NMDA receptors play important and diverse roles in CNS function, ranging from the regulation of synaptic plasticity and neuronal growth and survival to the initiation of cell death. It is generally agreed that mitochondrial calcium (Ca2+) overload and subsequent dysfunction, due to excessive Ca2+ entry through glutamate-overactivated NMDA receptors (NMDARs), are crucial early events in excitotoxic injury. However, the generally disappointing patient outcomes for anti-excitotoxic therapies targeted to glutamate receptors strongly suggests that in the real world additional factors are at play. This in turn prompts investigations into pathways of Ca2+-dependent excitotoxicity that are important in addition to, or in parallel with, early NMDAR-mediated events. The overall goals of this project are: 1) to establish the cellular mechanisms by which mitochondria mediate glutamate-induced, calcium-dependent toxicity;and 2) to determine the mechanism of mitochondrial injury per se and devise ways to prevent it. This project is generally informed by the unifying hypothesis that calcium-induced mitochondrial dysfunction is the indispensable, central event in excitotoxicity, so that the effects of other known factors can be explained by their common, convergent impact on calcium load-dependent mitochondrial injury.
Aim #1 : To determine whether mitochondrial calcium overload accounts for the role of NMDAR location and subunit composition in excitotoxic injury. Excitotoxicity depends on the activation of specific routes of Ca2+ entry. For example, NMDAR location and/or subunit composition appear to play important roles in specifying the induction of survival vs. death pathways. Our working hypothesis is that the effects of these factors can be accounted for by a common, convergent impact on calcium-induced mitochondrial injury. Thus, exposure of cultured rat hippocampal neurons to toxic concentrations of NMDA generally leads to massive calcium accumulation and cell death. In cells expressing comparable levels of NR2A- and NR2B-subunit-containing NMDARs, toxic calcium loading can be mediated by receptors of either subtype, and therefore subunit composition alone is not sufficient to specify signal coupling. Selective activation of synaptic vs. extrasynaptic NMDARs show that extrasynaptic NMDARs, because they are the dominant route of calcium entry, are the major mediator of excitotoxic stimuli. To this point, results continue to support the hypothesis that excessive calcium loading and mitochondrial dysfunction are common, obligatory steps along the pathway to excitotoxic injury.
Aim #2 : To test the hypothesis that calcium overload-induced mitochondrial damage is greater in CA1 neurons than in CA3 neurons, thereby explaining why CA1 neurons are more vulnerable to ischemic injury. Slice cultures of hippocampus are a good model for studying neuronal tolerance, since pyramidal neurons of the CA1 region are quite sensitive to excitotoxic stimuli, while neurons in the CA3 region show a high level of endogenous neuroprotection. Present data indicate that NMDA exposure selectively induces large calcium elevations in CA1 neurons, but not in CA3 neurons. Consistent with the general principle that mitochondrial damage is a key event in excitotoxic vulnerability, mitochondrial calcium overload and damage were also much more severe in CA1. NMDAR antagonists prevented CA1 calcium elevation and were neuroprotective. Considering results described in Aim #1, it is of interest to examine the role of route specificity in CA1 relative to CA3 to determine if mitochondrial calcium overload is generally responsible for the excitotoxic vulnerability of CA1 neurons.
Aim #3 : To determine, in various hippocampal models, the role of cytosolic Zn2+ elevations during glutamate excitotoxicity or oxygen-glucose deprivation. To establish the relationship to mitochondrial Ca2+ overload. Excessive elevation of intracellular Zn2+ following transient ischemia contributes to neuronal injury. It is thought that, similar to Ca2+, toxic levels of Zn2+ induce mitochondrial dysfunction and ROS production. However, it is not clear how Zn2+ accumulation relates to Ca2+-dependent dysfunction. Indeed, some recent studies suggest that excess Zn2+, not Ca2+, is the proximal trigger for excitotoxic death. Currently, whether and how Zn2+ contributes to excitotoxicity is controversial. Ongoing experiments in cultured hippocampal neurons suggest that NMDA induces Zn2+ elevations secondary to a prior rise in Ca2+. While this result may be clear-cut for isolated cells, the role of Zn2+ in OGD-induced cell death in intact tissues is a more interesting and relevant problem.
We aim to evaluate the impact of reported NMDA-evoked Zn2+ elevations and Zn2+-dependent toxicity in CA1, as well as the neuroprotective effects of Zn2+ chelation, in both acute hippocampal slices and slice cultures. The goal is to obtain a direct comparison of the effects of0 Zn2+ and Ca2+, so as to understand the relative roles of these two ions in neuronal injury.
Aim #4 : To further refine and apply a new technology -- collaboratively developed in this lab and based on energy filtering transmission electron microscopy (EFTEM) -- for the quantitative mapping of intracellular calcium distributions at the single organelle level. To apply this technology to elucidating spatio-temporal calcium dynamics in a model neuron. Because calcium plays such a central role in physiological and pathophysiological processes, quantitative mapping of Ca distributions in the analytical electron microscopy at the level of cellular organelles would be very helpful for elucidating mechanisms of calcium regulation and dysfunction. However, since physiological concentrations of calcium are very low this task is challenging. In this project we aim to quantitatively map intracellular calcium distributions using EFTEM. With EFTEM one can obtain megapixel images from cells with relatively short (<1 min) exposures, which greatly improves throughput and efficiency relative to alternative approaches. The main hurdle to producing reliable results from biological specimens is that thickness-dependent systematic errors in subtracting the spectral background from the weak, calcium-specific core edge signal must be eliminated. Ongoing studies show that by modeling the behavior of the spectrum background as a function of specimen thickness and inelastic mean free path, we can correct for plural scattering and detect calcium in neuronal preparations at concentrations as low as 10 mmol/kg dry weight, which is in the high physiological range. Results also suggest that it is feasible to further reduce these detection limits. The first proposed biological application of this methodology will be directed toward understanding calcium dynamics during recovery from stimulated calcium entry in frog sympathetic neurons, a model well studied in the past.

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Stanika, Ruslan I; Villanueva, Idalis; Kazanina, Galina et al. (2012) Comparative impact of voltage-gated calcium channels and NMDA receptors on mitochondria-mediated neuronal injury. J Neurosci 32:6642-50
Machado-Vieira, Rodrigo; Pivovarova, Natalia B; Stanika, Ruslan I et al. (2011) The Bcl-2 gene polymorphism rs956572AA increases inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum calcium release in subjects with bipolar disorder. Biol Psychiatry 69:344-52
Pivovarova, Natalia B; Andrews, S Brian (2010) Calcium-dependent mitochondrial function and dysfunction in neurons. FEBS J 277:3622-36
Stanika, Ruslan I; Winters, Christine A; Pivovarova, Natalia B et al. (2010) Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons. Neurobiol Dis 37:403-11
Stanika, Ruslan I; Pivovarova, Natalia B; Brantner, Christine A et al. (2009) Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity. Proc Natl Acad Sci U S A 106:9854-9
Aronova, M A; Kim, Y C; Pivovarova, N B et al. (2009) Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens. Ultramicroscopy 109:201-12
Pivovarova, Natalia B; Stanika, Ruslan I; Watts, Charlotte A et al. (2008) Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons. J Neurochem 104:1686-99