Stroke is a devastating neurological disease with high mortality rate and has profound implications for health economics and resources globally. There is a sense of urgency for scientists to discover a pharmacological target in stroke that could modulate multiple molecular cell injury mechanisms since many therapeutic agents that targeted only a single injury mechanism result in disappointing outcomes in human clinical trials. During stroke, cerebral ischemia triggers a number of membrane bound receptor-mediated signaling cascades that can activate many downstream kinases and transcription factors known to induce neuronal apoptosis. The activation of the membrane receptor Notch1 and its signaling pathway is seen in the infarcted brain, which is shown to deteriorate the outcome of ischemic stroke.
Notch1 is a transmembrane receptor that regulates cell fate decisions in the developing nervous system and adult brain. Binding of Notch’s ligands leads to the proteolytic cleavage of Notch1 by γ-secretase and the generation of Notch1 intracellular domain (NICD1), which is able to translocate to the nucleus and regulate transcription of its downstream genes. Although Notch1 has been demonstrated to worsen stroke outcome by stimulating the infiltration of pro-inflammatory leukocytes and microglia-induced inflammatory responses, the molecular mechanisms of Notch1 in neurons following ischemic stroke in the induction of pro-apoptotic cascades are still not yet fully established.
The present studies aim to investigate the role of γ-secretase-mediated Notch signaling pathway in the induction of neuronal cell death through its modulation with the nuclear factor-κB (NF-κB) pathway, the collaboration with hypoxia-inducible factor-1α (HIF-1α) pathway, and its contribution to the mitogen activated protein kinase (MAPK) pathway, which are all crucial in the induction of neuronal apoptosis. It also aims to observe the modulation of the pro-apoptotic proteins that are activated as a result of Notch1 activation in neurons following ischemic stroke.
The present studies demonstrate that the inhibition of Notch1 activation using γ-secretase inhibitors protect neuronal cells against ischemia-induced cell death by targeting an apoptotic marker, cleaved caspase-3, NF-κB subunits p65, p50 and pro-apoptotic BH3-only protein, Bcl-2 interacting mediator of cell death (Bim). In addition, treatment of mice with the γ-secretase inhibitor reduces NICD1, phosphorylated-p65 and Bim expression levels, with reduced infarct size and improved functional outcome in a mouse model of focal ischemic stroke. These findings suggest that γ-secretase-mediated Notch signaling endangers neurons after ischemic stroke by modulating the NF-κB-Bim pathway.
Additional findings suggest that the HIF-1α pathway, a global regulation pathway of cellular response to hypoxia, is able to interact with Notch1 and modulate its signaling during ischemic stroke. Treatment with either a HIF-1α inhibitor or a γ-secretase inhibitor protects neurons against ischemic stress and a combined inhibition of Notch1 and HIF-1α further decreases neuronal death. HIF-1α and NICD1 are co-expressed in the neuronal nucleus, and co-immunoprecipitated in cultured neurons and brain tissue from mice subjected to focal ischemic stroke. RNA interference- mediated endogenous Notch1 and HIF-1α depletion also decreases neuronal cell death in ischemic conditions. Finally, mice treated with HIF-1α and γ-secretase inhibitors exhibit improved outcome against stroke-induced injury, with combined treatment being superior to individual treatments. Evidence suggest that HIF-1α and NICD1 collaboration engage the apoptotic pathways via modulating the expression of cleaved caspase-3, phosphorylated-p65 and JNK MAP kinase, hence exacerbate the outcome in mice subjected to focal ischemic stroke.
Lastly, the role of γ-secretase-mediated Notch signaling in activating MAPK following ischemic stroke was investigated in a model of human neuronal death using human neuroblastoma cells (SH-SY5Y) subjected to stroke-like conditions. The γ-secretase inhibitor treated cells express decreased levels of phosphorylated JNK MAP kinase and downstream transcription factor c-Jun under ischemic conditions. Moreover, human embryonic kidney (HEK) cells overexpressing NICD1 express higher levels of phosphorylated JNK and c-Jun compared to wild type cells in response to ischemic conditions. Ischemic-induced cell death is significantly higher in NICD1 overexpressed cells and is reduced by JNK inhibition. These results suggest that Notch signaling induces cell death by modulating the JNK-c-Jun pathway following ischemic stroke.
In summary, these studies verify that γ-secretase-mediated Notch signaling regulates multiple molecular cell injury mechanisms during ischemic stroke and demonstrate for the first time that activation of Notch signaling pathway following ischemic stroke endangers neurons by activating the NF-κB, HIF-1α, and JNK pathways. Therefore, these studies will enhance the existing knowledge of the protective therapeutic effects of γ-secretase inhibitors in cerebral ischemia and indicate that γ-secretase inhibition as a potential pharmacological intervention in stroke therapy.