Abstract
Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.
Highlights
Rho GTPases belong to the Ras superfamily of monomeric G proteins
Inhibition of Rho, Rac, and Cdc42 with C. difficile Toxin B Induces Apoptosis of cerebellar granule neurons (CGNs)—Primary rat CGNs require survival signals derived from depolarization-induced calcium influx, and to a lesser extent serum, to be maintained in culture [19, 20]
To determine if signals originating from Rho family GTPases are required for CGN survival, the function of these G proteins was compromised by incubation with clostridial toxins that covalently modify and inhibit specific Rho family members [18]
Summary
Dominant-negative mutants of Rac and Cdc protect sympathetic neurons from nerve growth factor withdrawal-induced apoptosis by suppressing activation of apoptosis signal-regulated kinase and c-Jun-NH2-terminal kinase (JNK)1 [14, 15], suggesting that Rho GTPases can function as upstream activators of stressactivated protein kinase cascades during neuronal apoptosis. We utilized clostridial toxins, enzymes that covalently modify and inactivate Rho family GTPases in a highly specific manner [18], to investigate if Rho GTPases regulate the survival of primary rat cerebellar granule neurons (CGNs). Apoptosis occurs independently of actin cytoskeletal disruption and requires phosphorylation of the transcription factor c-Jun. The data are the first to establish clearly a prosurvival function for Rho. GTPases in a primary neuronal cell model
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