Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

Abstract

Loss of mitochondrial membrane integrity and release of apoptogenic factors are a key step in the signaling cascade leading to neuronal cell death in various neurological disorders, including ischemic injury. Emerging evidence has suggested that the intramitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and promotes caspase-independent cell death induced by glutamate toxicity, oxidative stress, hypoxia, or ischemia. However, the mechanism by which AIF is released from mitochondria after neuronal injury is not fully understood. In this study, we identified calpain I as a direct activator of AIF release in neuronal cultures challenged with oxygen-glucose deprivation and in the rat model of transient global ischemia. Normally residing in both neuronal cytosol and mitochondrial intermembrane space, calpain I was found to be activated in neurons after ischemia and to cleave intramitochondrial AIF near its N terminus. The truncation of AIF by calpain activity appeared to be essential for its translocation from mitochondria to the nucleus, because neuronal transfection of the mutant AIF resistant to calpain cleavage was not released after oxygen-glucose deprivation. Adeno-associated virus-mediated overexpression of calpastatin, a specific calpain-inhibitory protein, or small interfering RNAmediated knockdown of calpain I expression in neurons prevented ischemia-induced AIF translocation. Moreover, overexpression of calpastatin or knockdown of AIF expression conferred neuroprotection against cell death in neuronal cultures and in hippocampal CA1 neurons after transient global ischemia. Together, these results define calpain I-dependent AIF release as a novel signaling pathway that mediates neuronal cell death after cerebral ischemia.

An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death

“…It has been reported that both calpain and cathepsins can cleave AIF. [20][21][22] In the case of calpain, the subcellular localization of the enzyme responsible for AIF cleavage is not known. Although both m-and m-calpains are generally considered to be cytosolic, mitochondria were also shown to contain Ca 2 þ -dependent calpain 1.…”

Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia

“…Global cerebral ischemia (GCI) was induced for 12 min in isoflurane-anesthetized male Sprague-Dawley rats using the four-vessel occlusion method. Hippocampal CA1 cell death was quantified at either 4 or 60 days after GCI by stereological cell counting throughout the dorsal hippocampal formation (28). An in vitro model of neuronal ischemia was induced in primary cultures of hippocampal/cortical neurons by transient oxygen and glucose deprivation (OGD) for 1 h (28).…”

“…Mammalian expression plasmids directing the transcription of short hairpin (sh) small interfering RNAs (shRNAs) against rat APE1 or ATF2 were constructed using the pcDNA3.1 (+) backbone containing the U6 promoter (28). For each of the targeted genes, the scrambled sequence served as a control.…”

Recent Information on the Pathogenesis and Treatment of Hypoxic-Ischemic Brain Lesions in Newborns

“…В развивающемся мозге при гипоксии-ишемии к развитию МРТr приводят эксай-токсичность и оксидантный стресс; считается, что основ-ным механизмом этого является действие проапопто-тического белка X-protein (Bax) [33]. Вследствие МРТr митохондрии набухают и погибают, выделяя в цитоплазму ряд эффекторов апоптоза, включая цитохром С, апопто-зиндуцирующий фактор (Аpoptosis-inducing factor, AIF), прокаспазу-9 и эндонуклеазу G [34]. Цитохром С и про-каспаза-9, попадая в цитоплазму, в течение от 3 до 24 ч после повреждения приводят к активации каспазы-9, а в течение 6-48 ч -к переходу прокаспазы-3 в актив-ную каспазу-3 [18].…”