Validating FOXO4 as a therapeutic target for protecting against ischemia-reperfusion-caused neuronal injury
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Background
Previous data suggest that in the conditions of I/R in mice and the oxygen-glucose deprivation (OGD) in cell cultures, FOXO4 facilitates inflammation and oxidative stress in non-brain tissues , indicating that downregulation of FOXO4 may be neuroprotective in I/R-induced injury in the brain. However, this possibility has not been tested in the cerebral I/R condition.
Method
FOXO4 knockout (KO) and wild-type (WT) primary neuronal cultures were treated with an oxidative stress inducer, menadione (MD), or OGD, and then cell viability was assessed via ATP and MTT assays. The KO and WT mice at 2-3 months were subjected to one-hour (h) transient middle cerebral artery occlusion (tMCAO). Mice were sacrificed after 24 h for TTC staining or after 48 h for immunohistochemical staining. Alternatively, animals were allowed to survive 1-10 days after tMCAO to test their functional recovery. Furthermore, using a structure-based approach combined with cell-based assays, we screened FOXO4 inhibitors and identified actinomycin D (ActD) as a potent FOXO4 inhibitor. We also tested the therapeutic role of ActD in both in vitro and in vivo models of ischemic stroke.
Result
KO of FOXO4 reduced the infarct volume, improved animal survival, decreased neurological deficits, and enhanced functional recovery compared to WT mice. Immunohistochemical staining of astrocytes and microglia revealed that KO brains showed a reduced number of astrocytes and microglia in the peri-infarcted area two days after I/R. Western blot analysis of proinflammatory cytokines, IL-1β, IL-6, and TNF-α, indicated decreased levels of proinflammatory cytokines two days following I/R. The identified FOXO4 inhibitor, ActD, attenuated oxidative stress- and OGD-induced neuronal death. ActD also reduced neuronal injury of the brain and enhanced functional recovery in WT mice following tMCAO.
Conclusion
We conclude that disrupting FOXO4 is neuroprotective and the identified inhibitor, ActD, may be a therapeutic agent for treating ischemic stroke-induced brain injury.
