The NMDA subtype of the glutamate receptor plays important and diverse roles in CNS function. Mitochondrial calcium (Ca2+) overload and subsequent dysfunction, due to excessive Ca2+ entry through glutamate over-activated NMDA receptors (NMDARs), are crucial early events in excitotoxic injury. However, the generally disappointing patient outcomes for anti-excitotoxic therapies targeted to NMDARs strongly suggest that factors beyond the activation of these receptors are at play. This information prompts searches for other important Ca2+-dependent injury pathways. Recent progress and the Specific Aims of ongoing work are summarized next.
Aim #1 : To determine whether NMDAR activation is selectively toxic or whether equivalent calcium loading through voltage-gated calcium channels could be equally toxic. Although NMDARs clearly play the preeminent role in excitotoxic Ca2+ loading, evidence from our lab and others indicates that alternative routes of Ca2+ entry, for example, through voltage-gated calcium channels (VGCCs), can contribute significantly to toxicity in developmentally mature neurons. Thus, we find that in most neurons in hippocampal and cortical cultures even maximal VGCC activation does not promote significant cell death because it induces much lower calcium elevations than does toxic NMDAR activation. In a small subset of neurons, however, depolarization evokes much stronger calcium elevations, approaching those induced by toxic NMDA. These neurons are characterized by elevated expression of VGCCs, which leads to enhanced Ca2+ loading, mitochondrial dysfunction and cell death. The results indicate that neuronal vulnerability tracks the extent of Ca2+ loading, thereby reinforcing the idea that toxicity does not explicitly depend on the route of Ca2+ entry.
Aim #2 : To elucidate the cellular basis for the loss of calcium homeostatis in Bipolar Disorder. Bipolar Disorder is characterized by altered intracellular calcium homeostasis. The molecular mechanisms underlying this abnormality have been variously proposed to involve dysfunctions in endoplasmic reticulum (ER) and/or mitochondria calcium transport, potentially mediated by Bcl-2, a key protein that interacts with these organelles to regulate Ca2+ signaling. This study examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca2+ dynamics in patients with Bipolar Disorder. We observed increased basal cytosolic Ca2+ levels in risk variant AA and a general inverse correlation with Bcl-2 levels across all SNP variants. Measurements of intraluminal ER Ca2+ concentrations and release rates showed that Bcl-2 exerts its effect in variant AA cells by directly targeting ER Ca2+ release through the InsP3 receptor. The results reveal a critical mechanism by which abnormal Bcl-2 gene expression in a BPD-associated SNP variant leads to dysfunctional calcium dynamics.
Aim #3 : To determine the role of zinc in glutamate excitotoxicity and oxygen-glucose deprivation. Elevation of cellular zinc (Zn2+) concentrations following transient ischemia contributes to neuronal injury, but the mechanism(s) of Zn2+ toxicity remain unclear. Recent experiments reveal that Zn2+ can enter neurons via VGCCs, and that both Ca2+ and Zn2+ accumulate in mitochondria and contribute to mitochondrial swelling. Zinc co-precipitation with calcium within mitochondria, as well as zinc-induced mitochondrial swelling in Ca2+-free medium, suggest the possibility that mitochondrial dysfunction may be an early step in the mechanism of Zn2+ toxicity. We plan to further evaluate the relative contribution of Zn2+ and Ca2+ to mitochondrial dysfunction during glutamate-induced toxicity, with the near-term goal of understanding the interplay between the two cations.
Aim #4 : To apply a novel imaging technology one that quantitatively maps intracellular calcium at the single organelle level -- to elucidate the role of intramitochondrial calcium precipitate formation in Ca2+ signaling. Recently we have collaboratively developed an energy filtering transmission electron microscopy (EFTEM) technique capable of acquiring quantitative, megapixel images of intracellular calcium with subcellular resolution. This approach is being used to study in sympathetic neurons the functional significance of the intramitochondrial calcium-rich precipitates that form during depolarization-induced mitochondrial Ca2+ accumulation and persist for many minutes after repolarization. This issue is important because sustained post-stimulus cytosolic Ca2+ elevations contemporaneous with repolarization-induced mitochondrial Ca2+ release critically regulate many cell functions like gene expression, but the factors that control the time course of elevated Ca2+ are controversial. EFTEM calcium maps now indicate that the duration of mitochondrial Ca2+ release after repolarization is much longer than predicted by standard computational models. We find that this prolongation can be explained by considering the aforementioned mitochondrial calcium precipitates in an expanded model. In the current iteration of the model, discharge of the precipitates maintains mitochondrial Ca2+ release at a rate that appropriately accounts for the time course of Ca2+ recovery.

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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
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
Pivovarova, Natalia B; Andrews, S Brian (2010) Calcium-dependent mitochondrial function and dysfunction in neurons. FEBS J 277:3622-36
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
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
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