The roles of apoptosis signal-regulating kinase1 (ASK1) in cerebral ischemia

Abstract

Stroke is a worldwide social and economical issue. Ischemic stroke accounts for 85 percent of stroke in patients with disability and complications such as dementia and paralysis. Despite intensive efforts in rescuing stroke patients and increasing the treatment time window, clinical therapy is still limited. Along with advanced established methods, we also need to develop novel therapeutic targets for treating ischemic stroke. In cerebral ischemia, the underlying molecular mechanisms are still a question of debate due to its complex pathology. Cerebral ischemia is the most challenging disease with diverse biochemical and molecular pathophysiology. Calcium overload, ion imbalance, endoplasmic reticulum stress, excessive reactive oxygen species (ROS), and inflammatory signals are the main sources of cellular breakdown, which lead to cell death after interruption of circulation.Under pathological condition like ischemia, apoptosis signal-regulating kinase1 (ASK1) is an early responder of oxidative stress and has a pivotal role in intracellular signaling pathways of apoptosis, inflammation, and cell differentiation. ASK1 is a member of mitogen activated protein kinase kinase kinase (MAPKKK) family. ASK1 activates mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK) family, which play important roles in intracellular signaling even after various insults from extracellular to intracellular stimuli including oxidative stress, osmolality changes, endoplasmic reticulum (ER) stress, and immune response. Previous studies have shown that after cerebral ischemia, ASK1 causes neuronal cell death and oxidative stress-induced ASK1 leads to apoptosis. Previous studies demonstrate that silencing ASK1 by using small interfering RNA (siRNA) provides resistance to neuronal cell death and inhibition of ASK1 provides a neuroprotective role in renal

and myocardial ischemia. Given this, we believe that inhibition of ASK1 could be a novel therapeutic approach to neuroprotection after an ischemic stroke.For designing an effective therapeutic strategy, it is necessary that we analyze ischemic environments after cerebral ischemia in the deletion of ASK1. Identification of novel single gene, gene network or molecular pathway helps reveal the underlying complex mechanisms. However, very little is known about gene expression pattern of the brain in response to cerebral ischemia in the absence of ASK1. In the hope of identifying new molecular and cellular connections involved in cerebral ischemia, we used DNA microarray of about 7000 mouse genes after silencing ASK1.Together, we need different approaches to different cell types because cells have various roles and resistances thresholds for an injury. This study focused on the astrocytes, which are known for supporting neurons and are involved in ion homeostasis in the brain. Activated (reactive) astrocytes play an essential role in determining the tissue response to ischemia. Formation of glial scar functions as reducing the damage by inhibiting the spread of the ischemic boundary. However, this barrier can block neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important after cerebral ischemia. Although several studies have demonstrated the participation of astrocytes in glial scar formation, the mediators of this process has not been fully elucidated yet. Based on microarray analysis and previous studies, we hypothesized that ASK1 is the main mediator of reactive astrocytes and glial scar formation. To examine this hypothesis in cerebral ischemia models, we conducted experiments both in vivo and in vitro.Male C57BL/6 mice were subjected to blockade of the middle cerebral artery for 1 hour,

followed by reperfusion whereas cultured astrocytes were exposed to oxygen-glucose deprivation (OGD). It proved ASK1, induced by ischemic injury, was expressed in astrocytes and that upon silencing ASK1, astrocyte-associated genes and the glial fibrillary acidic protein (GFAP) were down-regulated seen with microarray. During the acute phase (8 hours), ablation of ASK1 resulted in decreased GFAP. During the sub-acute phase (7 days), GFAP-positive cells were decreased in the si-ASK1+MCAO group as compared to either the middle cerebral artery occlusion (MCAO) group or the si-con+MCAO group. During the chronic phase (30 days) after cerebral ischemia, si-ASK1+MCAO group had reduced glial scar formation and a well-conserved neuronal structure, compared with the MCAO group. Mice with reduced glial scar formation in the si-ASK1+MCAO group, had advanced functional outcomes, compared with the MCAO and si-con+MCAO groups. Moreover, ASK1 was expressed in astrocytes after OGD. Deletion of ASK1 results in improperly formed GFAP transcripts, which affected by p38 pathway and GFAP transcription factors, such as Smad1/5/8, Stat3, and p300/CBP. Repression of ASK1 retarded astrocytes migration rate in the cell scratch assay. Moreover, proper dendritic extension was inhibited in the neurons, cultured with astrocyte conditioned medium (ACM) of the OGD and si-con+OGD groups, more than ACM of the normal and si-ASK1+OGD groups.These data suggest that ASK1 may play important roles in cerebral ischemia, and through DNA microarray, we selected several novel genes that might play important roles in cerebral ischemia. Inhibition ASK1 contributes to reduced glial scar formation which leads to better functional recovery. Based on these results, ASK1 may provide a potential therapeutic insight for treatment after an ischemic stroke.