Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.     

Enhancement of (45)Ca(2+) influx and voltage-dependent Ca(2+) channel activity by beta-amyloid-(1-40) in rat cortical synaptosomes and cultured cortical neurons. Modulation by the proinflammatory cytokine interleukin-1beta

Beta-amyloid protein is thought to underlie the neurodegeneration associated with Alzheimer's disease by inducing Ca(2+)-dependent apoptosis. Elevated neuronal expression of the proinflammatory cytokine interleukin-1beta is an additional feature of neurodegeneration, and in this study we demonstrate that interleukin-1beta modulates the effects of beta-amyloid on Ca(2+) homeostasis in the rat cortex. beta-Amyloid-(1-40) (1 microM) caused a significant increase in (45)Ca(2+) influx into rat cortical synaptosomes via activation of L- and N-type voltage-dependent Ca(2+) channels and also increased the amplitude of N- and P-type Ca(2+) channel currents recorded from cultured cortical neurons. In contrast, interleukin-1beta (5 ng/ml) reduced the (45)Ca(2+) influx into cortical synaptosomes and inhibited Ca(2+) channel activity in cultured cortical neurons. Furthermore, the stimulatory effects of beta-amyloid protein on Ca(2+) influx were blocked following exposure to interleukin-1beta, suggesting that interleukin-1beta may govern neuronal responses to beta-amyloid by regulating Ca(2+) homeostasis.     

Ca(2+) and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III

Annexin 3 (ANX A3) represents approximately 1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca(2+)-dependent manner, via Ca(2+)-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca(2+), domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca(2+) ligand in domain III. Ca(2+) in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca(2+) and returns to its original value when interacting with membranes. Ca(2+) concentration for half binding of the W5A mutant to negatively charged membranes is approximately 0.5 mM while it increases to approximately 1 mM for the ANX A3 wild type and to approximately 3 mM for the W190 ANX A3 mutant. In addition to the expected perturbation of the W190 environment at the contact surface between the protein and the membrane bilayer, binding of the protein to Ca(2+) and to membranes modulates the flexibility of the ANX A3 hinge region at the opposite of this interface and might affect its membrane permeabilizing properties.     

2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanisms

Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2alpha. eEF-2 kinase may be activated both by Ca(2+)-independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na(+) pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca(2+)-dependent and Ca(2+)-independent eEF-2 phosphorylation.     

Ca2+-independent binding and cellular expression profiles question a significant role of calmyrin in transduction of Ca2+-signals to Alzheimer's disease-related presenilin 2 in forebrain

The interaction between the EF-hand Ca(2+)-binding protein calmyrin and presenilin 2 (PS2) has been suggested to play a role in Alzheimer's disease (AD). We now report that calmyrin binds specifically endogenous PS2 and not PS1. However, binding appears to be Ca(2+)-independent and calmyrin does not exhibit a Ca(2+)-dependent translocation to intracellular membranes as demonstrated in a Ca(2+)-myristoyl switch assay. Moreover, calmyrin is only present at very low levels in brain areas associated with the onset of AD. In rat, forebrain calmyrin is localized only in a subset of principal neurons, similarly as in human forebrain. Finally, subcellular fractionation demonstrates only a limited overlap of calmyrin and PS2 at neuronal membranes. We therefore conclude that calmyrin will not contribute significantly as a Ca(2+)-sensor that transduces Ca(2+)-signaling events to PS2 in forebrain.     

Calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture

In Alzheimer's disease, amyloid beta (Abeta) peptide is deposited in neuritic plaques in the brain. The Abeta peptide 1-42 or the fragment 25-35 are neurotoxic. We here review our recent explorations of the mechanisms of Abeta toxicity in hippocampal cultures. Abeta had no effect on intracellular calcium in neurons but caused striking changes in nearby astrocytes. The [Ca(2+)](c) signals started approximately 5-15 min after Abeta application and consisted of sporadic [Ca(2+)](c) pulses. These were entirely dependent on extracellular Ca(2+), independent of ER Ca(2+) stores and resulted from Ca(2+) influx, probably through Abeta-induced membrane channels. The Ca(2+) signals were closely associated with transient, episodic acidification which may reflect displacement of protons from binding sites or Ca(2+)/2H(+) exchange. Abeta caused an increased rate of generation of reactive oxygen species (ROS), also seen in astrocytes and not in neurons. The increased ROS generation was blocked by inhibitors of the NADPH oxidase, strongly suggesting that this enzyme, normally associated with immune cells, is expressed in astrocytes. ROS generation was also Ca(2+)-dependent, suggesting that Abeta activation of the enzyme may be secondary to the increase in [Ca(2+)](c). Abeta caused delayed neuronal death despite the fact that all responses were seen only in astrocytes. Neurons could not be protected by glutamate receptor antagonists, but were rescued by inhibition of the NADPH oxidase, by antioxidants and by increasing glutathione. These data suggest that Abeta causes Ca(2+)-dependent oxidative stress by activating an astrocytic NADPH oxidase, and that neuronal death follows through a failure of antioxidant support.     

Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane

Ca(2+)-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca2+ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca(2+)-dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca(2+)-induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca2+ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca(2+)-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.     

Specific localization of the annexin II heterotetramer in brain lipid raft fractions and its changes in spatial learning

Annexin-II (AII) is a Ca(2+)-dependent phospholipid-binding protein that is present in both intracellular and extracellular compartments. In the present study AII immunoreactivity was found in a subpopulation of neurons in specific brain regions, including the cerebral cortex and the surface of hippocampal pyramidal neurons from adult rats. AII from synaptic membranes was detected by immunoblotting as multiple species containing the monomer (AII36) and heterotetramer (AIIt). AIIt was resistant to beta-mercaptoethanol and dithiothreitol in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but was completely reduced to monomers (36 kDa) by two-dimensional electrophoresis. AIIt resided exclusively in the detergent-resistant lipid rafts concentrated in neuronal dendrites, and its recruitment to those structures was enhanced by antibody cross-link. AII abundantly distributed on the outer leaflet of neuronal membranes and between spaces of neurons appeared to be neuronal adhesive. The formation of AIIt required synthesis of sphingolipids and cholesterol, and its stability depended on Ca2+. Increases in neuronal activities such as depolarization and learning were shown to promote formation of AIIt. Our results suggest that, via a dynamic association with dendritic lipid rafts, AII may play a role in synaptic signal transduction and remodeling. This probably involves focal adhesion and interactions with actin that are associated with brain development and memory consolidation.     

The lack of annexin A7 affects functions of primary astrocytes

Annexin A7 is a Ca(2+)- and phospholipid-binding protein, which is thought to function in membrane organization and Ca(2+)-dependent signaling processes. It localizes to different cellular compartments and exists in a 47- and 51-kDa isoform with the large isoform being expressed in brain, skeletal, and heart muscle. In human temporal brain annexin A7 was found exclusively in astroglial cells. As astrocytes are thought to play key roles in several processes of the brain we focused on Ca(2+)-dependent signaling processes and astrocyte proliferation. Primary astrocytes from an anxA7(-/-) mouse exhibited an increased velocity of mechanically induced astrocytic Ca(2+) waves as compared to wild type. We also observed a remarkably increased proliferation rate in cultured mutant astrocytes. A search for annexin A7 binding partners with advanced biochemical methods confirmed sorcin as the major binding protein. However, in vivo GFP-tagged annexin A7 and sorcin appeared to redistribute mainly independently from each other in wild type and in mutant astrocytes. Our results favor an involvement of annexin A7 in Ca(2+)-dependent signaling or Ca(2+) homeostasis in astrocytes.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Unconventional neuroprotection against Ca2+ -dependent insults by metalloporphyrin catalytic antioxidants

We evaluated whether both inert and catalytically active metalloporphyrin antioxidants, meso-substituted with either phenyl-based or N-alkylpyridinium-based groups, suppress Ca(2+)-dependent neurotoxicity in cell culture models of relevance to cerebral ischemia. Representatives from both metalloporphyrin classes, regardless of antioxidant strength, protected cultured cortical neurons or PC-12 cultures against the Ca(2+) ionophores ionomycin or A23187, by suppressing neurotoxic Ca(2+) influx. Some metalloporphyrins suppressed excitotoxic Ca(2+) influx indirectly induced by the Ca(2+) ionophores in cortical neurons. Metalloporphyrins did not quench intracellular fluorescence, suggesting localization to the plasma membrane interface and/or interference with Ca(2+) ionophores. Metalloporphyrins suppressed ionomycin-induced Mn(2+) influx, but did not protect cortical neurons against pyrithione, a Zn(2+) ionophore. In other Ca(2+)-dependent paradigms, Ca(2+) influx via plasma membrane depolarization, but not through reversal of plasmalemmal Na(+)/Ca(2+) exchangers, was modestly suppressed by Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP) or by an inert analog, Zn(II)TBAP. Mn(III)TBAP and Zn(II)TBAP potently protected cortical neurons against long-duration oxygen-glucose deprivation (OGD), performed in the presence of antagonists of NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and L-type voltage-gated Ca(2+) channels, raising the possibility of an unconventional mode of blockade of transient receptor protein melastatin 7 channels by a metalloTBAP family of metalloporphyrins. The present study extends the range of Ca(2+)-dependent insults for which metalloporphyrins demonstrate unconventional neuroprotection. MetalloTBAPs appear capable of targeting an OGD temporal continuum.     

Role of catenins in the development of gap junctions in rat cardiomyocytes

Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of alpha-catenin, beta-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca(2+) (<5 microM) medium, then shifted to 1.8 mM Ca(2+) medium (Ca(2+) switch). Diffuse cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca(2+) medium. Staining of alpha-catenin, beta-catenin, and ZO-1 was detected at the plasma membrane of cell-cell contact sites 10 min after Ca(2+) switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca(2+) switch. Distinct junctional and extensive cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen 2 h after Ca(2+) switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-beta-catenin antibodies showed that beta-catenin was associated with alpha-catenin, ZO-1, and Cx43 at 2 h after Ca(2+) switch. Intracellular application of antisera against alpha-catenin, beta-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca(2+) medium inhibited the redistribution of Cx43 to the plasma membrane following Ca(2+) switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.     

Calcium ions in neuronal degeneration

Neuronal Ca(2+) homeostasis and Ca(2+) signaling regulate multiple neuronal functions, including synaptic transmission, plasticity, and cell survival. Therefore disturbances in Ca(2+) homeostasis can affect the well-being of the neuron in different ways and to various degrees. Ca(2+) homeostasis undergoes subtle dysregulation in the physiological ageing. Products of energy metabolism accumulating with age together with oxidative stress gradually impair Ca(2+) homeostasis, making neurons more vulnerable to additional stress which, in turn, can lead to neuronal degeneration. Neurodegenerative diseases related to aging, such as Alzheimer's disease, Parkinson's disease, or Huntington's disease, develop slowly and are characterized by the positive feedback between Ca(2+) dyshomeostasis and the aggregation of disease-related proteins such as amyloid beta, alfa-synuclein, or huntingtin. Ca(2+) dyshomeostasis escalates with time eventually leading to neuronal loss. Ca(2+) dyshomeostasis in these chronic pathologies comprises mitochondrial and endoplasmic reticulum dysfunction, Ca(2+) buffering impairment, glutamate excitotoxicity and alterations in Ca(2+) entry routes into neurons. Similar changes have been described in a group of multifactorial diseases not related to ageing, such as epilepsy, schizophrenia, amyotrophic lateral sclerosis, or glaucoma. Dysregulation of Ca(2+) homeostasis caused by HIV infection or by sudden accidents, such as brain stroke or traumatic brain injury, leads to rapid neuronal death. The differences between the distinct types of Ca(2+) dyshomeostasis underlying neuronal degeneration in various types of pathologies are not clear. Questions that should be addressed concern the sequence of pathogenic events in an affected neuron and the pattern of progressive degeneration in the brain itself. Moreover, elucidation of the selective vulnerability of various types of neurons affected in the diseases described here will require identification of differences in the types of Ca(2+) homeostasis and signaling among these neurons. This information will be required for improved targeting of Ca(2+) homeostasis and signaling components in future therapeutic strategies, since no effective treatment is currently available to prevent neuronal degeneration in any of the pathologies described here.     

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.     

Activity- and Ca(2+)-dependent modulation of surface expression of brain-derived neurotrophic factor receptors in hippocampal neurons

Brain-derived neurotrophic factor (BDNF) has been shown to regulate neuronal survival and synaptic plasticity in the central nervous system (CNS) in an activity-dependent manner, but the underlying mechanisms remain unclear. Here we report that the number of BDNF receptor TrkB on the surface of hippocampal neurons can be enhanced by high frequency neuronal activity and synaptic transmission, and this effect is mediated by Ca(2+) influx. Using membrane protein biotinylation as well as receptor binding assays, we show that field electric stimulation increased the number of TrkB on the surface of cultured hippocampal neurons. Immunofluorescence staining suggests that the electric stimulation facilitated the movement of TrkB from intracellular pool to the cell surface, particularly on neuronal processes. The number of surface TrkB was regulated only by high frequency tetanic stimulation, but not by low frequency stimulation. The activity dependent modulation appears to require Ca(2+) influx, since treatment of the neurons with blockers of voltage-gated Ca(2+) channels or NMDA receptors, or removal of extracellular Ca(2+), severely attenuated the effect of electric stimulation. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) significantly reduced the effectiveness of the tetanic stimulation. These findings may help us to understand the role of neuronal activity in neurotrophin function and the mechanism for receptor tyrosine kinase signaling.     

Rapid Ca2+-dependent increase in oxygen consumption by mitochondria in single mammalian central neurons

Oxygen consumption increases within a fraction of a second after the onset of neuronal activity, a phenomenon referred to as the "initial dip" in functional imaging studies of the living brain. The cellular mechanism that underlies this rapid increase in oxygen consumption has remained unclear, however. We have now used two-photon excitation imaging to characterize rapid activity-dependent mitochondrial responses in single neurons. This approach allowed simultaneous multicolor imaging of individual mitochondria in single mouse Purkinje neurons in culture. Mitochondrial depolarization was induced immediately when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) exceeded 15 microM and was associated with oxidation of mitochondrial NAD(P)H, suggesting that Ca(2+)-induced mitochondrial depolarization mediated by the Ca(2+) uniporter directly facilitated oxidation of NAD(P)H. With the use of a miniature oxygen electrode, we detected a burst of oxygen consumption within 0.2s after the onset of cell depolarization in single Purkinje neurons, and this rapid increase in oxygen consumption was dependent on the increase in [Ca(2+)](i). We have thus demonstrated a rapid Ca(2+)-dependent consumption of oxygen that is mediated by mitochondrial depolarization in mammalian central neurons. This process might function as a rapid feed-forward mechanism in homeostatic control of the cytosolic ATP concentration.     

Partitioning of the plasma membrane Ca2+-ATPase into lipid rafts in primary neurons: effects of cholesterol depletion

Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.