Differential expression of TRPM2 and TRPV4 channels and their potential role in oxidative stress-induced cell death in organotypic hippocampal culture
Introduction
Although it has been established that oxidative stress is a major factor in the cell death cascade in many acute and chronic neurodegenerative disorders (Barnham et al., 2004, Valko et al., 2007), the exact cellular mechanisms of the neuronal and astrocytic cell death are not well understood. In particular, it is not clear whether the direct action of reactive oxygen species (ROS) and other free radicals on lipids, proteins and DNA entirely accounts for cell damage, or whether there is also a contribution of other indirect mechanisms, for example excitotoxic damage of neurons resulting from ROS-induced inhibition of glutamate transporter (Trotti et al., 1998).
Indirect damage can also result from prolonged activation of calcium-permeable membrane channels, leading to intracellular calcium overload and cell death (e.g. Bernardi and Rasola, 2007, Berridge et al., 2000, Chakraborti et al., 1999, Miller, 2006). In this regards, one potentially important calcium influx pathway may be the activation of Ca2+-permeable Transient Receptor Potential (TRP) channels (Miller, 2006, Nilius et al., 2007, Ramsey et al., 2006). Of particular interest is a member of the melastatin subfamily TRPM2 (also known as TRPC7 or LTRPC2) that is ROS (and ADP-ribose) gated (Eisfeld and Luckhoff, 2007, Fonfria et al., 2004, Hara et al., 2002, Naziroglu and Luckhoff, 2008, Wehage et al., 2002). Another TRP channel that may contribute to neuronal damage under oxidative stress is a member of the vanilloid subfamily TRPV4 (also known as OTRPC4 or VR-AC) that opens in response to cell swelling, acidic pH and high temperature, factors that are often associated with acute stress (Nilius et al., 2004, Suzuki et al., 2003). TRPM2 and TRPV4 channels are widely expressed in the brain (Guler et al., 2002, Hara et al., 2002, Liedtke et al., 2000, Nagamine et al., 1998) and are known to be involved in intracellular signal transduction. TRPM2 channels have also been implicated in neuronal damage induced by oxidants, amyloid β-peptide and TNF-α (Eisfeld and Luckhoff, 2007, Fonfria et al., 2005, Hermosura and Garruto, 2007, Kaneko et al., 2006), and therefore are regarded as a potential therapeutic target in some neurological disorders including stroke (McNulty and Fonfria, 2005, Nilius et al., 2007). However, the link between activation of TRPM2 channels and cell viability remains controversial in light of a recent report that blockade of channel opening suppressed Ca2+ influx but did not correlate with protection from cell death (Wilkinson et al., 2008). The relationship between TRPV4 channel activation and cell death is still not clear.
Most of the previous studies on the role of TRP channels in cell death have used cell lines or primary cell cultures—models that may not replicate the features of cells in situ. The present study investigated the expression of TRPM2 and TRPV4 channels, and their effects on oxidative stress-induced cell death in organotypic hippocampal slice cultures, the model of choice in neurotoxicological and other studies requiring neuron–glia interaction (e.g. Noraberg et al., 2005). For a comparison, expression of these channels was also examined in the hippocampus obtained from juvenile rats. Oxidative stress was induced in hippocampal slice cultures in two ways: (a) by exogenous application of H2O2, and (b) by increasing endogenous ROS production through blocking glutathione synthesis with buthionine sulfoximine (BSO) or inhibiting glutathione peroxidase with mercaptosuccinate (MCS). The effects of pharmacological blockers of TRP channels on cell viability were compared with those evoked by antioxidants (Trolox and MitoE).
Section snippets
Organotypic hippocampal slice cultures
All procedures were approved by the Animal Ethics Committee of the University of Auckland, in accordance with the New Zealand Government Animal Welfare Act. Hippocampal slice cultures were established as described previously (Lipski et al., 2007, Stoppini et al., 1991). Briefly, P7–9 Wistar rats were deeply anesthetized with CO2 and their brains aseptically removed. Both hippocampi were isolated and sectioned (300 μm) in the coronal plane with a tissue chopper in an ice-cold dissection medium
TRPM2 and TRPV4 mRNAs and proteins are expressed in organotypic hippocampal culture and juvenile hippocampus
To determine whether the mRNAs encoding for TRPM2 and TRPV4 are expressed in organotypic hippocampal cultures, PCR primers flanking the specific TRP channel regions (see Section 2) were designed. RT-PCR was performed using total RNA extracted from the cultures. Gel analysis (Fig. 1A) revealed PCR products with an appropriate size (∼350 bp for TRPM2 and ∼370 bp for TRPV4), indicating the presence of both transcripts. No specific products were obtained from controls that did not contain RNA
TRPM2 and TRPV4 channels in the hippocampus
TRPM2 channels are known to be widely expressed in the brain (Hara et al., 2002, Nagamine et al., 1998) but their regional and cell-specific expression has not been fully characterized. In the hippocampus, the expression of these channels was demonstrated in CA1–CA3 pyramidal neurons with in situ hybridization (Uemura et al., 2005), and a recent study reported their functional expression in primary cultures of hippocampal pyramidal neurons and in acute hippocampal slices (Olah et al., 2009).
Conflicts of interest
None.
Acknowledgements
Funding provided by the Neurological Foundation of New Zealand, AMRF and the Marsden Fund.
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