Neurodegenerative diseases share a common mechanism of pathophysiology such as oxidative stress, mitochondrial aberrations, and inflammation, which lead to the degeneration and death of neurons. Developing therapeutics modulators of these universal mechanisms could have a significant impact in the management of these devastating diseases through delay of disease onset or disease progression. The interaction of glutamate with specific membrane receptors is responsible for many neurological actions mediated by neuronal cells in the CNS, including synaptic plasticity, sensation, and movement [32–34]. However, excessive glutamate can lead to neuronal cell death in a variety of pathological conditions which is thought to play a crucial role in the pathogenesis of many neuropsychiatric and neurodegenerative disorders . In a previous study, we described the synthesis and identification of a novel phenoxy thiophene sulphonamide small molecule (B355252) enhancer of neurotrophin-dependent signaling, which promotes neurite outgrowth, extension, and cell survival , functions under assault in many neurological disorders. In the present study we demonstrate the ability B355252 to rescue HT-22 neuronal cells from glutamate-induced neurotoxic injury and sort to define the cellular events that underlie the rescue. Our results strongly suggest that B355252 prevents glutamate neurotoxicity through multiple effects targeted at mitochondria-dependent events including inhibition of Ca2+ overload, depletion of ROS, restoration of glutathione, and expression of the apoptotic proteins AIF and Bax. In addition B355252 exerted its effect by modulating the activity of typical and atypical the Erk family members.
Glutamate induces apoptosis at high concentrations in neurons, and HT-22 cells provide a model system to study glutamate-evoked death signaling pathways that enhance ROS formation and oxidative stress independent of NMDA-receptor.
These cells lack ionotropic glutamate receptors, but are still sensitive to high concentration of extracellular glutamate, which depletes glutathione and causes oxidative toxicity in an Erk-dependent manner. There is wide variation in the literature on the concentration of glutamate that induces oxidative toxicity in HT-22 cells. In these studies, the dose of glutamate used to induce cell death following 24 h treatment varied between 1 mM and 10 mM, while the rate of induction of cell death varied between 10% and 90% [30, 31, 36]. Based on the variations in glutamate concentration in these studies we determined the effective concentration of glutamate for our experiment in a dose response assay. Prolonged treatment with glutamate for 10 h triggered significant concentration-dependent cell death in HT-22 as measured by decrease in cell viability and EB fluorescence staining (Figure 1; Figure 2B). The B355252 dose-dependently protected the cells and consequently prevented the harmful effect of glutamate on HT-22 cells by restoring the cell health of HT-22 to comparable level as that of naïve cells at a concentration of 8 μM (Figure 2A). In addition to its neuroprotective attributes B355252 also exhibited intrinsic proliferative activity by stimulating cell growth in HT-22 neurons. These results indicate that cell death promoted by glutamate toxicity could be ameliorated by B355252 and support a neuroprotective role and therapeutic potential for the compound.
Substantial controversy exists in the literature with regard to how glutamate mediates its toxic effect in HT-22. Glutamate evoked oxidative death result in a time and concentration-dependent manner from mechanisms that involve both necrotic and apoptotic processes [30, 37, 38]. However, apoptosis appears to be more intimately involved in the process at late time points. Previous studies suggest that glutamate induces a particular type of a caspase-independent cell death called oxytosis, which involves the translocation of AIF from the mitochondria to the nucleus [39, 40]. Immunoblot studies with anti AIF antibody show that AIF was marginally increased by glutamate treatment in the HT-22 cells (Figure 3B). This observation implies that AIF plays a role in glutamate-induced death but most likely does not represent a major cell death pathway in our current study, which is in agreement with previously published results . The glutamate induced expression of AIF was inhibited to background level in the presence of B355252 (Figure 3B). It is unclear whether B355252 acts by direct perturbation of AIF translocation, at upstream regulators of AIF such as PARP, or whether the compound destabilizes released AIF and promotes its clearance in the cytosol.
Recent evidence has shown that exposure to glutamate regulates the expression of the proapoptotic Bax protein in HT-22 . Bax specific inhibitors, antioxidants, and anti-inflammatory agents were capable of protecting against glutamate-induced cell death in neurons by blocking the expression of Bax [42, 43]. In our study, immunoblots probed with Bax specific antibody show that glutamate stimulated increased expression of Bax in HT-22 (Figure 3B), which supports the conclusion that prolonged treatment of HT-22 cells with glutamate leads to apoptosis. This observation is in agreement with widely published data on the mechanism of cell death caused by glutamate exposure. Our results show that the glutamate-evoked Bax expression was sharply blunted by B355252. Based on the expression level in the presence of B355252, the substantial reduction in the amount of Bax to a large extent suggests that B355252 is a highly effective inhibitor of a major glutamate cell death pathway caused by the accumulation of proapoptotic Bax protein.
A major event during programmed cell death is an increase in cytosolic Ca2+. Under normal physiological conditions glutamate-induced cell signaling intermediates such as Ca2+ influence a wide variety of cellular components and play a fundamental role in neuronal survival, differentiation, and development of synaptic circuits . However, it has been shown that Ca2+ is a key mediator of numerous cell death pathways and that a complex relationship exists between mitochondrial function, ROS, Ca2+, and cell death . Elevation of intracellular Ca2+ is a hallmark of excitotoxicity triggered by sustained or repeated glutamate exposure in neuronal cells. Ca2+ overload excessively activate Ca2+ signal transducers, which increase the vulnerability of neurons to cell damage or death. Previous studies have shown that inhibition of Ca2+ influx relieves glutamate neurotoxicity in HT-22 cells . In the present study, intracellular Ca2+ in HT-22 cells was significantly elevated by glutamate treatment in agreement with past research findings. Subsequent treatment with B355252 caused a marked decrease in glutamate-induced Ca2+ overload (Figure 4). Since disregulation of the Ca2+ homeostasis has been identified as a key factor in glutamate toxicity [10, 12], the observed effect of B355252 suggest that the compound interferes with glutamate activity or mechanistically restores Ca2+ balance in cells under glutamate assault leading to cell survival.
The production of ROS induced by oxidative stress has been noted in various studies of glutamate toxicity, suggesting that accumulation of ROS play a crucial role in the induction of cell death by glutamate. Previously studies show that protracted exposure of HT-22 to extracellular glutamate prevents cystine uptake into the cells via the cystine/glutamate antiporter, resulting in depletion of intracellular GSH. Both reduced GSH levels and increased ROS formation are established mechanisms that contribute to neuronal death in models of chronic and acute neurodegeneration. Reduced supply of glutathione, leads to influx of extracellular Ca2+ and accumulation of excessive amounts of ROS, which in turn leads to oxidative stress. Furthermore, elevated ROS level results in damage to macromolecules neurons. Excessive ROS must be promptly eliminated from the cell by a variety of antioxidant defense mechanisms that scavenge ROS if cells are to be protected from oxidative damage. In this study we observed that treatment of HT-22 with glutamate resulted in oxidative stress characterized by depletion of GSH, elevated production of ROS, and changes in cell morphology as reported in the literature. Pre-exposure of HT-22 cells to B355252 blocked glutamate-induced death through mechanisms that involve both increase in cellular GSH (Figure 3A) and reduction of ROS (Figure 5). Antioxidant scavengers such as N-acetylcysteine (NAC) and trolox prevent glutamate-induced cell death in HT-22 by sustaining cellular glutathione and reduction of ROS [30, 47]. Thus, the present finding support the conclusion that B355252 acts as oxidant scavenger and the neuroprotection conferred on HT-22 may be dependent in part on its antioxidant attributes.
The superfamily of mitogen-activated protein kinases (MAPKs) which include extracellular signal-regulated kinases (Erks), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase modulate in a variety of cellular function in many cell types . The Erk subfamily comprises 5 different isoforms, Erk1 to Erk5. Although Erks are traditionally viewed as a survival factor recent reports have demonstrated a death-promoting role for Erks in neuronal cells [30, 31, 49]. Erk1/2 has been implicated in glutamate-induced neuronal oxidative toxicity based upon the observation that U0126, a specific inhibitor of the Erk-activating kinase, MEK-1/2, protects both HT-22 cells and immature primary cortical neuron cultures from glutamate toxicity . Administration of U0126 following focal ischemia in rodents led to a reduction in brain injury suggesting that Erk1/2 may also promote neuronal cell death as a consequence of acute injury in vivo. Our results confirmed that U0126 could prevent glutamate-induced cell death in HT-22 by reduction of Erk phosphorylation (data not shown). Similarly, B355252 protected against glutamate toxicity via inhibition of Erk activation (Figure 6), but not JNK or P38 activation (data not shown), clearly demonstrating the involvement of Erk1/2 activation in the protection conferred by B355252. Furthermore, the activation of Erk1/2 in the glutamate excitotoxic model has been tightly linked to ROS production partly through Ca2+-sensitive signals . These Ca2+-permeable pathways upregulates Ca2+ influx, which in turn activates several Ca2+-dependent kinases to increase Erk phosphorylation. However, some research reports have indicated that activation of Erk in HT-22 is independent of ROS accumulation. This conclusion is supported by the observation that U0126 was unable to block the generation of intracellular ROS during activation of Erk 1/2 in a glutamate excitotoxic model . In the current study the production of intracellular ROS by glutamate and activation of Erk1/2 were significantly reduced in cells that are protected by B355252. These data support the view that B355252 unlike U0126 exerts it effects through multiple functional pathways, which influence glutamate-evoked activation of Erk1/2 and accumulation of ROS in promoting cell survival during glutamate toxicity. The mechanisms by which B355252 exerts these actions remain to be determined.
Erk3 is an atypical member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases. Little is known about the biological function of Erk3 and even less about its regulation, substrate specificity, and cellular targets. Erk3 is abundantly expressed in neurons were it is found in both the cytoplasm and nucleus. Although its physiological functions remain to be established, signaling by Erk3 kinase has been theorized to play a role in neuronal morphogenesis and survival and in the regulation of cell growth and differentiation [52, 53]. Recent work has shown that Erk3 interacts with and activates the MAP kinase-activated protein kinase MK5 and has been reported to inhibit S-phase transition in fibroblasts upon serum activation, which suggest that Erk3 may negatively regulate the cell-cycle depending on cellular conditions. However, it is unclear whether Erk3 regulates cell proliferation under physiological conditions. Research has shown that Erk3 kinase increases during differentiation of PC12 into neuronal lineage and that Erk3 mRNA is tightly regulated during mouse development, suggesting a role for Erk3 in embryogenesis [54, 55]. Recently, Erk3 was found to form a ternary complex with MK5 and septin7 to promote dendrite development and spine formation in MK5 mouse knockout suggesting a role in the regulation of neuronal morphogenesis and survival . In our study, glutamate treatment substantially blunted the expression of Erk3 in contrast to increased phosphorylation of Erk1/2. Treatment of cells with B355252 led to increase in the magnitude of Erk3, restoring the expression of the kinase (Figure 7). B355252 alone had no effect on the expression of pERK3, which suggests that pERK3 does not play a role in B355252-dependent cell proliferative activity. Taken together, the results of Erk regulation signify that B355252 protects HT-22 from glutamate-evoked neurotoxicity by opposing the deleterious effects of glutamate through coordinated restoration of typical and atypical Erk kinases.