Statins are widely used cholesterol-lowering drugs that act by inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol biogenesis. Recent evidence suggests that statin use may be associated with a decreased risk for Alzheimer's disease, although the mechanisms underlying this apparent risk reduction are poorly understood. One popular hypothesis for statin action is related to the drug's ability to activate α-secretase-type shedding of the Alzheimer amyloid precursor protein (APP) ectodomain (sAPPα). In the present study, we demonstrate that, like statin's effect on reducing Aβ production, which is likely through its stimulating effect on α-secretases and sAPPα secretion, this stimulating action also mediates its excitoprotective mechanism.
Statins have been documented to exhibit potent neuroprotective effects in stroke, both for prevention and for recovery. Therefore, they are expected to exert protective actions against a broad spectrum of neuronal insults. Interestingly, our results indicate a rather selective action on excitotoxicity. We report here that the two CNS-permeable statins can selectively protect cultured cortical neurons against NMDA excitotoxic challenge, and that this protection requires chronic pretreatment of neurons with statins, which is consistent with their anti-cholesterol action. Moreover, statins are found to selectively modulate the calpain-mediated truncations on a series of molecules crucial for the neuronal survival pathways. These findings greatly expand our knowledge on statin's neuroprotective mechanisms and the signaling survival pathways involved, which will be instrumental to future therapeutic design. In particular, given the fact that there is currently no success with various pharmacological calpain inhibitors, these clinically approved statins represents a significant and safe option.
Although the calpain-mediated truncation was individually identified on CDK5 coactivator p35 to p25 conversion, GSK3 and β-catenin (Goni-Oliver et al., 2007
; Abe and Takeichi, 2007
), our work, for the first time, suggests a strong link for these events in a unified pathway. We showed that all these molecules are truncated by NMDA and that statins can prevent these truncations and thus suppress the NMDA-induced GSK3 β activation and further preserve β-catenin. These findings identify the nuclear β-catenin as the major mediator of survival in statin-treated neurons. Specific phosphorylation events triggered by an imbalance of kinases and phosphatases are known to be the major regulatory mechanisms on the biological activities of both the GSK3 β and β-catenin. Our work now suggests that calpain-mediated truncation can be the additional crucial mechanism regulating the biological activity of GSK3 β. This truncation of GSK3 takes place in parallel to its Ser-9 phosphorylation, which is known to inversely correlate with its kinase activity. Interestingly, statins appear to be able to modulate on both events. Moreover, the NMDA-induced calpain-mediated truncation appears to be the sole mechanism regulating the nuclear translocation of β-catenin, which determines its biological activity on transcription, whereas other apoptotic stimulus, such as sturosporine, only elicits alterations on the phosphorylation state of β-catenin. It remains to be determined whether this novel regulation of β-catenin mediated by calpain cleavage is an independent event to its upstream modulator GSK3 β and how would this truncation event further regulate β-catenin translocation into the nucleus.
Our work also brings another crucial player, CDK5/p35/p25, into the paradigm. Despite its ubiquitous expression, Cyclin-dependent kinase 5 (CDK5) activity is almost exclusively restricted to postmitotic neurons because of the neuron-specific expression of its regulators p35/p39. During neuronal insult and subsequent disruption of calcium homeostasis, conversion of inactive p35 to active p25 is mediated via proteolytic cleavage by the calcium-regulated calpains. Recently, CDK5 has received considerable attention as a regulator of neuronal death (Cruz and Tsai, 2004
). In particular, studies using apoptotic and excitotoxic death models demonstrate that p25/CDK5 complexes accumulate within the nucleus and that this activity is required in excitotoxic death but not apoptotic death (O'Hare et al., 2005
). It is worth stressing that statins can modulate both CDK5 activation and β-catenin via a unified protein truncation mechanism, though it is not clear whether the CDK5-mediated pathway converges with the Wnt signaling pathway.
One important question remains as to how statins suppress calpain activation. As a major family of calcium-induced proteases, calpains are activated in response to calcium flux as a result of overactivated NMDA receptor channel. Given the well-defined role of soluble APP in suppressing calcium influx upon excitotoxic conditions (Mattson et al., 1993
; Mattson, 1997
), along with statins' stimulating action on α-secretase-mediated shedding of APP (Kojro et al., 2001
, Parvathy et al., 2004
), we speculated that sAPP plays a key role in statins' excitoprotection. Indeed, results from a number of experimental designs all pointed to a conclusion that statins' excitoprotection is largely dependent on its stimulating effect on sAPP. In addition, we also identified the PI3K/Akt pathway as one of the crucial upstream signaling pathways activated by insulin receptor, which is preserved by statins upon NMDA challenge. Interestingly, the specific pharmacological inhibitor LY 294002 not only abolishes statin's excitoprotective effect against NMDA, but also attenuates its effect on sAPP secretion (). It is not clear whether and how this insulin signaling-regulated PI3K pathway affects statin's modulation on the sAPP secretion directly or indirectly. Taken together, the notions that statins can also modulate on APP processing, resulting in reduced Aβ production, likely through its stimulating effect on sAPP production which later is found to be modulated on protein isoprenylation (Buxbaum et al., 2001
; Puglielli et al., 2001
; Ostrowski et al., 2007
), it appeared that sAPP is a key executor in the pluripotent actions of statins in CNS. Therefore, we continued to investigate the upstream signaling events that mediate statins' stimulation on sAPP production upon excitotoxic conditions.
The primary action of statins is the inhibition of HMG-CoA reductase, blocking the de novo
synthesis of cholesterol and resulting in lower plasma cholesterol levels. However, recent observations demonstrate that statins have pleiotropic actions that are not dependent on cholesterol reduction (Liao and Laufs, 2005
; Cole and Vassar, 2006
). Specifically, statins have been shown to inhibit vascular inflammation, enhance endothelial function, inhibit the proliferation of vascular smooth muscle, reduce platelet activation and aggregation, and increase athereosclerotic plaque stability. Many of these functions were postulated to arise from disruption of the actions of small G-proteins. Although blockade of HMG-CoA reductase prevents de novo
synthesis of cholesterol, it also decreases the pools of intermediate metabolites, namely the two isoprenoids farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP), in the biosynthetic pathway that have ancillary functions (Supplemented Fig. 1
Statins are known to inhibit the isoprenoid pathway, thereby modulating the activities of the Rho family of small GTPases-Rho A, B, and C as well as the activities of Rac and cdc42. Rho proteins, in turn, exert many of their effects via Rho-associated protein kinases (ROCKs/ROCK1 and ROCK2). Since it is known that the statin-activated shedding of APP ectodomain can be modulated by ROCKs (Pedrini et al., 2005
), we therefore examined whether statin's excitoprotective mechanism requires Rho-ROCK signaling. Our findings using a ROCK specific inhibitor Y27632, as well as direct testing of the two isoprenoids (FPP and GGPP), confirmed the involvement of both the protein isoprenylation and the Rho-ROCK signaling in statin-mediated excitoprotective mechanism. However, it is still not clear how the negative regulation by the Rho-ROCK modulates sAPP production and its interplay with the insulin signaling-mediated PI3K pathway. In several reports, the insulin/IGF-mediated signaling appears to be able to activate RhoA and thus negatively regulates on Rho-ROCK signaling in cancer and in metabolic settings (Zhang et al., 2005
; Tapia, 2006
). It is not clear whether the insulin/IGF-mediated survival signaling is upstream to the Rho-ROCK in neurons under excitotoxic stressed conditions.
ROCK family members (ROCK1 and ROCK2) were initially identified as a Rho-binding protein with serine/threonine protein kinase activity. They have been generally implicated in cell death and survival processes in many cell types (Shi and Wei., 2007
) and reported to counter-regulate insulin signaling and the subsequent PI3-K activation in adipocytes and muscle cell lines (Negum et al., 2002
; Furukawa et al., 2005
). Moreover, PTEN, the phosphatase and tensin homologue, is a newly identified ROCK substrate (Li et al., 2005
) and ROCK phosphorylates and stimulates its phosphatase activity, which in turn antagonizes PI3K activity, resulting in reduced Akt phosphorylation/activation. Reduced PTEN is typically found to be associated with excitotoxic response in neurons, accompanied by elevated Akt activation (Liao, unpublished data), which may contribute to the protective effect of ROCK inhibition seen with either its inhibitor Y27632 or with statin-mediated inhibition on isoprenylation (). Together with the notion that Rho-ROCK signaling is required for soluble APP production, the latter is being recognized to be one of the most potent neurotrophic and neuroprotective factors in CNS and the beneficial therapeutic potentials of Rho-ROCK inhibitors are further warranted.