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Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases. In vitro modeling of excitotoxic injury has shown that activation of G-protein coupled receptors (GPCRs) counteracts such injury through modulation of neuronal pro-survival pathways and/or NMDA receptor signaling. We have previously demonstrated that the GPCR APJ and its endogenous neuropeptide ligand apelin can protect neurons against excitotoxicity, but the mechanism(s) of this neuroprotection remain incompletely understood. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that (i) apelin activates pro-survival signaling via inositol trisphosphate (IP3), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase-1/2 (ERK1/2) to protect against excitotoxicity, and (ii) apelin inhibits excitotoxic signaling by attenuating NMDA receptor and calpain activity, and by modulating NMDA receptor subunit NR2B phosphorylation at serine 1480. These studies delineate a novel apelinergic signaling pathway that concurrently promotes survival and limits NMDA receptor-mediated injury to protect neurons against excitotoxicity. Defining apelin-mediated neuroprotection advances our understanding of neuroprotective pathways and will potentially improve our ability to develop therapeutics for excitotoxicity-associated neurodegenerative disorders.
Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases, and further understanding of endogenous mechanisms that regulate NMDA receptor signaling is critical for the development of effective therapeutics (Hardingham & Bading 2010). NMDA receptors include two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits of four types (NR2A - NR2D; (Cull-Candy & Leszkiewicz 2004), and these subunits demonstrate well-defined developmental and regional distribution in rodent models and in humans (Monyer et al. 1994, Standaert et al. 1996, Conti et al. 1999, Law et al. 2003). Phosphorylation of these subunits, particularly within the C-terminus of NR2B, can differentially regulate receptor function and susceptibility to excitotoxicity (Waxman & Lynch 2005, Chen & Roche 2007). Specifically, Src family kinases phosphorylate NR2B tyrosine residues Y1336 and Y1472, and in general, potentiate NMDA receptor currents to enhance excitotoxicity (Salter & Kalia 2004). Casein kinase-2 (CK2) phosphorylates NR2B serine residue S1480 (Chung et al. 2004, Sanz-Clemente et al. 2010), which has been indirectly implicated in neuroprotection against excitotoxicity (Clapp et al. 2009). Our studies have focused on HIV infection of the CNS as an excitotoxicity disease model, and we have shown that neuronal vulnerability to HIV neurotoxicity is dependent upon developmentally-regulated expression of NR2B (O’Donnell et al. 2006). Additionally, NR2B phosphorylation has been implicated in susceptibility to excitotoxicity in various HIV neurotoxicity models (Haughey et al. 2001, Viviani et al. 2006, Eugenin et al. 2007).
Accordingly, G-protein coupled receptors (GPCRs) may protect against HIV-induced excitotoxicity via NMDA receptor modulation (Kaul & Lipton 1999, Meucci et al. 2000, Bruno et al. 2000). The GPCR APJ receptor and its endogenous neuropeptide ligand apelin are highly expressed in the CNS, especially in cortical, hippocampal, and hypothalamic neurons, with comparable distribution between rodents and humans (De Mota et al. 2000, Lee et al. 2000, Reaux et al. 2002, Kleinz & Davenport 2005). We have shown that treatment of rodent hippocampal neuronal cultures with several native apelin isoforms, including apelin-36, phosphorylates extracellular signal-regulated kinase-1/2 (ERK1/2) and protects against excitotoxicity (O’Donnell et al. 2007). However, the mechanism(s) of apelin-mediated neuroprotection remain largely unknown, including clear delineation of the neuronal apelinergic G-protein coupled signaling pathway and potential modulation of NMDA receptors. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that apelinergic signaling concurrently promotes survival via inositol trisphosphate (IP3), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and ERK1/2 activation and limits excitotoxicity by modulating NR2B S1480 phosphorylation and attenuating NMDA receptor-mediated ionic currents, Ca2+ accumulation, and calpain activation. Targeting apelinergic signaling may have therapeutic value for disorders involving excitotoxicity, including HIV-associated neurocognitive disorders, ischemia, epilepsy, Huntington’s disease, Parkinson’s disease and Alzheimer’s disease (Waxman & Lynch 2005, Hardingham & Bading 2010).
Detailed information regarding materials, quantification of excitotoxicity in primary rat brain cultures, calcium imaging, electrophysiology and Western blotting can be found as Supplemental Methods.
Primary rat cerebrocortical cultures were prepared from embryonic day 17 Sprague-Dawley rat pups as previously described (Brewer 1995, Wilcox et al. 1994). All procedures were within the ARRIVE guidelines for animal research, and in accordance with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Cells were plated in tissue culture dishes pre-coated with poly-L-lysine (Peptides International, Louisville, KY) and maintained in neurobasal media plus B27 supplement (Invitrogen, Carlsbad, CA) at 37 °C/5% CO2. Half of the media was replaced every 7 days, and cultures were used between 14 days in vitro (DIV) and 18 DIV.
HIV infection of monocyte-derived macrophages was performed as previously described (Chen et al. 2002, O’Donnell et al. 2006), and in accordance with protocols approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Briefly, macrophages were infected with a CSF-derived, macrophage-tropic HIV-1 isolate (HIV-Jago) from a patient with confirmed HIV-associated dementia. Viral stocks were prepared by the University of Pennsylvania Center for AIDS Research Virology Core. Supernatants from HIV-infected or non-infected (vehicle) macrophages were collected and monitored for HIV replication by quantifying viral reverse transcriptase activity as the amount of radiolabeled deoxythymidine incorporation (Ho et al. 1992).
Cell survival was quantified by three methods: (i) cell counting of microtubule associated protein-2 (MAP-2)- and glial fibrillary acidic protein (GFAP)-reactive cells (O’Donnell et al. 2006); (ii) cell-based MAP-2 ELISA assay (Wang et al. 2007, White et al. 2011); and (iii) lactate dehydrogenase (LDH) assay.
Primary rat cerebrocortical cells were plated at a density of 2×105 cells per 35mm glass bottom dish (MatTek Corporation, Ashland, MD), and intracellular neuronal Ca2+ concentration was determined as previously described (Wu et al. 2007, Lynch et al. 2001).
Primary rat cerebrocortical cells were plated at a density of 2–4×105 cells per 35mm dish with glass coverslips, and standard whole-cell voltage clamp techniques were used for recording NMDA-induced ionic currents. Treatments applications were performed under the following paradigm: 2sec agonist, 1min wash, 2min treatment, 2sec agonist, 3min wash.
NR2B at serine 1480 was mutated to alanine (S1480A) in the mammalian expression vector pRK7-NR2B using the QuikChange II Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA) as previously described (Wu et al. 2007) using published primers (Chung et al. 2004).
HEK293 cells were plated in tissue culture dishes pre-coated with poly-D-lysine (Sigma) and maintained in minimum essential media plus 7.5% fetal bovine serum, 2.5% horse serum, 1% penicillin/streptomycin, and 1% L-glutamine at 37 °C/5% CO2. Transfections were performed using calcium phosphate in the presence of ketamine (500μM, Sigma) to prevent NMDA receptor activation (Grant et al. 1997). Treatments were administered 16–18hr post-transfection.
HEK293 cells were plated in 24-well plates with glass coverslips, and following transfection and experimental treatments, cultures were incubated in Hoescht 33342 (5μg/ml) for 20min at room temperature and fixed in 4% paraformaldehyde in PBS for 20min at 4°C. The number of surviving GFP-positive cells was estimated by blinded counting of GFP- and Hoescht-positive cells from 3–5 randomly selected fields per coverslip at 20x magnification.
HEK293 cells or primary rat cerebrocortical cells were plated at a density of 1×106 cells per 60mm dish or 4×105 cells per 35mm dish. Following experimental treatments, whole cell lysates were subjected to SDS-PAGE as previously described (O’Donnell et al. 2006).
For quantification of excitotoxicity, Western blotting, and electrophysiology, values are expressed as mean ± standard error of mean, and statistical comparisons were made by Student’s t-test or one-way ANOVA plus Newman-Keuls post hoc testing as indicated in the figure legends. For Ca2+ imaging, statistical comparisons for populations of individuals neurons were made by the Kruskal-Wallis test plus Dunn’s Multiple Comparison post hoc testing, and correlations were made by Spearman’s test. All graphs were generated and statistical analyses were performed using GraphPad Prism software (San Diego, CA), and values of p<0.05 were considered significant.
Several in vitro models have shown that HIV infection in the CNS causes release of soluble excitotoxins, especially glutamate, from productively infected macrophages (Kaul et al. 2005). Glutamate and supernatants from HIV-infected macrophages cause neurotoxicity in primary rat hippocampal and cerebrocortical cultures dependent upon developmentally-regulated expression of NR2B (14 days in vitro and older; (O’Donnell et al. 2006)). To define the mechanism(s) of apelin-mediated neuroprotection, we used 14–18 days in vitro fetal rat cerebrocortical neuronal/glial cultures exposed to glutamate or HIV-infected macrophage supernatants, each of which caused dose-dependent excitotoxicity (Fig. 1; Supplemental Fig. 1; Supplemental Fig. 2). Treatment with a native apelin peptide (apelin-36), but not a negative control peptide (apelin-36 scramble), showed robust, dose-dependent neuroprotection against both glutamate and HIV supernatant insults, while apelin in the absence of insult had no effect on neuronal survival (Fig. 1b, 1d). Interestingly, MK801 in the absence of insult induced some neurotoxicity (Fig. 1d), consistent with studies suggesting that elimination of all NMDA receptor activity can cause cell death (Papadia et al. 2005). Together, these results demonstrate that apelin protects cerebrocortical neurons against excitotoxicity in a dose-dependent manner.
We next sought to identify signaling pathways underlying apelin-mediated neuroprotection using specific pharmacologic inhibitors (Fig. 2). Apelin caused phosphorylation of ERK1/2 at the kinases’ active sites (Fig. 2a). Apelin-induced ERK1/2 phosphorylation was blocked by 2-APB, which inhibits Ca2+ release from IP3 receptors, and by MEK1/2 inhibitors U0126 and PD98059, consistent with Ca2+-dependent GPCR activation of ERK1/2 (Fig. 2a, Fig. 8, (Gutkind 2000)). Apelin also caused PKC phosphorylation at its active site, which is required prior to Ca2+ and/or lipid second messenger binding for PKC activation (Gould & Newton 2008). As expected, apelin-induced PKC phosphorylation was blocked by GF109203X and chelerythrine chloride, which inhibit conventional and novel PKC isoforms, but not by 2-APB, as PKC phosphorylation occurs before its activation by Ca2+ (Fig. 2b). Notably, 2-APB, U0126, PD98059, GF109203X, and chelerythrine chloride all blocked apelin neuroprotection against glutamate, while a negative control epidermal growth factor tyrosine kinase inhibitor AG1478 did not (Fig. 2c). Notably, similar effects were seen at apelin concentrations of 20μM (Fig. 2c) and 2μM (data not shown). Together, these studies demonstrate that apelin promotes neuronal survival against excitotoxicity by activating IP3, PKC, MEK1/2, and ERK1/2 intermediaries.
Additional studies investigated other pathways implicated in apelinergic signaling, including the phosphoinositide 3-kinase–Akt–mammalian target of rapamycin pathway (Masri et al. 2005, Japp & Newby 2008), but pharmacologic inhibitors caused significant neurotoxicity (data not shown), thereby preventing conclusive analysis.
In addition to activation of pro-survival signaling pathways, some GPCR ligands can induce neuronal Ca2+ transients and inhibit NMDA receptor-mediated Ca2+ influx and accumulation to protect against excitotoxicity (Meucci et al. 2000, Deiva et al. 2004, Limatola et al. 2005, Yao et al. 2009). Similarly, apelin induced dose-dependent, oscillatory Ca2+ transients in cerebrocortical neurons (Fig. 3a-d), consistent with our previous findings in human NT2.N neuronal cell lines (Choe et al. 2000). Expanding on these findings, apelin-induced Ca2+ transients were below the maximum Ca2+ concentration, as determined by exposure to the Ca2+ ionophore ionomycin (Fig. 3a-d), and decayed within 10min post-treatment (data not shown), suggesting that apelin causes sub-maximal and reversible Ca2+ accumulation. Furthermore, apelin inhibited Ca2+ accumulation induced by NMDA plus glycine (Fig. 3e), and neurons with the largest Ca2+ response to apelin showed the greatest attenuation of NMDA receptor-mediated Ca2+ accumulation (Fig. 3f). This result directly links apelin induction of Ca2+ transients with attenuation of NMDA receptor-mediated Ca2+ accumulation. Additional studies demonstrated that apelin can attenuate Ca2+ accumulation at higher NMDA receptor agonist concentrations (200μM NMDA plus 100μM glycine), and that apelin did not affect potassium-depolarization-induced Ca2+ accumulation (data not shown), suggesting that apelin modulation of NMDA receptors is not due to non-specific inhibition of neuronal Ca2+ responses.
As apelin effectively blocks NMDA receptor-mediated Ca2+ accumulation, we reasoned that apelin might also directly attenuate NMDA receptor-mediated ionic currents. Using whole-cell voltage clamp electrophysiology in cerebrocortical neurons, apelin demonstrated a dose-dependent inhibition of NMDA-induced currents, while apelin-36 scramble showed no effect (Fig. 4). These studies demonstrate that rapid apelin-mediated signaling events can directly inhibit NMDA receptor activity as measured by ionic currents and Ca2+ accumulation.
Excessive NMDA receptor activity can ultimately lead to Ca2+-dependent activation of calpains (Goll et al. 2003), and calpain inhibitors can provide neuroprotection against glutamate- or HIV-induced excitotoxicity (Supplemental Fig. 3a, (O’Donnell et al. 2006, Wang et al. 2007)). As expected, treatment with apelin inhibited NMDA-induced calpain activation, as measured by calpain-specific cleavage of the cytoskeletal protein spectrin (Fig. 5). Moreover, apelin in the absence of NMDA did not modulate calpain activation (Fig. 5), again suggesting that apelin causes sub-maximal and reversible Ca2+ accumulation that is insufficient to either activate calpain or cause excitotoxicity. Together, these studies demonstrate that apelin attenuates excessive NMDAR activity and calpain activation, suggesting that apelin promotes neuronal survival by inhibiting excitotoxic NMDA receptor signaling.
NR2B-containing NMDA receptors are the primary mediators of excitotoxicity in our model system (Supplemental Fig. 3b–c, (O’Donnell et al. 2006)), and NR2B phosphorylation can differentially regulate susceptibility to excitotoxicity (Salter & Kalia 2004, Chen & Roche 2007). Thus, we sought to identify the effects of apelin on NR2B phosphorylation as a mechanism for neuroprotection. Apelin increased NR2B phosphorylation at S1480 in the presence of NMDA (Fig. 6a) without altering NR2B phosphorylation at Y1336 or Y1472 (data not shown). Treatment with 2-APB or TBB, a competitive inhibitor of CK2, prior to apelin and NMDA exposure blocked S1480 phosphorylation (Fig. 6b), consistent with Ca2+-dependent CK2 phosphorylation of S1480 (Chung et al. 2004, Sanz-Clemente et al. 2010). Neither PKC inhibitor GF109203X nor MEK1/2 inhibitor PD98059 blocked apelin-induced phosphorylation of S1480 (Fig. 6b). Additionally, TBB blocked apelin-mediated neuroprotection against glutamate, while the Src family kinase inhibitor PP2, which blocks phosphorylation at Y1336 and Y1472, had no effect (Fig. 6c). Similar effects were seen at apelin concentrations of 20μM (Fig. 2c) and 2μM (data not shown). Together, these studies demonstrate that apelin-induced phosphorylation of NR2B S1480 during NMDA receptor activation may contribute to apelin neuroprotection.
To further validate the role of NR2B S1480 phosphorylation in apelin-mediated neuroprotection, we transiently transfected HEK293 cells with expression plasmids containing green fluorescent protein (GFP), APJ, NR1, postsynaptic density-95 (PSD-95), and wild-type (WT) NR2B or NR2B with S1480 mutated to alanine (S1480A), which cannot be phosphorylated (Lim et al. 2002). Consistent with our studies in neurons, exposure to apelin and NMDA induced S1480 phosphorylation in WT NR2B-expressing cells, but not S1480A-expressing cells (Fig. 7a). Similarly, apelin protected WT NR2B-expressing cells, but not S1480A-expressing cells, against glutamate-induced excitotoxicity (Fig. 7b). These studies further suggest that apelin-induced phosphorylation of NR2B S1480 contributes to apelin neuroprotection.
Previous work in our laboratory demonstrated that apelin can protect neurons against excitotoxicity (O’Donnell et al. 2007), and in this study, we sought to more fully define the mechanism(s) of this neuroprotection. We now propose a novel pathway in which apelin triggers signaling through IP3, Ca2+, CK2, PKC, MEK1/2, ERK1/2, and NR2B S1480 phosphorylation to concurrently promote survival and limit NMDA receptor-mediated excitotoxic activity (Fig. 8). Therefore, targeting the apelinergic system in the CNS may have significant and novel therapeutic value, especially for HIV and other neurodegenerative disorders in which excitotoxicity is implicated.
We have shown that apelin dose-dependently protects cerebrocortical neurons against HIV- or glutamate-induced excitotoxicity via IP3-, PKC-, MEK1/2- and ERK1/2-mediated signaling pathways (Fig. 1–2). Although the G-protein coupling of APJ, apelin’s receptor, is unknown in neurons, apelinergic signaling in heterologous systems and in non-neuronal cells occurs through Gi/o or Gq (Masri et al. 2005, Japp & Newby 2008). Gi/o and Gq can activate phospholipase C (PLC) to hydrolyze phosphatidylinositol bisphosphate (PIP2) into IP3 and diacylglcerol (DAG), causing Ca2+ release through endoplasmic reticulum IP3 receptors and subsequent activation of several PKC isoforms, MEK1/2, and ERK1/2 (Fig. 8; (Gutkind 2000)). Thus, our findings are consistent with Gi/o- or Gq-coupling of apelinergic signaling in primary neurons (Fig. 2).
Apelin triggers neuronal Ca2+ transients typical of GPCR signaling, and our inhibitor studies indirectly implicate IP3 receptor activation in these transients (Fig. 2–3). However, we have not ruled out the contribution of other Ca2+ channels in apelinergic signaling. Recent studies suggest that neuroprotective chemokine GPCR ligands that trigger IP3 signaling can activate transient receptor potential canonical channels, a superfamily of Ca2+-permeable channels that shunt out intracellular Ca2+ during NMDA receptor activation (Yao et al. 2009). Apelin modulation of such channels remains a possibility for further investigation.
Our inhibitor studies also indicate apelin activation of conventional (α, βI/βII, γ) and/or novel (δ, ε, η, θ) PKC isoforms (Fig. 2). PKC activation requires sequential phosphorylation at three sites followed by binding of Ca2+ and/or lipid second messengers, such as DAG. Conventional PKC isoforms require Ca2+ and DAG for activation, novel isoforms require DAG only, and atypical isoforms (ι/λ, ζ) require neither (Gould & Newton 2008). Apelin-mediated PKC phosphorylation was blocked by inhibitors of conventional and novel PKC isoforms, GF109203X and chelerythrine chloride, but not by 2-APB, since PKC phosphorylation occurs prior to its activation by Ca2+ (Fig. 2). However, 2-APB blocked apelin-mediated ERK1/2 phosphorylation (Fig. 2), suggesting that Ca2+-dependent conventional PKC isoforms may underlie apelin activation of ERK1/2 (Fig. 8). These findings are inconsistent with studies in APJ-transfected cells in which an inhibitor of atypical PKCs blocked apelin-stimulated ERK1/2 activation (Masri et al. 2004), indicating that apelinergic signaling in primary neurons might be distinct from that observed in cell lines.
We have also shown that apelin induces Ca2+ transients and attenuates excitotoxic NMDA receptor and calpain activity (Fig. 3–5). Furthermore, we correlate increased Ca2+ response to apelin with decreased response to NMDA, directly linking apelin-induced Ca2+ transients to NMDA receptor attenuation (Fig. 3f). Recent work has implicated apelin at lower concentrations than used in our study in modulation of NMDA-mediated Ca2+ accumulation and injury (Zeng et al. 2010). However, several key disparities suggest a substantially different mechanism of low-dose apelin neuroprotection, including no apelin-induced Ca2+ transients, greatly delayed kinetics of NMDA-mediated Ca2+ accumulation, decreased severity of the excitotoxic insult, and diminished rescue with apelin (Zeng et al. 2010). Moreover, neuronal signaling pathways activated by low-dose apelin have not been addressed, whereas our study outlines a detailed cascade linked to direct modulation of NMDA-induced Ca2+ accumulation as well as ionic currents, calpain activity and phosphorylation state (Fig. 8). Nonetheless, difference in dose-dependent apelin signaling can inform development of apelinergic therapeutics and remain an area of ongoing study in our laboratory.
Apelin can induce phosphorylation of NR2B S1480 through Ca2+- and CK2-dependent mechanisms to provide protection against excitotoxicity (Fig. 6–7), and the phosphorylation of S1480 is of emerging importance in the regulation of synaptic plasticity and excitability (Chung et al. 2004, Clapp et al. 2009, Li et al. 2009, Sanz-Clemente et al. 2010). Recent work demonstrates that CK2 preferentially phosphorylates S1480, disrupting NR2B-PSD-95 interactions and causing decreased surface NR2B expression (Chung et al. 2004, Sanz-Clemente et al. 2010). Moreover, S1480 phosphorylation has been indirectly implicated in neuroprotection against excitotoxicity following ethanol withdrawal (Clapp et al. 2009). Therefore, S1480 phosphorylation may decrease NR2B surface expression to limit NMDA receptor-mediated injury, although the specific effects of S1480 phosphorylation on NMDA receptor and calpain activity are yet to be defined. An alternative hypothesis is that CK2 may have direct, PSD-95-independent effects on NMDA receptor-mediated Ca2+ permeability and conductance, as has been shown for other serine/threonine kinases like PKA (Skeberdis et al. 2006). Whether apelin-mediated S1480 phosphorylation modulates NR2B trafficking and/or activity is unknown, and our preliminary studies indicate no consistent effect of apelin on NR2B surface expression (Cook DR and Gleichman AJ, unpublished observations). Studies are currently underway in our laboratory to define the functional consequences of apelin-induced S1480 phosphorylation on NMDA receptors.
The relative contributions of increased pro-survival signaling and decreased excitotoxic NMDA receptor signaling are not clearly distinguished in our current study, and indeed they could be directly linked. Our proposed mechanism suggests that apelin-induced Ca2+ transients can activate canonical GPCR pro-survival signaling through PKC, MEK1/2, and ERK1/2 in conjunction with activation of CK2 and S1480 phosphorylation which may attenuate excitotoxic signaling (Fig. 8). Moreover, events such as PKC and ERK1/2 activation have pleiotropic effects on multiple signaling pathways, and these could certainly impact NMDA receptor modulation, either directly or indirectly. Excitotoxic NMDA receptor activity has also been shown to directly inhibit pro-survival signaling pathways, including ERK1/2 activation (Soriano & Hardingham 2007). Together, these findings suggest that increased pro-survival signaling and decreased excitotoxic NMDA receptor activity are overlapping pathways for apelin-mediated neuroprotection that combine to prevent neuronal injury.
Our study focuses on apelin modulation of excitotoxic NMDA receptor activity, and we have no evidence for complete suppression of NMDA receptor activity by apelin. Indeed, NMDA receptor activity is essential for normal neuronal function and survival, and preservation of physiologic activity is a key therapeutic consideration (Hardingham & Bading 2010). Moreover, physiologic NMDAR activity can actually mitigate excitotoxic cell death. Referred to as preconditioning, a low dose of NMDA (~10μM) prior to an excitotoxic dose (≥20μM) promotes neuronal survival by preferentially activating synaptic NMDA receptors to cause ERK1/2 phosphorylation (Ivanov et al. 2006, Soriano et al. 2006) and attenuation of Ca2+ accumulation and calpain activity caused by the excitotoxic dose (Tauskela et al. 2001). NMDA preconditioning is contingent on the neuroprotective dose enhancing neuronal excitability, characterized by oscillatory, sub-maximal Ca2+ transients, while the toxic NMDA dose triggers non-oscillatory, maximum, and sustained Ca2+ accumulation (Soriano et al. 2006). These preconditioning mechanisms are strikingly similar to our findings with apelin (Fig. 2–5), including our observation that apelin only decreases calpain activity or increases NR2B S1480 phosphorylation in the presence of NMDA (50μM; Fig. 5–7). Furthermore, preliminary studies indicate that apelin increases excitatory postsynaptic potential frequency in a dose-dependent and reversible manner (Cook DR and Gleichman AJ, unpublished observations), suggesting that apelin may cause a large but transient increase in synaptic glutamate that preconditions neurons against excitotoxicity. Overall, our results suggest that apelin does not diminish NMDA receptor activity under physiologic conditions, but only under conditions of excitotoxicity. Further studies to determine the role of apelin in pro-survival NMDA receptor activity are crucial, as effective therapeutics must selectively inhibit excitotoxic signaling while preserving cell survival signaling.
Much current effort is directed towards targeting ubiquitous GPCRs for therapeutics against both CNS and systemic diseases, and similar efforts are underway for targeting the apelinergic system (Lee et al. 2006, Sorli et al. 2006, Ladeiras-Lopes et al. 2008). Because of the remarkable redundancy of GPCR/ligand expression and function, ‘off target effects’ for any such pharmacologic activation of GPCRs are being considered, but are not seen as deterrents for investigating APJ agonists as therapeutics. Further studies in vitro and in vivo are needed to determine the effects of longer-term APJ activation, and we hypothesize that chronic APJ activation would provide persistent neuroprotection via upregulation of pro-survival genes without prolonged and potentially detrimental induction of ERK1/2 signaling or suppression of NMDA receptor signaling due to ligand-induced APJ internalization (Zhou et al. 2003, Lee et al. 2010). Additionally, a neuroactive, nonpeptidic APJ agonist has recently been identified (Iturrioz et al. 2009), and our studies suggest that such small molecule agonists should be considered as potential neuroprotectants for excitotoxicity-associated neurodegenerative disorders.
This work was supported by NIH grants NS043994 (DLK), NS27405 (DLK), NS45956 (DRL), and MH083395 (AJG). DRC was supported by the University of Pennsylvania Training Grant T32 AI07632 in HIV Pathogenesis and by NIH grant F31 NS066791. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. The authors declare no conflicts of interest. We are grateful to Patricia Vance and Lorraine Kolson for expert preparation of human monocyte-derived macrophages and HIV infections, Margaret Maronski for expert preparation of primary rodent cultures, Evan Eisler and Siddharth Kishore for technical assistance with the HEK293 studies, and Samantha Soldan and Lauren O’Donnell for critical review of the manuscript.