Feb 15, 2017 - Plamena R. Angelova1, Andrey Y. Abramov1. 1Molecular ... Megan R. McCarthy, Robyn T. Rebeck, Razvan L. Cornea,. David D. Thomas.
Wednesday, February 15, 2017 2657-Pos Board B264 Tonic Activation of Extrasynaptic NMDA Receptors Decreases Neuronal Excitability in Alzheimer’s Disease David Gall1, Antonio Lobo-Antunes2, Genevie`ve Dupont2. 1 Laboratoire de Physiologie et Pharmacologie (CP604), Universite´ Libre de Bruxelles, Bruxelles, Belgium, 2Unite´ de Chronobiologie The´orique (CP231), Universite´ Libre de Bruxelles, Bruxelles, Belgium. Amyloids b (Ab) are a hallmark of Alzheimer’s disease. They affect the communication between neurons. They can also bind to neuronal targets and thereby affect both intracellular signalling and neuronal electrical activity. During the onset of Alzheimer’s disease, a positive feedback loop between Ab40/ 42 and cytosolic calcium is thoughtto accelerate the progression of the disease. If intracellular calcium and Ab reinforce themselves through this mechanism, one would expect that the neurons targets of Ab may display an altered electrical activity caused by the increase in cytoplasmic calcium as it is known that there is a tight coupling between calcium dynamics and the electrical excitabilty. The aim of this work is to test this assumption when considering one of the privileged target of Ab, the activity of extrasynaptic NMDA receptors. Our theoretical model is a simple description of neuronal electrical activity based on the Hodgkin-Huxley like formalism, including a term that corresponds to the activity of the NMDA receptor and a cytosolic calcium compartment. When the tonic activity of extrasynaptic NMDA receptors is increased, neurons are less excitable. This is a counterintuive result as NMDA receptors exert an excitatory effect. Further analysis show that this inhibitory effect is due to the activation of calcium-dependent potassium channels, which hyperpolarize the neurons. Activation of extrasynaptic NMDA receptors also provokes a marked increase in intracellular calcium concentration, thus reinforcing the feed-forward relation between Ab production and calcium. 2658-Pos Board B265 Regulation of Axon Growth by Alpha 7 Nicotinic Receptor Calcium Transients at the Growth Cone E. Bak, J. Jedrzejewska-Szmek, J. King, K. Blackwell, N. Kabbani. Krasnow Institute for Advanced Study, George Mason University, Fairfax, VA, USA. Cell surface receptors of the growth cone (GC) transmit extracellular information that is essential for synaptogenesis and proper brain wiring. Various external cues including neurotransmitter gradients serve to modulate the turning, extension, and retraction of the GC by targeting intracellular calcium signaling pathways. Both the magnitude of the intracellular calcium rise as well as its source encode specific information leading to cytoskeletal remodeling during axon growth or retraction. We have shown that calcium conducting alpha7 nicotinic acetylcholine receptor (a7nAChR) channels bind Gaq thereby promoting IP3 receptor mediated calcium store release during nAChR channel desensitization. Here we examine the role of a7 nAChR-mediated calcium transients in neurite growth and retraction using a stochastic reaction-diffusion model of calcium gradients, cAMP, and Gaq pathways within the GC. The model allows for prediction of intracellular calcium dynamics via the entry of calcium through the a7 nAChR, activation of voltage gated channels channels, and the metabotropic signaling properties of the a7nAChR on local ER. We show that the identity of the calcium source impacts the dynamics of non-linear interactions between a cohort of calcium sensitive effectors such as PP2B and PP1, CaMKII, PKA, and calpain. Our model begins to explain experimental observations on neurite growth in cultured PC12 cells and hippocampal neurons suggesting non-monotonic dependence of structural growth on calcium levels, where both high and low calcium can inhibit growth. Elucidating the mechanisms of calcium signaling within the GC yields a better understanding of synaptic growth and plasticity, as well as an opportunity for fostering regeneration. 2659-Pos Board B266 Inorganic Polyphosphate Protects Neurons against Glutamate-Induced Excitotoxicity Marta Maiolino1, Vincenzo Lariccia2, Salvatore Amoroso2, Plamena R. Angelova1, Andrey Y. Abramov1. 1 Molecular Neuroscience, UCL Institute of Neurology, London, United Kingdom, 2Biomedical Science, UNIVP Universita’ Politecnica delle Marche, Ancona, Italy. Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma, and neurodegenerative diseases. Loss of calcium homeostasis and mitochondrial dysfunction are the key mediators of glutamate induced cell death. Recently, we found that inorganic polyphosphate (Poly P) can act as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in regulation of neuronal activity remains still undefined. Considering the number of studies which demonstrate the close interaction between neurons and glia in physiology and pathology, we studied the effect of the polyP on glutamate induced calcium signal in neurons, in
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physiological and pathological conditions. We used fluorescence imaging to measure mitochondrial membrane potential and intracellular calcium concentration ([Ca2þ]i) in primary cultures of hippocampal or cortical neurons (10-15 days in vivo), using Rhodamine123 and Fura-ff respectively. Application of glutamate (50-100mM) causes a stereotypical response consisting of an initial transient spike in [Ca2þ]i followed by a secondary increase in [Ca2þ]i which is coincident with a delayed mitochondrial collapse. Pretreatment of the neurons and astrocytes with different length of polyP significantly reduced the number of cells with a secondary delayed calcium deregulation induced by high concentration of glutamate. Moreover, glutamateinduced mitochondrial depolarization was also prevented by PolyP addition. Importantly, long chain PolyP successfully protected cells against glutamate induced cell death. Thus, polyP protects neurons against glutamate-induced excitotoxicity by reduction of the calcium overload and restoring mitochondrial function.
Intercellular Calcium Channels and Calcium Sparks and Waves II 2660-Pos Board B267 ATP Release through Gap Junction Hemichannels Increases Ca2D Spark Occurrence via P2Y Purinoceptor Signaling in Rat Ventricular Myocytes under Shear Stress Jun Wang, Joon-Chul Kim, Sun-Hee Woo. College of Pharmacy, Chungnam National University, Daejeon, Korea, Republic of. Shear stress in ventricles increases under pressure/volume overload caused by valve diseases, heart failure, and hypertension and enhances Ca2þ transients in ventricular myocytes. We have previously proposed that the shear-mediated Ca2þ transient increase is in part due to sensitization of Ca2þ release sites through ROS generation by NADPH oxidase (NOX) and NO synthase (NOS). Here, we investigated remaining mechanism for the activation of Ca2þ release sites under shear stress in rat ventricular myocytes. Shear stress of ~16 dyn/cm2 was applied onto single cells using micro-jet apparatus. Two-dimensional confocal Ca2þ imaging was performed at 30 Hz. The frequency of resting Ca2þ sparks was immediately increased to ~180% and further increased to ~250% by prolonged exposure (20 s). Pretreatment of cells with inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor 2-APB (2 mM) or protein kinase C (PKC) inhibitor chelerythrine (2 mM) partly (about 50%) suppressed both immediate and prolonged shear effects on the spark occurrence. Blockade of P2 purinoceptors (30 mM suramin) almost completely suppressed shear-induced spark enhancements. Inhibition of phospholipase C (PLC) using U73122 (5 mM) significantly suppressed (~80%) shear-induced Ca2þ spark increases. Pretreatment of P2Y1 receptor antagonist MRS2179 (400 nM) diminished immediate and late increases in spark occurrence during shear stimulation by 60-70%. Inhibition of gap junction hemichannels using carbenoxolone (50 mM) or external treatment of ATP metabolizing apyrase (2 units/ml) eliminated the stimulatory effects of shear stress on the spark occurrence. Consistently, luciferin-luciferase assay revealed ATP release from these myocytes by shear stress within 2 s. These results suggest that shear stress may enhance Ca2þ spark occurrence partly via activation of P2Y-PLC-IP3R/PKC signaling by connexin hemichannel-mediated ATP release in rat ventricular myocytes. Possible link between this signaling and NOX/NOS upon shear-mediated spark enhancement in ventricular myocytes needs further investigation. 2661-Pos Board B268 Structural Dynamics of Calmodulin in Regulation of Calcium Release in Health and Disease Megan R. McCarthy, Robyn T. Rebeck, Razvan L. Cornea, David D. Thomas. Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. We have used time-resolved fluorescence (FRET) and EPR (DEER) spectroscopies to study the structural changes in calmodulin (CaM) that are relevant to regulation of the muscle calcium release channel, the ryanodine receptor (RyR). Regulation of RyR by CaM is disrupted by oxidation and diseasecausing mutations. However, the structural basis for these regulatory changes, and the role CaM plays in the development of heart failure and arrhythmias, is not well understood. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the correlation between structure and function in physiologically relevant conditions is largely unknown. To test the hypothesis that the modulatory action of CaM on RyR is caused by structural changes in the CaM-RyR complex, we use site-directed spectroscopy to determine the structural changes that contribute to calcium regulation in skeletal and cardiac muscle. The approach is to prepare CaM mutants that contain a single Cys on each of the two lobes (N and C), then attach
