[Show abstract][Hide abstract] ABSTRACT: Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.Cell Death and Differentiation advance online publication, 24 April 2015; doi:10.1038/cdd.2015.39.
Cell death and differentiation 04/2015; DOI:10.1038/cdd.2015.39 · 8.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the CNS, renders the brain more tolerant to lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and still remain undefined. Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation, we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance. To this aim, we set up an in vitro model of IPC by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD) followed by 3-h OGD plus reoxygenation. IPC was able to stimulate NCX activity, as revealed by Fura-2AM single-cell microfluorimetry. This effect was mediated by the NO/PI3K/ Akt pathway since it was blocked by the following: (a) the NOS inhibitors L-NAME and 7-Nitroindazole, (b) the IP3K/Akt inhibitors LY294002, wortmannin and the Akt-negative dominant, (c) the NCX1 and NCX3 siRNA. Intriguingly, this IPC-mediated upregulation of NCX1 and NCX3 activity may control calcium level within endoplasimc reticulum (ER) and mitochondria, respectively. In fact, IPC-induced NCX1 upregulation produced an increase in ER calcium refilling since this increase was prevented by siNCX1. Moreover, by increasing NCX3 activity, IPC reduced mitochondrial calcium concentration. Accordingly, the inhibition of NCX by CGP37157 reverted this effect, thus suggesting that IPC-induced NCX3-increased activity may improve mitochondrial function during OGD/reoxygenation. Collectively, these results indicate that IPC-induced neuroprotection may occur through the modulation of calcium homeostasis in ER and mitochondria through NO/PI3K/Akt-mediated NCX1 and NCX3 upregulation.
Cell death and differentiation 03/2014; 21(7). DOI:10.1038/cdd.2014.32 · 8.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial influx and efflux calcium pathways play a relevant role in cytosolic and mitochondrial calcium homeostasis and contribute to the regulation of mitochondrial functions in neurons. The mitochondrial Na(+)/Ca(2+) exchanger, although hypothesized in 1974, has been primarily investigated only from a functional point of view and its identity and localization in the mitochondria have been a matter of debate over the last three decades. Recently, a lithium-dependent sodium/calcium exchanger extruding calcium from the matrix has been found in the inner mitochondrial membrane of neuronal cells. However, evidence has been provided that the outer membrane is impermeable to calcium efflux into the cytoplasm.In this study, we have demonstrated for the first time that the nuclear encoded NCX3 isoform (a) is localized on the outer mitochondrial membrane (OMM) of neurons, (b) co-localizes and immunoprecipitates with AKAP121, a member of the protein kinase A anchoring proteins (AKAPs) present on the outer membrane, (c) extrudes calcium from mitochondria through AKAP121 interaction in a PKA-mediated manner, both under normoxia and hypoxia, and (d) improves cell survival when it works in the Ca(2+) efflux mode at the level of the OMM.Collectively, these results suggest that, in neurons, NCX3 regulates mitochondrial calcium handling from the OMM through an AKAP121-anchored signalling complex, thus promoting cell survival during hypoxia.
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are now recognized as one of the main intracellular calcium-storing organelles which play a key role in the intracellular calcium signalling. Indeed, besides performing oxidative phosphorylation, mitochondria are able to sense and shape calcium (Ca(2+)) transients, thus controlling cytosolic Ca(2+) signals and Ca(2+)-dependent protein activity. It has been well established for many years that mitochondria have a huge capacity to accumulate calcium. While the physiological significance of this pathway was hotly debated until relatively recently, it is now clear that the ability of mitochondria in calcium handling is a ubiquitous phenomenon described in every cell system in which the issue has been addressed.In this chapter, we will review the molecular mechanisms involved in the regulation of mitochondrial calcium cycling in physiological conditions with particular regard to the role played by the mitochondrial Na(+)/Ca(2+) exchanger.
Advances in Experimental Medicine and Biology 01/2013; 961:203-9. DOI:10.1007/978-1-4614-4756-6_17 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Besides performing oxidative phosphorylation, mitochondria are able to sense and shape calcium (Ca2+) transients, thus controlling cytosolic Ca2+ signals and Ca2+-dependent protein activity. Indeed, it has been well established for many years that mitochondria have a huge capacity to accumulate calcium. While the physiological significance of this pathway was hotly debated until relatively recently, it is now clear that the ability of mitochondria in calcium handling is an ubiquitous phenomenon described in every cell system in which the issue has been addressed. Therefore, mitochondria are now recognized as one of the main intracellular calcium storing organelles which play a key role in the intracellular calcium signaling. In this chapter, the molecular mechanisms involved in regulation of mitochondrial calcium cycling both in physiological and in pathological conditions are described. A particular emphasis is devoted to the understanding of the mitochondrial responses occurring in cerebral ischemia and to the discussion of the contribution played by these organelles to tissue damage. Finally, the role of the newly identified mitochondrial proteins in the regulation of mitochondrial calcium dynamics is also explored as a starting point for investigation of new molecular target responsible for mitochondrial dysfunctions leading to cell death.
Metal Ion in Stroke, 01/2012: pages 41-67; , ISBN: 978-1-4419-9662-6
[Show abstract][Hide abstract] ABSTRACT: Activation of G-protein-coupled receptors (GPCRs) mobilizes compartmentalized pulses of cyclic AMP. The main cellular effector of cAMP is protein kinase A (PKA), which is assembled as an inactive holoenzyme consisting of two regulatory (R) and two catalytic (PKAc) subunits. cAMP binding to R subunits dissociates the holoenzyme and releases the catalytic moiety, which phosphorylates a wide array of cellular proteins. Reassociation of PKAc and R components terminates the signal. Here we report that the RING ligase praja2 controls the stability of mammalian R subunits. Praja2 forms a stable complex with, and is phosphorylated by, PKA. Rising cAMP levels promote praja2-mediated ubiquitylation and subsequent proteolysis of compartmentalized R subunits, leading to sustained substrate phosphorylation by the activated kinase. Praja2 is required for efficient nuclear cAMP signalling and for PKA-mediated long-term memory. Thus, praja2 regulates the total concentration of R subunits, tuning the strength and duration of PKA signal output in response to cAMP.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to investigate the molecular mechanisms underlying the neuroprotective effect of the hydrophilic statin rosuvastatin on cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation. Rosuvastatin (RSV), at concentrations ranging from 10 nM to 1μM, was able to ameliorate the survival of cortical neurons exposed to OGD followed by reoxygenation. This effect was observed either if neurons were pretreated with RSV 24 hrs before OGD/reoxygenation exposure or if RSV was added during the OGD or the reoxygenation phase. Moreover, RSV was also able to improve mitochondrial oxidative capacity in basal conditions, an effect that was already observed at 10 nM either after 24 or after 48 hrs of treatment. These neuroprotective actions were not counteracted by mevalonate, an intermediate of cholesterol biosynthesis that bypasses RSV induced blockade of cholesterol synthesis. Furthermore, the hypothesis that RSV might affect neuronal nitric oxide synthase (nNOS) activity during OGD/reoxygenation was explored. RSV was able to reduce the increase of NO occurring during the reoxygenation phase, an effect prevented by NPLA, the selective inhibitor of nNOS. Finally, the possibility that RSV-induced NO reduction during OGD/reoxygenation might involve ERK1/2 activation was also investigated. The treatment of neurons with PD98059, an ERK1/2 kinase inhibitor, abolished the neuroprotective effect exerted by RSV in cortical neurons exposed to OGD/reoxygenation. In conclusion, these results demonstrated that RSV-induced neuroprotection involves an impairment of constitutive and inducible NOS activity which in turn causes the improvement of mitochondrial function and the stimulation of ERK1/2 via H-Ras activation.
International Journal of Physiology, Pathophysiology and Pharmacology 01/2011; 3(1):57-64.