Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens.
BACKGROUND
Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22.


RESULTS
Glutamate-induced apoptotic cell death in neuronal cells was associated with characteristic DNA fragmentation, morphological changes, activation of calpain and caspase-3 as well as the upregulation and/or translocation of AIF from mitochondria into cytosol and nuclei. Our results reveal that primary cortical cells and HT22 cells display different patterns of regulation of these genes/proteins. In primary cortical cells, glutamate induces activation of calpain, caspase-3 and translocation of AIF from mitochondria to cytosol and nuclei. In contrast, in HT22 cells, only the activation of calpain and upregulation and translocation of AIF occurred. In both cell types, these processes were inhibited/reversed by 17beta-estradiol and Delta8,17beta-estradiol with the latter being more potent.


CONCLUSIONS
Depending upon the neuronal cell type, at least two mechanisms are involved in glutamate-induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpain and AIF. Since HT22 cells lack caspase-3, glutamate-induced apoptosis is mediated via the caspase-independent pathway in this cell line. Kinetics of this apoptotic pathway further indicate that calpain rather than caspase-3, plays a critical role in the glutamate-induced apoptosis. Our studies further indicate that glutamate- induced changes of these proteins can be inhibited by estrogens, with Delta8,17beta-estradiol, a novel equine estrogen being more potent than 17beta-estradiol. To our knowledge, this is the first demonstration that glutamate-induced apoptosis involves regulation of multiple apoptotic effectors that can be inhibited by estrogens. Whether these observations can help in the development of novel therapeutic approaches for the prevention of neurodegenerative diseases with estrogens and calpain inhibitors remains to be investigated.
Zhang
YueMei
Y
Department of Obstetrics and Gynecology, University of Toronto, Institute of Medical Sciences, Canada. ymzhang@hotmail.com
Bhavnani
Bhagu R
BR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000494:pharmacology*; D018698:Glutamic Acid / Q000633:toxicity*; D006736:Horses; D051379:Mice; D009410:Nerve Degeneration / Q000139:chemically induced / Q000188:drug therapy / Q000503:physiopathology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D051033:Apoptosis Inducing Factor; D061945:Caspase Inhibitors; D004967:Estrogens; D018696:Neuroprotective Agents; C494830:AIFM1 protein, mouse; D018698:Glutamic Acid; D004958:Estradiol; D002154:Calpain; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases