Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Abstract

Proteolytic cleavage of the Na þ /Ca 2 þ exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca 2 þ dynamics, calpain activation and cell viability, in a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. The sodium-calcium exchanger (NCX) plays a fundamental role in controlling Na þ and Ca 2 þ homeostasis. 1,2 NCX primarily extrudes Ca 2 þ in exchange for Na þ , whereas upon neuronal depolarization, Na þ is pumped out by NCX, while Ca 2 þ is pumped in. In pathophysiological conditions, overactivation of glutamate receptors can cause the reversal of NCX, leading to Ca 2 þ entry into the cell. 3 Three NCX genes have been identified, NCX1, 4 NCX2 5 and NCX3. 6 In excitotoxic cell death, an increase in intracellular free calcium concentration ([Ca 2 þ ] i ) may directly cause activation of Ca 2 þ -dependent cysteine proteases, the calpains. Calpains modulate a variety of physiological processes, 7 and are important mediators of cell death. 8,9 Calpains mediate the neurotoxic effect of N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons by a caspase-independent cell death mechanism of excitotoxicity. 10 Calpains are also involved in the neurotoxic effect caused by a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons, 11 and in hippocampal slice cultures. 12 During excitotoxic neurodegeneration, calpains are responsible for the proteolysis of several cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters. 13 Recently, the involvement of calpains in the cleavage of NCX was described in cultured cerebellar granule neurons exposed to glutamate and following brain ischemia. 14 The NCX3 subtype is inactivated by proteolytic cleavage by calpains, and is no longer able to pump Ca 2 þ out of the cell, thus enhancing cell death. Furthermore, NCX3 was shown to be more relevant for cell survival than NCX1 or NCX2, namely in cultured cerebellar granule neurons. 14, 15 We recently reported that neurotoxicity induced by activation of AMPA receptors is characterized by calpain activation, lack of caspase activation, nuclear condensation/fragmentation, release of cytochrome c from mitochondria, decreased intracellular ATP levels, production of nitric oxide, moderate superoxide production and increased levels of peroxynitrite. [16][17][18] In this in vitro model of excitotoxicity, the cell

Evaluation of neurotoxic and neuroprotective pathways affected by antiepileptic drugs in cultured hippocampal neurons

“…Lower concentrations of OXC (0.01 and 0.03 mM) did not affect cell viability. The observed toxic effects caused by high concentrations of OXC on the hippocampal neurons are in agreement with previous studies (Ambrosio et al, 2000;Araujo et al, 2007).…”

“…In control cultures, 21.4 ± 1.4% of total cells presented condensed/fragmented nuclei, which is in agreement to previous studies showing similar basal levels of cell death in hippocampal cultures (Almeida et al, 2005;Araujo et al, 2007). Exposure to ESL, S-Lic, RLic, CBZ, LTG or VPA did not increase the percentage of condensed/ fragmented nuclei (p > 0.05) (Supplementary data, Fig.…”

“…Cell viability was assessed in cultured hippocampal neurons, following treatment with different concentrations of AEDs for 24 h, using the resazurin reduction assay, as described previously (Araujo et al, 2007). Briefly, resazurin reduction was measured spectrophotometrically at 570 and 600 nm, following incubation of the cells with 300 ll of 10% resazurin in Krebs buffer (132 mM NaCl, 4 mM KCl, 1.4 mM MgCl 2 , 1 mM CaCl 2 , 6 mM glucose, 10 mM Hepes-Na, pH 7.4), for 2 h at 37°C, in the dark.…”

Loss-of-function of inositol polyphosphate-4-phosphatase reversibly increases the severity of allergic airway inflammation

“…As PEST sequences are calpain protease target sites, and a characteristic PEST motif was also seen in mouse INPP4A, we hypothesized that increased calpain-protease-mediated degradation of INPP4A in mouse lungs may be important in experimental asthma pathogenesis. As an increase in the intracellular free calcium is known to activate the calpain family of proteases 30 , and abnormal calcium homeostasis has been observed in asthma 31,32 , it is very likely to be one of the underlying mechanisms for calpain-mediated INPP4A degradation. Various types of calpain inhibitors have been known to alleviate a myriad of disorders 33,34 .…”

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca2+]i Changes Evoked by Kainate in Hippocampal Neurons

“…The recordings shown above are representative of each cell population, with [Ca 2+ ] i responses below 60% of the control (A), between 60% and 90% of the control (B), and above 90% of the control (C). A total of 58 cells, from four independent cell cultures, were analyzed, and the quantification of the data is shown in [17].…”