hoffmann A ，Hofmann F ，Mucke C ，Grosse G ，Van Rooijen N ，Kettenmann H ，Just I ，Ahnert-Hilger G ... -  《JOURNAL OF NEUROCHEMISTRY》

Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells.

Ikeda H ，Nagashima K ，Yanase M ，Tomiya T ，Arai M ，Inoue Y ，Tejima K ，Nishikawa T ，Omata M ，Kimura S ，Fujiwara K ... -  《AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY》