Recently, studies have shown that STI1 protein secretion from astrocytes is significantly increased following oxygen glucose deprivation (OGD) (Beraldo et al., 2013). In fact, 9 hours following OGD, there is a 3-fold increase in extracellular STI1 levels (Beraldo et al., 2013). This increased STI1 expression during hypoxic conditions has been found to result from direct binding of the hypoxia inducible factor-1-alpha onto the promoter of STI1 gene (Lee at al., 2013).
The recruitment of bone marrow-derived endothelial cells has been described as an endogenous recovery mechanism in the brain following stroke (Hess et al., 2002). Recently, STI1 has also been shown to recruit bone marrow-derived stem cells (BMDCs) into ischemic brains in vivo (Lee et al., 2013). Furthermore, STI1 signalling promoted the proliferation and migration of BMDCs in vitro (Lee et al., 2013), suggesting that the recruitment of BMDCs into hypoxic areas of the brain by STI1 may be an endogenous response to facilitate recovery following ischemia.
Apoptosis and necrosis are two forms of cell death with distinct morphological features. While necrosis is a passive, irreversible process, apoptosis is much more active and requires the action of numerous apoptotic regulatory proteins (Walker et al., 1988). There are two types of apoptosis. The first type is intrinsic and involves the mitochondria. The second type depends on the action of membrane death receptors, and is thus extrinsic (Walker et al., 1988). In intrinsic apoptosis, the balance of both pro-apoptotic proteins
(such as BIM and Bax) and anti-apoptotic proteins (such as Bcl-2 and BclXL) regulate the
release of cytochrome c from the mitochondria. In both forms of apoptosis, caspase enzymes are activated and damages occur to the DNA, chromatin, and the plasma membrane (Walker et al., 1988). Cyclic AMP (cAMP) is one of the most well studied second messengers, especially in regulation of apoptosis. In neuronal cell types, cAMP has been found to have an anti-apoptotic effect (Insel et al., 2012). Activation of PKA, an effector of cAMP, has been found to protect cells against TNF-alpha-induced apoptosis (Kragsted et al., 2004).
Anisomycin induces apoptosis by inhibition of protein synthesis (Croons et al., 2009). In early studies, activation of PrPC using a PrPC-binding peptide modeling STI1 was found to transduce neuroprotective signals against anisomycin-induced apoptosis in retinal cells (Chiarini et al., 2002). While activation of PrPC has been found to activate both cAMP/PKA and ERK pathways, inhibition of PKA activity blocked the protective effect of PrPC (Chiarini et al., 2002). Treatment of hippocampal neurons with STI1 led to a PrPC-dependent increase of PKA activity to a level similar to that observed with forskolin, a potent activator of PKA (Lopes et al., 2005). Furthermore, PrPC has been documented to modulate calcium signalling (Whatley et la., 1995). In fact, the interaction between STI1 and PrPC triggers calcium influx via physical interaction with the α7nAChR, leading to protection against staurosporine-induced apoptosis (Beraldo et al., 2010). Unlike the massive influx of calcium during excitotoxicity, this controlled increment of calcium influx via α7nAChR is upstream of PKA activation and is subsequently protective against staurosporine (Cooper et al., 1995, Beraldo et al., 2010).
1.4
Alpha-7 nicotinic acetylcholine receptor
In intra-cerebral haemorrhage, the development of brain edema induces apoptosis in surrounding neurons, extending the damage beyond the initial site of ischemia (Matsushika et al., 2000). Secondary injury mechanisms, such as the inflammatory cascade, are significant contributors to neuronal ischemic damage (Keep et al., 2012, Lakhan et al., 2009). Excessive inflammation and the production of tumour necrosis factor (TNF) leads to tissue injury and cell death (Tracey et al., 1986). The α7nAChR is a member of the nicotinic acetylcholine receptor family and has been shown to have an important role in anti-inflammatory pathways (Rosas-Ballina et al., 2009). These receptors are ligand-gated pentameric ion channels that transmit signals for acetylcholine at neuromuscular junctions in both the central and peripheral nervous systems (Leonard and Bertrand, 2001, Marubio and Changeux, 2000). In human macrophages, acetylcholine has been found to target nicotinic receptors and inhibit the release of inflammatory cytokines such as TNF (Borovikova et al., 2000). Acetylcholine agonists such as nicotine have anti-inflammatory effects by inhibition of cytokine synthesis in numerous cell types (Sadis et al., 2007). However, the role of the α7nAChR during ischemia in the central nervous system is not completely understood.
In rats, stimulation of the α7nAChR has been shown to attenuate apoptosis induced by subarachnoid haemorrhage by down-regulation of caspase-3 activity via increased Ak2 phosphorylation (Duris et al., 2011). More specifically, in neurons, activation of Akt leads to inhibition of glycogen synthase kinase-3β (GSK-3β) (Grimes and Jope, 2001), an enzyme that shows increased expression following oxygen-glucose deprivation in ischemic stroke (Valerio et al., 2011). During ischemia, phosphorylation of
GSK-3β in specific residues involved in its activity is increased, and the activated GSK- 3β induces caspase-3 activation and subsequent apoptosis (King et al., 2001). In addition, inhibition of GSK-3β also increases the expression of β-catenin, an important component of the blood brain barrier (Nelson and Nusse, 2004). Hence, increased phosphorylation of Akt by α7nAChR activation leads to decreased GSK-3β activity, thereby attenuating apoptosis and blood brain barrier degradation following ischemia.
Furthermore, studies using murine models of intra-cerebral haemorrhage have shown that activation of the α7nAChR via PHA-543613 (an agonist of the α7nAChR) leads to attenuation of behavioural deficits such as sensorimotor function, reflexive motor ability, and spatial exploratory and cognitive performance (Krafft et al., 2012). In contrast, treatment with MLA (a selective α7nAChR antagonist) leads to exacerbation of these behavioural deficits (Krafft et al., 2012). The authors suggested that the activation of the α7nAChR lead to both anti-apoptotic and anti-inflammatory effects via the inhibition of pro-inflammatory cytokines. More recently, studies have shown that PNU 282,987, a selective α7nAChR agonist, protects against hepatic ischemia-reperfusion injury (Li et al., 2013). It was found that treatment with PNU 282,987 attenuated liver injury following ischemia-reperfusion by suppression of cytokine and NF-κB activation. These findings further suggest that activation of the α7nAChR leads to attenuation of apoptosis and inflammation.
Interestingly, a recent study found that hippocampal cultures treated with melatonin during reperfusion protected against OGD (Parada et al., 2014). The protective effect of melatonin was prevented when co-treated with α-bungarotoxin, a selective α7nAChR antagonist, indicating that the α7nAChR plays a critical role in the protective
effect of melatonin against OGD (Parada et al., 2014). The authors suggest that the activation of the α7nAChR and subsequent overexpression of heme oxygenase-1, a key enzyme in the protection against oxidative and inflammatory stress (Kim et al., 2005), greatly contributes to the neuroprotective effect of melatonin (Parada et al., 2014). In vivo
studies demonstrate that melatonin administration following stroke results in reduced infarct size and attenuated behaviour deficits, while administration of MLA partially blocks the protective effect of melatonin (Parada et al., 2014).
Recently the α7nAChR has been identified in signalling pathways of STI1 and PrPC in hippocampal neurons (Beraldo et al., 2010, Ostapchenko et al., 2013a). STI1 binding to PrPC leads to a physical interaction with the α7nAChR, inducing calcium influx required for protection against staurosporine-induced apoptosis via PKA activity (Beraldo et al., 2010). Since calcium influx induced by STI1 is abolished in both PrPC- null neurons or with α7nAChR inhibition, STI1-PrPC interaction is suggested to be upstream of α7nAChR-induced calcium influx (Beraldo et al., 2010). One possible mechanism could be that STI1-PrPC modulates the responsiveness of the α7nAChR to choline present in the extracellular milieu, since the concentration of choline is too low to activate the α7nAChR under basal conditions (Beraldo et al., 2010). The signalling pathways activated by STI1 and α7nAChR overlap, including ERK1/2 inducing neuritogenesis and PKA inducing neuroprotection (Caetano et al., 2008, Beraldo et al., 2010). However, the role of the α7nAChR in OGD-induced apoptosis is not fully understood.