Inhibition of autophagy has been shown to

Inhibition of autophagy has been shown to alleviate neuronal damage after cerebral ischemia, both in cell culture and rodent models (Koike et al., 2008, Li et al., 2015, Wang et al., 2016, Zhang et al., 2014, Zheng et al., 2014). Therefore, blockage of autophagy is a potential target for prevention of I/R injury-induced neuronal damage. Maintenance of cytoplasmic Ca2+ homeostasis is another strategy to prevent I/R injury, as abnormal influx of Ca2+ through Ca2+ ampk inhibitor contributes to neuronal toxicity (Tuttolomondo et al., 2009). In the current study, increased cytoplasmic Ca2+ concentration was detected in hippocampal neurons 24h after OGD/R. Similar in vitro results have been reported earlier (He et al., 2014). Of note, pretreatment with 3-MA effectively attenuated the increase in cytoplasmic Ca2+ levels, which suggests that prolonged autophagic stress may lead to Ca2+ toxicity and neuronal cell death in OGD/R model. Molecular mechanisms that mediate the interplay between autophagy and calcium homeostasis are not clear. Endoplasmic reticulum (ER) stress is thought to be a common upstream factor that modulates both Ca2+ levels and autophagy (Dubois et al., 2016, Su and Li, 2016). Moreover, abnormal calcium homeostasis has also been reported to alter the autophagic flux (Sasaki et al., 2015), which suggests a loop control between autophagy and calcium homeostasis, and that maintenance of both autophagic activity and intracellular calcium level at an appropriate level is important for maintenance of neuronal cell integrity. In a previous study, Ca2+-permeable AMPA receptor was shown to trigger motor neuron death in sporadic amyotrophic lateral sclerosis (ALS) (Kwak et al., 2010). Consistent with this finding, neuronal damage in our study was accompanied by enhanced mRNA and protein expression of Ca2+-permeable AMPA receptors, including GluR1, GluR2 and GluR3. It is possible that in OGD/R treated neurons, increased GluRs level leads to excitotoxicity and subsequently results in Ca2+ overload. Importantly, the upregulation of GluR1, GluR2 and GluR3 caused by OGD/R was effectively reversed by autophagy inhibition, which indicates that autophagy may mediate the degradation of GluRs in hippocampal neurons. Consistent with this hypothesis, Shehata et al. detected an autophagy-mediated GluR1 clearance in hippocampal neurons (Shehata et al., 2012). To confirm the role of autophagy in modulating GluRs and cytoplasmic Ca2+ homeostasis in damaged neurons, further studies are required to investigate the autophagic flux in cells exposed to OGD/R. In this study, OGD/R induced upregulation of GluR, increased cytoplasmic Ca2+ levels, and reduced neuronal viability in cultured hippocampal neurons. These events appear to be regulated by enhanced autophagy. Autophagy may prove to be a potential target for prevention of hypoxic-ischemic brain injury.
Experimental procedures
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Authors’ contributions
Introduction AMPA-type glutamate receptors underlie most excitatory synaptic transmission in brain. In addition to mediating moment-to-moment signaling, AMPARs undergo activity-dependent functional changes, which mediate aspects of the synaptic plasticity that underlies learning and memory (Anggono and Huganir, 2012; Ehlers, 2000; Huganir and Nicoll, 2013; Malinow and Malenka, 2002; Nicoll et al., 2006; Sheng and Kim, 2002). Molecular manifestations of this plasticity include changes in AMPAR protein synthesis, post-translational modification, channel trafficking, and subunit composition. Assembly of neuronal AMPAR complexes is precisely controlled. AMPARs comprise heterotetramers of the glutamate-binding, pore-forming subunits GluA1–4 (Boulter et al., 1990; Seeburg, 1993). Distinct combinations of GluA subunits and their alternative splicing and post-transcriptional editing impart differential physiological properties to AMPARs (Boulter et al., 1990; Seeburg, 1993). Additionally, AMPAR complexes often contain multiple classes of auxiliary subunits (Kato et al., 2010; Yan and Tomita, 2012). The auxiliary subunit composition and stoichiometry of AMPARs varies, even within a single neuronal type, and this imparts differential properties at specific synaptic types (Coombs and Cull-Candy, 2009; Jackson and Nicoll, 2011). Furthermore, the molecular composition of neuronal AMPARs dynamically changes as part of synaptic plasticity (Bats et al., 2013; Jackson and Nicoll, 2011). Molecular mechanisms that control assembly of AMPARs remain poorly understood.