This project studies physiological and cellular aspects of neuronal calcium signaling, with long-range emphasis on postsynaptic responses in large central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration ([Ca2+]i) that is strongly modulated by the activity of intracellular Ca stores. This activity plays an important role in spatio-temporally shaping Ca2+ signals that regulate important processes such as gene expression and LTP induction. We had earlier shown that stimulus-induced increases in [Ca2]i in sympathetic neurons are accompanied by large, reversible elevations in mitochondrial calcium concentration. We have now explored, in hippocampal pyramidal neurons, analogous mitochondrial responses to a range of synaptic stimuli, and show that in these cells, mitochondrial Ca2+ transport activity has important physiological and pathophysiological effects. In hippocampal pyramidal neurons, large increases in [Ca2+]i activate several key kinases, whereas lower [Ca2+]i enhances protein phosphatase activity. This Ca2+-dependent rebalancing of the phosphorylation status of certain key enzymes, e.g., Ca/calmodulin-dependent kinases (CaMKs), controls the activity of pathways central to neuronal plasticity. We previously found that mitochondrial Ca accumulation mediated by strong Ca2+ entry leads to an increase in the production of superoxide radicals (O2-), and that this activity up-regulates the phosphorylation of CaMKII and CaMKIV-dependent CREB by inhibiting an array of phosphatases (PP1, PP2A and/or PP2B). Conversely, we now find that weak [Ca2+]i increases induced by prolonged low-frequency stimulation preferential enhance phosphatase activity, thereby leading to ERK1/2 activation because PP2A activates Raf-1, an up-stream mediator of ERK. Following strong [Ca2+]i increases induced by short high-frequency (HF) stimulation, however, the onset of ERK activity is delayed by mitochondrially-produced O2- inhibition of PP2A. This increase in ERK is maximal at 10-min post-stimulation, and depends on CaMKII activity, which is, in turn, enhanced by mitochondrial O2-mediated inhibition of CaMKII dephosphorylation. In contrast, NADPH oxidase, a plasma membrane-bound source of O2-, enhances ERK activation during early HF-stimulated ERK induction, but the effect is much weaker and the mechanism does not involve suppression of PP2A. The results indicate that O2- produced by mitochondria and NADPH oxidase modulate specific and distinct steps in Ras/Raf/MEK/ERK1/2 pathway. Mitochondrial dysfunction plays a central role in glutamate-induced excitotoxicity, but mechanisms leading to cell death remain controversial. Earlier work had shown that in hippocampal neurons mitochondrial dysfunction depends on the size of the calcium load, since co-application of agents that inhibit mitochondrial Ca2+ uptake, e.g., FCCP, greatly improve cell viability even though [Ca2+]i elevations are larger in the presence of such drugs. Thus, [Ca2+]i elevation alone, without an increase in mitochondrial Ca, is not sufficient to induce cell death. We have continued to investigate mechanisms of mitochondrial involvement in excitotoxic death by studying ionic, structural and functional changes in mitochondria following injurious stimuli. Following strong NMDA stimulation, the Ca content of mitochondria is in general very high, but more importantly, the content of individual mitochondria is highly variable. There is parallel heterogeneity among mitochondria with regard to swelling and membrane rupture, accompanied by the loss of mitochondrial membrane potential. Surprisingly, the majority of mitochondria tolerate these high levels of Ca without permanent damage, recovering their prestimulus structure, composition and membrane potential within 2h. In some cells, however, small subsets of mitochondria do not re-establish normal Ca2+ and volume regulation. In these mitochondria Ca overload leads to functional and morphological changes resulting in loss of membrane integrity and the probable release of apoptogenic proteins; by 6-8h post-stimulation, typical apoptotic features had developed in ~35% of cells. Presumably, enough mitochondria in those cells were injured by Ca overload to trigger an effective proapoptotic signal. These results support the hypothesis that excitotoxic injury to only a few mitochondria within a neuron can lead to apoptotic cell death. In other progress driven by technical requirements and a longstanding interest in molecular motors, we have used scanning transmission electron microscopy (STEM) and diffraction analysis to determine the hierarchical and spatial organization of cytoskeletal ribbon of the bacterium Spiroplasma, which acts as a linear, contractile motor. The structural unit of this ribbon appears to be a fibril, ~5 nm wide, composed of dimers of a 59 kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit is a pair of aligned fibrils along which pairs of dimers form tetrameric ring-like repeats. This organization explains how the cytoskeletal ribbon functions as a linear motor: Force is generated by a circular-to-elliptical conformational change in the tetrameric subunits, which differentially changes the length of its fibril components. In addition, we have developed and implemented several technical and instrumental refinements that significantly improve sensitivity for mapping intracellular Ca by electron energy loss spectrum imaging.
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