Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death upon various brain injuries. However, the role of Omi/HtrA2 in neuronal death induced by status epilepticus (SE) in the immature brain has not been reported. In this study, we analyzed the contribution of serine protease Omi/HtrA2, its substrate X-linked inhibitor of apoptosis protein (XIAP) and the caspase-3 activation to damage of hippocamplal CA1 cells following lithium-pilocarpine SE in P14 rat pups. Status epilepticus in the immature brain significantly induced translocation of Omi/HtrA2 from mitochondria into the cytosol, increased cytosolic accumulation of Omi/HtrA2, induced appearance of XIAP-breakdown products and enhanced caspase-3 activity in the selectively vulnerable hippocampal CA1-subfield. Taken together, these results demonstrate for the first time that SE in the immature brain results in Omi/HtrA2 accumulation in the cytosol, where it probably promotes neuronal death by neutralizing and cleaving XIAP, one of the most potent endogenous inhibitors of apoptosis.

Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.

HS1-associated protein X1 (HAX-1) is a mitochondrial protein which interacts with a diverse group of molecules such as inflammatory cytokines; interleukin-1, hematopoietic lineage specific protein-1 and vimentin. It has been reported that HAX-1 may act as antiapoptotic protein in HeLa- and Jurkat cells after Fas-treatment, irradiation or serum deprivation. This underlines the evidence that HAX-1 might be involved in both receptor- and mitochondria-mediated apoptosis pathways. However, the role of HAX-1 in neuronal death induced by status epilepticus in the immature brain has not been reported. In this study, we performed a status epilepticus in rats and investigated the dynamic changes of HAX-1 expression, HtrA2 distribution and caspase-3 activation in the hippocampus. Western blot and immunohistochemistry analysis revealed that HAX-1 was expressed at very low levels in the hippocampus. Status epilepticus in the immature brain significantly induced increased cytosolic accumulation of HAX-1 in a biphasic manner, induced an upregulation of HtrA2 and enhanced caspase-3 activity in the selectively vulnerable hippocampal CA1-subfield. Taken together, these results suggested that HAX-1 is probably involved in the pathophysiology of cell death induced by epilepsy.