N-methyl-D-aspartate (NMDA) neurotoxicity in primary rat retinal cultures: neuroprotective action of curcumin

The use of plant-derived supplements for improving health is gaining popularity because most people consider these natural products to be safer and produce less side effects than synthetic drugs (Raskin et al., 2002). Plants contain numerous bioactive molecules that can improve the body’s resistance to cellular stress and prevent the cytotoxicity of various agents. Among them, polyphenols such as curcumin, resveratrol (nonflavonoids), and flavonoids have received much attention for their ability to reduce cellular stress-induced injury (Mattson et al., 2007). Polyphenols inhibit toxin-mediated stress responses via their antinflammatory and antioxidant properties in addition to inducing the expression of cytoprotective proteins (Aggarwal and Sung, 2009; Shakibaei et al., 2007). There are more than 50 different plant species and over 8000 phenolic compounds identified either in single, pure molecular form or in specific proportions of differing plant extracts. Investigating the health benefits of these natural compounds is an enormous challenge to modern medicine. Besides scavenging free radicals, many phenolics also exhibit multiple biological properties, e.g. antinflammatory, anticancer, antiviral, antimicrobial, vasorelaxant, and anticlotting activities (Rahman et al., 2007). In general, these phenolic compounds are rapidly converted to their glucuronide derivatives upon ingestion and are transported to the circulatory system and different body organs including the brain. In recent years, a number of reviews have reported on neuroprotective effects of polyphenols in cell and animal models (Wang et al., 2001; Dajas et al., 2003; Mandel and Youdim, 2004; Simonyi et al., 2005). Curcuminoids, the main components in Curcuma species, share a common unsaturated alkyl-linked biphenyl structural feature responsible for their major pharmacological effects. Curcuminoids in Curcuma longa and other Curcuma species are mainly curcumin, bis-demethoxycurcumin and demethoxycurcumin, among which curcumin is the most studied and shows a broad range of biological activities. Curcumin (1,7-bis(4-hydroxy 3-methoxy phenyl)-1,6-heptadiene3,5-dione), a phenolic compound extracted from the rhizome of Curcuma longa, has a unique conjugated structure including two methylated phenols linked by the enol form of a heptadiene-3,5-diketone that gives the compound a bright yellow color. Curcumin is used worldwide as spice, flavoring agent, food preservative and coloring agent and is currently used also as a key ingredient in both cosmetics and pharmaceuticals. In Asian countries, curcumin is regarded as an herbal medicine to treat different diseases such as chronic inflammation, hepatic diseases and anorexia. Curcumin is non-toxic even at high dosages and has been classified by the United States Food and Drug Administration as GRAS (Generally Recognized As Safe) (Chainani-Wu N., 2003). A large body of evidence suggests that curcumin has various biological activity and potential therapeutic effects on numerous pathological disorders including type II diabetes, rheumatoid arthritis, multiple sclerosis and cancer (Aggarwal et al., 2003; Aggarwal and Haikumar, 2009). In the central nervous system, curcumin administration has been reported to attenuate cognitive deficits, neuroinflammation, and plaque pathology in Alzheimer disease models (Frautschy et al., 2001; Yang et al., 2005; Garcia-Alloza et al., 2007). Moreover it shows neuroprotective potential in cerebral ischemia (Thiyagarajan and Sharma, 2004; Zhao et al., 2008) and against excitotoxicity in cerebral cortical neurons and retinal cultures (Wang et al., 2008; Matteucci et al., 2005). To conduct more detailed studies about this molecule could render in the future curcumin a safe and inexpensive drug contributing to the prevention and treatment of various neurodegenerative disorders. However its efficacy has been object of controversy, and its mechanism of action remains obscure. Glutamate receptors are categorized into two main classes: ionotropic and metabotropic glutamate receptors. The ionotropic glutamate receptors are described as either NMDA (NMDAr) or non-NMDA subtypes; both receptor types are glutamate-gated cation channels that convert a chemical signal (glutamate released from presynaptic terminals) to an electric signal (a membrane voltage change due to cation flow through the channels). NMDArs are characterized by a high permeability to Ca2+, voltage-dependent block by Mg2+, and slow gating kinetics. These receptors are known to be involved in a variety of physiological processes in the CNS. Because NMDArs are highly permeable to Ca2+, in addition to their physiological roles, the activity of NMDArs is closely related to glutamatecaused excitotoxicity in central nervous system. Excitotoxicity is thought to be a major mechanism in many human disease states such as ischemia (Doyle et al. 2008), trauma, epilepsy and chronic neurodegenerative disorders. Briefly, synaptic overactivity leads to the excessive release of glutamate that activates postsynaptic cell membrane receptors, which upon activation open their associated ion channel pore to produce an abnormal ion influx and the resulting neuronal damage and death. In the retina, diseases that are associated with excitotoxicity include glaucoma, retinal ischemia, and diabetic retinopathy (Wax and Tezel, 2002; Kim et al., 2000; Neal et al., 1994; Quigley, 1999; Ng et al., 2004), in which excessive stimulation of glutamate receptors, including NMDArs, has been demonstrated to result in the degeneration of retinal neurons. Retina is generally thought to be an excellent model for the understanding of the neural mechanisms underlying elementary neural information processing in the brain. It is easily accessible and it comprises only a few classes of neurons, which are organized into clearly distinct sublayers. We performed our experiment in primary rat retinal cell cultures, which are considered a useful tool, with broad applications in the field of ophthalmology. With a balanced approach, in conjunction with animal studies, in vitro studies can provide important basic information about normal retinal functions. In a previous work, we have described the protective effect of curcumin against NMDA-induced excitotoxicity in rat retinal cultures, suggesting a mechanism involving modification of NMDAr activity. Here we have further investigated curcumin modulation of NMDAr using electrophysiological and biochemical approaches. We have identified in the increased level and functional activity of the NMDAr subunit type 2A (NR2A), related to CaMKII activation, a mechanism of curcumin neuroprotection against excitotoxicity.