Tissue kallikrein protects cortical neurons against in vitro ischemia-acidosis/reperfusion-induced injury through the ERK1/2 pathway

Abstract

Human tissue kallikrein (hTK) gene transfer has been shown to protect neurons against cerebral ischemia/reperfusion (I/R) injury, and exogenous tissue kallikrein (TK) administration can enhance neurogenesis and angiogenesis following focal cortical infarction. Previous studies have reported that acidosis is a common feature of ischemia and plays a critical role in brain injury. However, little is known about the role of TK in ischemia-acidosis-induced injury, which is partially caused by the activation of acid-sensing ion channels (ASICs). Here we report that pretreatment of cultured cortical neurons with TK reduced cell death induced by either acidosis or oxygen and glucose deprivation-acidosis/reoxygenation (OGD-A/R). Immunocytochemical staining revealed that TK largely prevented OGD-A/R-induced neuronal morphological changes. We also observed that TK treatment protected cultured neurons from acidosis and OGD-A/R insults. TK exerted the neuroprotective effects by reducing production of reactive oxygen species (ROS), stabilizing the mitochondrial membrane potential (MMP) and inhibiting caspase-3 activation, and thereby attenuating oxidative stress and apoptosis. In addition, we found that activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade but not the PI3K/Akt signaling pathway was required for the survival-promoting effect of TK on neurons exposed to OGD-A/R. Moreover, blockade of ASICs had effects similar to TK administration, suggesting direct or indirect involvement of ASICs in TK protection. In conclusion, TK has antioxidant characteristics and is capable of alleviating ischemia-acidosis/reperfusion-induced injury, inhibiting apoptosis and promoting cell survival in vitro through activating the ERK1/2 signaling pathways. Therefore, TK represents a promising therapeutic strategy for ischemic stroke.

Introduction

Ischemic stroke is the second leading cause of death in China and the most common cause of disability in modern world (Chalela et al., 2004). Enormous efforts have been made to identify ways to attenuate neural cell injury induced by cerebral ischemia. However, thrombolysis with rt-PA remains the only approved treatment for ischemia within 3 h after stroke onset. For various reasons, the overwhelming majority of patients do not benefit from this therapeutic strategy (Fisher, 2002). Therefore, identifying novel, safe, and therapeutic agents to rescue ischemic tissues after ischemia continues to be a major research endeavor.

Tissue acidosis is a well established feature of cerebral ischemia (Nemoto and Frinak, 1981). Initially, a drop in extracellular pH was considered neuroprotective due to subsequent inhibition of NMDA receptors (Giffard et al., 1990, Tang et al., 1990). However, this pH decrease is now believed to drive activation of acid-sensing ion channels (ASICs), resulting in subsequent intracellular Ca²⁺ accumulation (Xiong et al., 2004, Yermolaieva et al., 2004) and enhancement of AMPA-kainate receptor-mediated injury, despite NMDA blockade (McDonald et al., 1998). ASICs belong to the degenerin/epithelial Na⁺ channel (Deg/ENaC) superfamily. Six ASIC subunits, including 1a, 1b, 2a, 2b, 3, and 4 have been cloned, among which 1a, 2a and 2b are primarily expressed in neurons in the brain. Different ASIC subunits can form complexes which are activated at different pH levels (Krishtal, 2003, Waldmann et al., 1996, Waldmann et al., 1997a, Waldmann et al., 1997b). The homomeric channel composed of ASIC1a has been increasingly believed to mediate glutamate-independent Ca²⁺ toxicity during brain ischemia/reperfusion due to its high Ca²⁺ permeability (Yermolaieva et al., 2004, Xiong et al., 2004). Recent studies have demonstrated that, following ischemia, brain tissue pH changes dramatically prior to the development of the ischemic infarction, reaching a level sufficient to activate ASIC1a, which begins to open at pH 7.0 and reaches half maximal activation at pH 6.2 (Waldmann et al., 1997a). During ischemic injury, blockade of ASIC1a or bicarbonate administration provided neuroprotection (Pignataro et al., 2007). Additionally, a specific ASIC1a antagonist psalmotoxin1 (PcTX) alleviated the neurotoxic effects on neurons exposed to pH 6.0 (Xiong et al., 2004). These results suggest that developing therapeutic agents against ASIC activation, or re-categorizing existing neuroprotective agents to recognize those with anti-acidotoxicity characteristics may provide a novel and more appropriate strategy for ischemic stroke.

Tissue kallikrein (TK), an important component of the kallikrein/kinin system, is a serine proteinase capable of cleaving low molecular weight kininogen to release vasoactive kinins, which in turn activate bradykinin B1 and B2 receptors (B1R and B2R) and trigger a series of biological effects (Emanueli and Madeddu, 2003, Chao and Chao, 2005). All components of the kallikrein/kinin system are expressed and widely distributed throughout many mammalian tissues and are up-regulated by ischemic stroke (Walker et al., 1995, Wagner et al., 2002). Previous studies have demonstrated that systemic or local delivery of human TK (hTK) gene protects against mouse myocardial and cerebral ischemia/reperfusion (I/R) injury through inhibition of oxidative stress and apoptosis, enhancement of glial cell survival, and migration and promotion of angiogenesis and neurogenesis (Xia et al., 2004, Xia et al., 2006). Based on these observations, we hypothesized that TK may protect neurons from injury induced by extracellular acidity occurring in ischemic brain regions.

To test this hypothesis, we investigated the potential role of TK in ischemia-acidosis/reperfusion-induced neuronal injury in vitro. The oxygen and glucose deprivation (OGD) model has been widely used in cultured neurons and brain slices to simulate brain ischemia (Gwag et al., 1995, Velazquez et al., 1997). The present study characterizes a new in vitro model of ischemia, OGD combined with acidosis (OGD-A) or in vitro acidosis (pH 6.0) alone. To eliminate potential secondary activation of glutamate NMDA and AMPA receptors and voltage-gated Ca²⁺ channels, all experiments are conducted in the presence of glutamate, as well as voltage-gated Ca²⁺ channels blockers (Xiong et al., 2004).

Numerous studies have reported that mitochondria are involved in a variety of key events in apoptosis (Chan, 2004). Cerebral I/R can induce mitochondrial disturbances and promote excessive production of reactive oxygen species (ROS) to activate pro-apoptotic caspase pathways, thus directly or indirectly causing cell death (Lièvre et al., 2000, Chan, 2001, Sugawara and Chan, 2003). We therefore examined the effects of TK on apoptotic-like death and oxidative stress injury during acidosis or combined with ischemia/reperfusion in vitro. To probe the underlying signaling mechanism of TK neuroprotection, we further evaluated the effect of TK on activation of MAPK and PI3K/Akt signaling cascades. TK plays a neuroprotective role in ischemia-acidosis/reperfusion injury in vitro involving ERK signaling pathways.