A novel phenoxy thiophene sulphonamide molecule protects against glutamate evoked oxidative injury in a neuronal cell model

Abstract

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.

Neuronal Nitric Oxide Synthase-Mediated Genotoxicity of 2-Methoxyestradiol in Hippocampal HT22 Cell Line

“…HT22 cells have often been used as a tool to screen for novel neuroprotective reagents [39,40]. Moreover, this cell line does not express functional ionotropic glutamate receptors, thus cell death excitotoxicity can be excluded when using HT22 cells [41].…”

Researching glutamate – induced cytotoxicity in different cell lines: a comparative/collective analysis/study

“…This toxicity is exerted by reduction in GSH production through the CySS/glutamate exchanger ( Tan et al, 1998 ; Zaulyanov et al, 1999 ; Xu et al, 2007 ; Grohm et al, 2010 ; Chen et al, 2011 ; Tobaben et al, 2011 ; Poteet et al, 2012 ; Figure 4 ). Mitochondrial oxidative stress and dysfunction are important preceding events promoting glutamate induced cell death in HT-22 ( Suh et al, 2007 ; Gliyazova et al, 2013 ; Shah et al, 2014 ). Treatment with glutamate (i) alters MMP (ii) induces mitochondrial cytochrome c release (iii) release of mitochondrial AIF, which catalyzes DNA fragmentation and apoptosis.…”

“…In vitro evidence support that glutamate treated HT22 cells, exhibit a delayed and persistent activation of ERKs which contributes to oxidative toxicity ( Stanciu et al, 2000 ). In HT-22 cell line glutamate significantly up regulates the phosphorylation of ERK1/2 while decreasing Erk3 ( Gliyazova et al, 2013 ). Furthermore glutamate treated HT22 increase intracellular Ca 2+ levels by means of activation of cobalt-sensitive channels ( Finkbeiner and Greenberg, 1996 ).…”

Orexin A attenuates palmitic acid-induced hypothalamic cell death

“…Cells were pretreated with OXA (or vehicle) for 24 h,then challenged with PA (or vehicle) plus OXA (or vehicle) for 2 h. During the last hour of challenge, the cell-permeable deep red dye was added to cells and allowed to incubate at 37°C and 5% CO 2 . Intracellular superoxide and hydroxyl radicals oxidize the deep red dye producing a fluorescent signal (Gliyazova et al, 2013). Fluorescence was measured at 650 EX /675 EM using a spectrophotometer (SpectraMax-M5).…”