We presently show that intracellular dsRNA activates the intrinsic pathway of apoptosis in pancreatic beta cells. Triggering of apoptosis by dsRNA is secondary to an early and sustained downregulation of Mcl-1 protein expression (), resulting in the release of the proapoptotic protein Bim. The unbound Bim then activates BAX translocation to the mitochondria, mitochondrial permeabilization, cytochrome c release, caspases 9 and 3 activation and beta cell apoptosis. Key results obtained with dsRNA, an intermediary product of viral replication, where partially confirmed in the context of a beta cell infection by the diabetogenic enterovirus CVB5.
Schematic representation of dsRNA- and virus-induced beta cell apoptosis.
Apoptosis is the main form of cell death in T1D 
. Several virus-induced human diseases are associated with increased apoptosis in the target cells, including HIV1-associated dementia 
, cytomegalovirus encephalitis 
and viral myocarditis 
. Coxsakieviruses can induce beta cell death by different mechanisms, depending on the strain and multiplicity of infection (MOI) used 
. At higher MOIs (>100) necrosis is the preferential mechanism of cell death, but at lower MOIs a shift towards apoptosis is observed 
. Viral triggering of apoptosis depends on both the host and the virus 
. The host regulates viral-induced apoptosis trough the local production of pro-inflammatory cytokines and chemokines 
. Viral factors leading to apoptosis include dsRNA 
. This viral nucleic acid is recognized by receptors such as the toll-like receptor 3 (TLR3), the kinase PKR, the helicases melanoma differentiation-associated gene 5 (MDA5), (which is also a candidate gene for T1D 
) and retinoic-acid-inducible protein 1 (RIG-I); these receptors activate genes involved in both antiviral responses and apoptosis 
. All these receptors are expressed in pancreatic beta cells 
. The pathways downstream of viral recognition/signaling leading to beta cell apoptosis, however, remained to be clarified. The present observations provide a coherent hypothesis for the mechanisms triggering beta cell apoptosis following a viral infection ().
Mcl-1 is a pro-survival Bcl-2 family member with a short half-life (30–180 min), which makes it specially susceptible to changes in protein translation 
. Mcl-1 is degraded in the proteasome 
, and use of a proteasome inhibitor prevented, at least in part, dsRNA-induced decrease in Mcl-1 expression (present findings). We also observed that intracellular dsRNA induces an early and sustained phosphorylation of eIF2α which, as previously shown by our group, leads to a decrease in total protein translation 
. This eIF2α phosphorylation is mediated by PKR, a protein kinase mainly activated by dsRNAs produced during viral replication 
, but not by PERK (present data). Protein translation inhibition contributes to an early and progressive decrease in Mcl-1 protein levels, which is reverted by knocking down PKR. PKR silencing actually increases Mcl-1 protein expression, probably due to the “release” of protein translation combined with an increase in Mcl-1 mRNA induced by dsRNA (). PKR silencing also prevents dsRNA-induced apoptosis, at least partially due to this increase in Mcl-1 protein. Mcl-1 protein stability is partly regulated by multiple sites of phosphorylation, and JNK-mediated phosphorylation of Mcl-1 increases the rate of protein degradation 
. We recently described that JNK activation contributes to Mcl-1 degradation in beta cells exposed to the cytokines interleukin-1β + interferon-γ 
. Here, we observed that dsRNA induces JNK phosphorylation in beta cells, but JNK inhibition does not prevent Mcl-1 degradation. These findings indicate that dsRNA mainly regulates Mcl-1 protein expression via inhibition of protein translation.
Mcl-1 functions as a prosurvivor factor by neutralizing specific propapoptotic BH3-only proteins; Bim has been described as a preferential target of Mcl-1 in other cell types 
. Bim is a BH3-only protein that presents features of both a “sensitizer”, i.e. it binds to antiapoptotic Bcl-2 protein and displaces the effectors BAX and BAK 
, and also as an “activator”; i.e. through directly binding to BAX and BAK it promotes their activation and the induction of the mitochondrial apoptosis pathway 
. An imbalance between Mcl-1 and Bim has been shown to trigger apoptosis in other cell types in the context of cytokine deprivation 
and granzyme B 
activation. We presently observed that Bim is neutralized by Mcl-1 under basal condition. After dsRNA exposure or the use of specific siRNAs against Mcl-1, however, there is an increase in “free” Bim that subsequently activates BAX and triggers apoptosis (). This is suggested by the observation that a double knockdown of Bim plus Mcl-1 () reverts the proapoptotic effects of Mcl-1 silencing. Bim can also mediate the pro-apoptotic effects of type I interferons produced during viral infections 
and it is targeted by viruses to evade apoptosis in the host cells 
. Type I interferons were induced during both dsRNA and CVB5 exposure (Supplemental Figure S6
). Importantly, inhibition of Bim by specific siRNAs prevented caspases 9/3 activation and apoptosis in beta cells infected with the diabetogenic virus CVB5, confirming the key role of Bim during viral infection of beta cells.
Apoptosis is one of the mechanisms by which the host eliminates virus-infected cells and blocks viral spread 
. In postmitotic and poorly proliferating cells such as neurons and beta cells, apoptosis might also promote functional loss and disease. Indeed, studies in viral-induced neuronal disease demonstrate that inhibition of apoptosis may reduce disease severity without changes in virus titers 
. In line with these findings, apoptosis prevention in beta cells by knocking down Bim did not modify viral replication in vitro
In conclusion, we presently show that decreased expression of the anti-apoptotic protein Mcl-1, coupled to activation of the pro-apoptotic protein Bim, contributes to beta cell apoptosis during in vitro
exposure to the viral mimetic dsRNA. In case these observations can be confirmed in future in vivo
experiments, novel strategies to increase Mcl-1 or decrease Bim expression in pancreatic beta cells 
may turn to be interesting approaches to protect beta cells during infection by putative “diabetogenic” viruses.