BackgroundGlutamate is one of the major neurotransmitters in the central nervous system. It is a potent neurotoxin capable of neuronal destruction through numerous signal pathways when present in high concentration. Glutamate-evoked excitotoxicity has been implicated in the etiology of many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and ischemic stroke. Increasing evidence has shown that reactive oxygen species (ROS) provoked by glutamate-linked oxidative stress plays a crucial role in the pathogenesis of these disorders. We previously reported the discovery of an aryl thiophene compound, 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B355252) from a proprietary library of small molecules. We showed that this compound was capable of potentiating nerve growth factor (NGF)-primed neurite outgrowth in neuronal cell models in a low NGF environment. In the present study we investigated the neuroprotective effects and signaling pathways of B355252 on glutamate-evoked excitotoxicity in HT-22, a murine hippocampal neuronal cell line.ResultsGlutamate significantly decreased HT-22 neuronal cell viability in a concentration-dependent manner as measured by the MTT assay. Co-treatment with 2, 4, and 8 μM B355252 protected against cell death caused by glutamate-induced toxicity by 9.1% (p<0.01), 26.0% (p<0.001), and 61.9% (p<0.001) respectively, compared to glutamate-treated control group. B355252 at a concentration of 8 μM fully rescued HT-22 from the neurototoxic effects of glutamate, and by itself increased cell viability by 16% (p<0.001) above untreated control. Glutamate enhanced reduction in glutathione (GSH) synthesis was reversed by 15% (p<0.01) in the presence of B355252. B355252 reduced the expression of apoptosis inducing factor (AIF) by 27%, while the proapoptotic Bcl-2 associated X protein (Bax) was strongly attenuated 3-fold. Glutamate-evoked increase in intracellular calcium (Ca2+) load and subsequent ROS production was inhibited by 71% (p<0.001) and 40% (p<0.001) respectively, to comparable level as untreated control in the presence of B355252. Glutamate significantly upregulated the phosphorylation of extracellular signal regulated kinase Erk1/2 (pERK1/2), while decreasing Erk3. In contrast, B355252 potently attenuated the glutamate-dependent activation of Erk1/2 and robustly increased the level of ERK3 in HT-22.ConclusionsA novel phenoxy thiophene small molecule, B355252, suppresses glutamate-evoked oxidative stress in HT-22 neurons by blocking Ca2+ and ROS production, and altering the expression or phosphorylation states of Erk kinases. This molecule previously reported to enhance neurite outgrowth in the presence of sub-physiological concentrations of NGF appears to be a promising drug candidate for development as a potential therapeutic and neuroprotective agent for various neurodegenerative disorders.

6-Hydroxydopamine (6-OHDA) is a neurotoxin frequently used to create in vitro and in vivo experimental models of Parkinson's disease (PD), a chronic neurodegenerative disorder largely resulting from damage to the nigrostriatal dopaminergic pathway. No effective drugs or therapies have been developed for this devastating disorder, and current regimens of symptomatic therapeutics only alleviate symptoms temporarily. Therefore, effective treatments that reverse or cure this disorder are urgently needed. The aim of the study described in this report was to investigate the therapeutic impact of B355252, an aryl thiophene sulfonamide chemical entity, in the widely recognized in vitro model of PD, and to characterize the molecular signaling pathways. We show here that 6-OHDA-induced cell death in HT22, a murine neuronal cell model, through a pathway that involves the mitochondria by increasing the levels of reactive oxygen species (ROS), raising intracellular calcium ([Ca(2+)]i), enhancing the release of cytochrome c to the cytosol, and promoting activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway. More importantly, we found that B355252 protected HT22 neurons against 6-OHDA toxin-induced neuronal cell death by significant attenuation of ROS production, blocking of mitochondrial depolarization, inhibition of cytochrome c release, sequestration of [Ca(2+)]i, modulation of JNK cascade, and strong inhibition of caspase 3/7 cleavage. Overall, this study demonstrates that death of neurons under toxic conditions characteristic of PD can be efficiently halted by B355252 and suggests that further development of the molecule could be potentially beneficial as a therapeutic prevention or treatment option for PD.

Glutamate neurotoxicity has been implicated in the initiation and progression of various neurological and neurodegenerative disorders. Therefore, it is necessary to develop therapeutics for the treatment of patients with these devastating diseases. Mitochondrial fission plays an import role in the mediation of cell death and survival. The objective of the present study was to determine whether B355252, a phenoxythiophene sulfonamide derivative, reduces glutamateinduced cell death by inhibiting mitochondrial fission and the nuclear translocation of apoptosis-inducing factor (AIF) in glutamate-challenged HT22 neuronal cells. The results revealed that glutamate treatment led to large increases in the mitochondrial levels of the major fission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1), but only small elevations in the fusion proteins mitofusin 1 and 2 (Mfn1/2) and optic atrophy 1 (Opa1). In addition, glutamate toxicity disrupted mitochondrial reticular networks and increased the translocation of AIF to the nucleus. Pretreatment with B35525 reduced glutamate-induced cell death and prevented the increases in the protein levels of Drp1, Fis1, Mfn1/2 and Opa1 in the mitochondrial fraction. More importantly, the architecture of the mitochondria was protected and nuclear translocation of AIF was completely inhibited by B35525. These findings suggest that the regulation of mitochondrial dynamics is central to the neuroprotective properties of B355252, and presents an attractive opportunity for potential development as a therapy for neurodegenerative disorders associated with mitochondria dysfunction.

Reactive oxygen species (ROS) play a central role in oxidative stress-associated neuronal cell death during ischemia. Further investigation into the inhibition of excessive ROS generation post-stroke is urgently required for the treatment of ischemic stroke. In the present study, the neuroprotective properties of the blood-brain barrier (BBB) penetrant B355227 were investigated. B355227 is a chemical analogue of B355252, and the role of the phenoxythiophene sulfonamide compound B355227 was further investigated in a glutamate-induced oxidative injury model. An in vitro model of the BBB was established in the immortalized mouse brain capillary endothelial cell line, bEnd.3. Formation of barrier in Transwell inserts was confirmed using EVOM resistance meter and Caffeine, Imatinib and Axitinib were used to validate the efficacy of the model. The validated BBB assay in combination with high performance liquid chromatography were used to analyse and verify the permeability of B355227 through the barrier. The integrity of the cell junctions after the BBB assays were confirmed using immunofluorescence to visualize the expression of the barrier junction protein zonula occludens-1. Cell survival was measured with Resazurin, a redox indicator dye, in HT22, a hippocampal neuronal cell treated with 5 mM glutamate or co-treated with the B355227 recovered from the BBB permeability experiment. Changes in glutathione levels were detected using a glutathione detection kit, while analyses of ROS, calcium (Ca 2+ ), and mitochondrial membrane potential (MMP) were accomplished with the fluorescent dyes 2',7'-dichlorofluorescein diacetate, Fura-2 AM and MitoTracker Red dyes, respectively. Immunoblotting was also performed to detect the expression and activation of Erk1/2, p-38, JNK, Bax and Bcl-2. The results of the present study demonstrated that B355227 crossed the BBB in vitro and protected HT22 from oxidative injury induced by glutamate exposure. Treatment of cells with B355227 blocked the glutamate-dependent depletion of intracellular glutathione and significantly reduced ROS production. Increased Ca 2+ influx and subsequent collapse of the MMP was attenuated by B355227. Furthermore, the results of the present study demonstrated that B355227 protected against oxidative stress via the MAPK pathway, by increasing the activation of Erk1/2, JNK and P38, and restoring anti-apoptotic Bcl-2. Collectively, the results of the present study indicate that B355227 has potent antioxidant and neuroprotective attributes in glutamate-induced neuronal cell death. Further investigation into the role of B355227 in the modulation of glutamate-dependent oxidative stress is required.

No abstract