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 test the hypothesis that calcium overload-induced mitochondrial damage is greater in hippocampal CA1 neurons than in CA3, thereby explaining why the CA1 region is more vulnerable to ischemic injury. Pyramidal neurons of the CA1 region are highly vulnerable to excitotoxic injury, while neurons of the CA3 region show a high level of endogenous neuroprotection. Recent data indicate that NMDA exposure selectively induces large Ca2+ elevations in CA1 neurons, but not in CA3. Mitochondrial calcium overload and damage were also much more severe in CA1, which us consistent with the general principle that mitochondrial damage is a key event in excitotoxicity. This study is the first to demonstrate a direct relationship between selective neuronal vulnerability and mitochondrial dysfunction in an intact brain preparation.
Aim #2 : To determine whether NMDAR activation is selectively toxic or whether equivalent calcium loading through voltage-gated calcium channels could be equally toxic. The prominent role of NMDARs in excitotoxic Ca2+ loading is thought to reflect the fact that the biophysical properties of alternative routes of entry such as voltage-gated calcium channels (VGCCs) restrict the amount of Ca2+ that can be transported. However, recent results from our lab and others provide evidence that there is a significant contribution of VGCCs to toxicity in developmentally mature neurons. Compared to NMDARs, activation of VGCCs in young (14 DIV) hippocampal neurons induces very small Ca2+ elevations. However, in mature neurons VGCC-dependent Ca2+ entry was significantly higher and amplified NMDA-evoked elevations, resulting in accelerated mitochondrial calcium accumulation, dysfunction and cell death. The results indicate that neuronal vulnerability correlates well with the extent of Ca2+ loading and reinforce the idea that toxicity does not explicitly depend on the route of calcium entry.
Aim #3 : To elucidate the cellular basis for the loss of calcium homeostatis that characterizes Bipolar Disorder. Bipolar Disorder is characterized by altered intracellular calcium homeostasis;the molecular mechanisms underlying this abnormality may involve endoplasmic reticulum (ER) and/or mitochondria calcium transport dysfunction, potentially mediated by Bcl-2, a key protein that regulates Ca2+ signaling through direct interactions with these organelles. This study examines the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular calcium dynamics in patients with Bipolar Disorder. In these patients, abnormal Bcl-2 gene expression in the AA variant of the target SNP contributed to dysfunctional calcium homeostasis through a specific ER InsP3R-dependent mechanism.
Aim #4 : To apply a new imaging technology for quantitatively mapping intracellular calcium at the single organelle level an approach collaboratively developed in this lab and based on energy filtering transmission electron microscopy (EFTEM) -- to elucidate mechanisms by which stimulus-induced intramitochondrial calcium precipitates influence Ca2+-dependent signal transduction. We have recently developed a new EFTEM technique capable of acquiring quantitative, megapixel images of intracellular calcium with greatly improved throughput and efficiency. This approach, together with computational modeling, is being used to study in sympathetic neurons the functional significance of the intramitochondrial calcium-rich precipitates that form during depolarization-induced Ca2+ accumulation. The specific problem is that EFTEM calcium maps indicate that the model-predicted duration of mitochondrial Ca2+ release after repolarization is too short, implying that continuous mitochondrial Ca2+ release cannot account for the post-stimulus Ca2+ plateau. It is important to understand the origin of prolonged post-stimulus Ca2+ elevations these critically regulate multiple cell functions such as gene expression and kinase activation. Now, we find that theory and experiment can be brought into line by considering the mitochondrial calcium precipitates as a nonlinear buffer, in which case discharging the precipitates maintains mitochondrial Ca2+ release at an constant rate, thereby appropriately extending the duration of Ca2+ recovery.

Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
2010
Total Cost
$1,339,703
Indirect Cost
City
State
Country
Zip Code
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
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