Cyclin D1, cdk4, and Bim are involved in thrombin‐induced apoptosis in cultured cortical neurons

Abstract

Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombininduced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.

Src kinase inhibition decreases thrombin-induced injury and cell cycle re-entry in striatal neurons

“…Sporadic expression of cyclin D1 was observed in control neurons in our study and in previous reports (Rao et al, 2007). However, there was no active Cdk4 detected in the control neurons in our study and in the previous reports (Rao et al, 2007).…”

“…In addition, thrombin causes apoptosis possibly by stimulating post-mitotic cortical neurons to re-enter the cell cycle (Rao et al, 2007). These observations suggest that mitogenic signaling by thrombin and aberrant cell cycle re-entry may be one mechanism by which thrombin produces neuronal apoptosis.…”

“…Thrombin actions in brain are associated with mitogen-activated protein kinase (MAPK) signaling (Fujimoto et al, 2007). In cortical neuronal cultures, the mitogenic signaling by thrombin causes neurons to re-enter the cell cycle, and result in their apoptotic death (Rao et al, 2007). These results support a possible thrombin-induced "mitogenic signaling/cell cycle/neuronal death hypothesis."…”

“…Although it appears that they face continuing stress to re-enter the cell cycle, a series of anti-growth factors prevent them from normally doing so (Copani and Nicoletti, 2005). A growing number of in vivo and in vitro studies, however, show that post-mitotic neurons can re-enter the cell cycle after brain injury in rat and humans (Hayashi et al, 2000, Copani et al, 2001, Love, 2003, Becker and Bonni, 2004, Greene et al, 2004, Herrup et al, 2004, Kuan et al, 2004, Di Giovanni et al, 2005, Copani et al, 1999, Liu and Greene, 2001, Rao et al, 2007. This cell cycle re-entry is a critical element of the DNA damage response of post mitotic neurons leading to apoptosis (Kruman et al, 2004).…”

“…Four hours after thrombin incubation, the Cdk4 assay was performed according to published methods (Rao et al, 2007). Briefly, the retinoblastoma (Rb) peptide is phosphorylated as a result of Cdk4 activity, which is measured using luminometric estimation of ATP depletion.…”