Receptor‐interacting protein 3‐phosphorylated Ca²⁺/calmodulin‐dependent protein kinase II and mixed lineage kinase domain‐like protein mediate intracerebral hemorrhage‐induced neuronal necroptosis

Abstract

Necroptosis‐mediated cell death is an important mechanism in intracerebral hemorrhage (ICH)‐induced secondary brain injury (SBI). Our previous study has demonstrated that receptor‐interacting protein 1 (RIP1) mediated necroptosis in SBI after ICH. However, further mechanisms, such as the roles of receptor‐interacting protein 3 (RIP3), mixed lineage kinase domain‐like protein (MLKL), and Ca2+/calmodulin‐dependent protein kinase II (CaMK II), remain unclear. We hypothesized that RIP3, MLKL, and CaMK II might participate in necroptosis after ICH, including their phosphorylation. The ICH model was induced by autologous blood injection. First, we found the activation of necroptosis after ICH in brain tissues surrounding the hematoma (propidium iodide staining). Meanwhile, the phosphorylation and expression of RIP3, MLKL, and CaMK II were differently up‐regulated (western blotting and immunofluorescent staining). The specific inhibitors could suppress RIP3, MLKL, and CaMK II (GSK'872 for RIP3, necrosulfonamide for MLKL, and KN‐93 for CaMK II). We found the necroptosis surrounding the hematoma and the concrete interactions in RIP3‐MLKL/RIP3‐CaMK II also both decreased after the specific intervention (co‐immunoprecipitation). Then we conducted the short‐/long‐term neurobehavioral tests, and the rats with specific inhibition mostly had better performance. We also found less blood–brain barrier (BBB) injury, and less neuron loss (Nissl staining) in intervention groups, which supported the neurobehavioral tests. Besides, oxidative stress and inflammation were also alleviated with intervention, which had significant less reactive oxygen species (ROS), tumor necrosis factor (TNF)‐α, lactate dehydrogenase (LDH), Iba1, and GFAP surrounding the hematoma. These results confirmed that RIP3‐phosphorylated MLKL and CaMK II participate in ICH‐induced necroptosis and could provide potential targets for the treatment of ICH patients.

Acute neuroinflammation promotes a metabolic shift that alters extracellular vesicle cargo in the mouse brain cortex

“…Increased phosphorylation and expression of CaMKII is induced through LPS treatment resulting in the formation of N‐methyl D‐aspartate receptor subtype 2B‐CaMKII‐Postsynaptic density protein 95 in the frontal cortex and hippocampus of mice where the proteins’ presence is associated with neuronal damage (Song et al., 2019 ). In addition to mediating inflammation, CaMKII is known to be involved in apoptotic pathways and its increased phosphorylation and expression has been observed in mouse brain tissues undergoing necroptosis after intracerebral haemorrhage (Sun et al., 2023 ; Yuan et al., 2023 ).…”

Cell type‐specific molecular mechanisms and implications of necroptosis in inflammatory respiratory diseases

“…37 Phosphorylated CaMKII influences mitochondrial permeability transition pore to modulate necroptosis and apoptosis. [38][39][40] F I G U R E 1 Key mediators of necroptotic signaling. Necroptotic signaling is evoked upon activated death receptors: TNFR, FASL, DR4/5, etc.…”

“…Apart from MLKL, Ca 2+ /calmodulin‐dependent protein kinase (CaMKII) functions as another substrate for RIP3 37 . Phosphorylated CaMKII influences mitochondrial permeability transition pore to modulate necroptosis and apoptosis 38–40 . Table 2 summarizes the key mediators of necroptosis.…”

MIC19 Exerts Neuroprotective Role via Maintaining the Mitochondrial Structure in a Rat Model of Intracerebral Hemorrhage

“…We utilized an autologous blood model because this model can closely simulate clinical ICH [22,35,36]. During the establishment of the ICH model, the vital signs of each rat were monitored by the researchers, and their rectal temperature was kept at 36.5-37.5 • C. Briefly, rats were anesthetized (isoflurane gas anesthesia) and fastened via a stereotaxic apparatus.…”