Rapid Degradation of Bim by the Ubiquitin-Proteasome Pathway Mediates Short-term Ischemic Tolerance in Cultured Neurons

Abstract

A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke.Tolerance is the phenomenon whereby previous exposure to a subtoxic insult conditions the cell or organism against a more severe toxic challenge (1). Tolerance appears to be a conserved endogenous phenomenon and has been described in multiple organisms from simple cells to more complex systems (1, 2). Ischemic tolerance has been observed in the brain; a brief preconditioning ischemic insult results in a reduced activation of cell death pathways that occur following a harmful ischemic event (1,(3)(4)(5)(6).Two types of ischemic tolerance, delayed and rapid, have been reported for the brain. Delayed ischemic tolerance develops over 1-3 days in vivo (7,8) or 24 h in vitro (9 -11), is mediated by a gene-based mechanism, and requires new protein synthesis (1,6,12). In contrast, rapid ischemic tolerance is protein synthesis-independent and occurs within 1 h of the preconditioning ischemia (13-17). Rapid ischemic tolerance can be blocked by adenosine A 1 receptor antagonists (17, 18) and mimicked by K ATP channel openers (13,19,20). The above suggests that different molecular mechanisms contribute to each type of tolerance.The Bcl-2 family of genes regulates cell death processes and consists of two groups, the pro-cell death family members, for example Bax, and the pro-survival members, for example, Bcl-2 (21). The ratio between pro-apoptotic and pro-survival members of the Bcl-2 family determines the fate of the cell (22, 23). Bim (also called Bod) is a member of the pro-apoptotic Bcl-2 family that contains only a BH3 domain (e.g. Bim, Bad, and Bid) (24). Bim is expressed as at least three...

BAG-1 overexpression attenuates luminal apoptosis in MCF-10A mammary epithelial cells through enhanced RAF-1 activation

“…Its expression is regulated by ERK-mediated phosphorylation on serine 69 (S69), which subsequently targets it for proteasomal degradation (Ley et al, 2003;Luciano et al, 2003). BIM has a relatively short half-life (Meller et al, 2006), thus to enable optimal observation of BIM S69 phosphorylation, we blocked the activity of the proteasome using the proteasome inhibitor MG132 and examined expression of BIM at the earlier time points of days 5 and 6 of 3D culture. The cell cycle regulator, p21 WAF1/CIP1 , was used as a positive control in these experiments, as it is known to be rapidly degraded by the proteasome (Blagosklonny et al, 1996).…”

In vivoContributions of BH3-Only Proteins to Neuronal Death Following Seizures, Ischemia, and Traumatic Brain Injury

“…The proapoptotic functions of Bim are triggered when its expression is increased or it is released from sequestration by dynein LC8 light chain (Puthalakath et al, 1999). Bim is also posttranslationally regulated by phosphorylation, which can result in activation (Putcha et al, 2003) or targeting to the proteasome for degradation (Meller et al, 2006).…”

Ubiquitin–Proteasome-Mediated Synaptic Reorganization: A Novel Mechanism Underlying Rapid Ischemic Tolerance

“…Classic, or delayed, ischemic tolerance requires new protein synthesis and results in protection 24 -72 h after the preconditioning stimulus (Barone et al, 1998;Dirnagl et al, 2003;Meller et al, 2005). In contrast, rapid ischemic tolerance does not require new protein synthesis and produces neuroprotection within 1 h of the preconditioning event (Perez-Pinzon et al, 1996;Reshef et al, 1996;Perez-Pinzon and Born, 1999;Meller et al, 2006). The relatively short time required for induction of rapid ischemic tolerance suggests that it is regulated by posttranslational neurochemical events.…”

“…The relatively short time required for induction of rapid ischemic tolerance suggests that it is regulated by posttranslational neurochemical events. In addition, rapid ischemic tolerance reduces the activation of programmed cell death-associated caspases (caspase3) after normally harmful ischemia (Meller et al, 2006).…”

“…Tolerance to ischemia can occur via the selective ubiquitination and degradation of a cell death-associated protein, Bcl-2-interacting mediator of cell death (Bim). Rapid ischemic tolerance and Bim degradation occurs independently of new protein synthesis and is blocked by the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) (Meller et al, 2006). …”