Cytosolic Calcium Homeostasis in Neurons — Control Systems, Modulation by Reactive Oxygen and Nitrogen Species, and Space and Time Fluctuations
Cytosolic calcium plays a major and central role in neuronal activity and functions both in brain and in peripheral nervous systems, and its sustained alteration is a critical event that leads to neuronal death. On these grounds, it is not surprising that a sustained alteration of intracellular calcium homeostasis in neurons is a point of convergence of the cellular mechanisms underlying many neurodegenerative processes in the brain. Indeed, this has been shown to be the case for the brain s
... rodegenerative diseases of higher incidence to humans, like "lzheimer s and Parkinson s, or in the acute neurodegeneration observed in amyotrophic lateral sclerosis, and also for major brain insults, such as excitotoxicity in trauma and ischemiareperfusion, inflammation and neurotoxicity by drugs and environmental chemicals. Sustained deregulation of cytosolic calcium concentration have been reported in neuronal apoptosis and necrosis, the two major cellular death pathways involved in brain neurodegeneration. It has been experimentally demonstrated and confirmed by many investigations using cell cultures that a sustained rise of cytosolic calcium concentration in the neuronal soma within the range . -μM elicits a rapid necrotic neuronal death, mediated by calciumdependent proteases activation, like calpains. On the other hand, long-term sustained cytosolic calcium concentrations below -nM in the neuronal soma promote the slow development of apoptotic neuronal death of neurons in culture [ , ]. Since the central role of calcium in neurotransmitter secretion and neuronal plasticity is also well known, the basal steady state cytosolic calcium concentration in the neuronal soma can be considered as a bioenergetics marker of neuronal activity and survival. We shall then present the major calcium transport systems that control the cytosolic calcium homeostasis in the wider space within neurons, i.e. in the neuronal soma. Owing to the large subcellular regionalization of neuronal processes essential for the normal activity of neurons and especially in neuronal signal transduction pathways, we shall also place a particular emphasis in the subcellular compartmentation of these calcium transport systems. Noteworthy, neurodegenerative processes in the brain also share another common metabolic deviation, namely, that neurons are also exposed to an enhanced oxidative stress in the brain. Using different types of neuronal cultures, many investigators have shown during the last years that the cellular oxidative stress produced by reactive oxygen species ROS and reactive nitrogen species RNS and a sustained alteration of the intracellular calcium homeostasis are metabolic deregulations usually observed during the early stages of the development of the process of neuronal death and before the cell viability loss induced entry in the irreversible steps characterized by the activation of proteases. In addition, it has been proposed that alterations of the intracellular calcium homeostasis of glial cells can also contribute to inflammation and damage in the brain in neurodegenerative processes [ ]. Therefore, a better knowledge of the major molecular pathways contributing to induce the oxidative stress in the brain and the deregulation of intracellular calcium homeostasis in neurons should be expected to lead to the development of novel and more efficient therapies against brain neurodegeneration. The fact that the most relevant calcium transport systems for the fine tuning of cytosolic calcium homeostasis in neurons have been shown to be molecular targets for ROS/RNS generated in neurodegenerative insults and diseases will be analyzed next in this context. "s most of neurotoxic ROS/RNS species react with many intracellular molecules and these species are short-lived within the cells, the extent of chemical modification of each calcium transport system by ROS/RNS is strongly dependent on its relative proximity to the ROS/RNS source. In functional terms, it has been shown by many studies that ROS/RNS can elicit estimulation or inhibition of key proteins of calcium signalling pathways in neurons, and that these effects are strongly dependent on the specific protein, on the ROS/RNS concentration in the microenvironment and on the accummulated dose of ROS/RNS time of exposure . Therefore, clustering of these systems within subcellular microdomains plays a major role in crossmodulation between calcium and ROS/RNS intracellular signalling, and this point will be specifically addressed thereafter in this chapter. Furthermore, the accummulated experimental evidences pointing out that there is an intimate cross-talk between calcium and ROS/RNS intracellular signalling pathways are now ovelwhelming, including the modulation of ROS/RNS sources by calcium in neurons and the redox modulation of calcium transport systems. "oth, calcium and ROS/RNS intracellular signalling show a clear pattern of local and focalized transients of intracellular concentration peaks . Therefore, clustering of calcium transport systems responsible of the rise of cytosolic calcium and ROS/RNS sources within the same subcellular microcompartments will generate overlapping focalized points of high concentration of calcium and ROS/RNS. In addition, this clustering will produce transient and highly focalized cytosolic calcium concentration peaks near the calcium entry points and associated calcium concentration waves owing to the rapid Neurochemistry 60 diffusion coefficient of calcium ions. Thus, we shall discuss the space and time fluctuations of cytosolic calcium concentrations that are known to be produced by the activity of calcium transport systems more relevant for the control of cytosolic calcium homeostasis in the neuronal soma. Finally, the last section of this chapter is focussed in the most relevant calcium buffering systems expressed in neurons and their modulation by oxidative stress, since calcium buffering systems of the neuronal cytosol play a major role to attenuate the local gradients of calcium concentration. . Neuronal cytosolic calcium homeostasis is attained by functional coupling between different types of calcium transport systems " highly efficient spatial and temporal coupling between the activity of transport systems producing calcium entry to the cytosol and those extruding calcium out of the cytosol is a basic bioenergetics need for brain neurons, as they establish many functional synapses and have to maintain and rapidly restore cytosolic calcium in the neuronal soma within the narrow concentration window that allows for neuron survival. Extensive experimental studies carried out during last thirty years have settled the major molecular actors that allow neurons to achieve this goal, see for example the reviews [ -], and these are schematically presented in the diagram of the Figure . Thus, the control of cytosolic calcium homeostasis in neurons is primarily the result of the activity of transport systems at the plasma membrane acting in concert, with the help of calcium transport systems located in intracellular stores, mainly in the endoplasmic reticulum and mitochondria. The concentration gradient of calcium ions across the neuronal plasma membrane in the brain is by far larger than the concentration gradients of other ions involved in the control of neuronal excitability, like potassium, sodium and chloride. In addition, cytosolic calcium binding proteins provide the neurons with buffering capacity to attenuate the peak height of free cytosolic calcium concentration spikes after focal neuronal stimulation by some neurotransmitters or after high frequency repetitive neuronal stimulation [ ]. In primary cultures of cerebellar granule neurons, calcium entry through L-type voltageoperated calcium channel L-VOCC accounts for more than % of the increase of the steadystate cytosolic calcium in the neuronal soma after partial depolarization of the plasma membrane upon raising the extracellular potassium concentration from to mM [ ]. The particular relevance of this observation for neuronal survival is highlighted by the fact that the apoptosis of these neurons induced by low potassium mM in the extracellular medium can be blocked simply by raising the extracellular potassium concentration up to mM [ , ]. Many other experimental data accumulated along the last two decades point out that the transport systems more potent to elicit a fast and sustained increase of cytosolic calcium in neurons are located at the plasma membrane, i.e. ionotropic receptors and VOCC. These calcium transport systems are activated by extracellular stimuli, neurotransmitters or neuromodulators, either directly or indirectly through plasma membrane depolarization. Let us recall here, for example, that high frequency stimulation of neurons by application of electrical Cytosolic Calcium Homeostasis in Neurons -Control Systems, Modulation by Reactive Oxygen and... http://dx.doi.org/10.5772/57576 61 depolarizing pulses or of the appropriate neurotransmitter chemical stimulation can lead to neuronal tetanic activity. "ecause of their focalized distribution pattern in the neurons and also because of the large differences in the intensity of calcium currents across activated ionotropic receptors and VOCC, significant calcium concentration gradients between different cytosolic regions of the neuron during normal neuronal activity are expected to develop at least transiently. In contrast, the rise of IP following activation of phospholipase C after stimulation of members of the large family of G-coupled neurotransmitter receptors [ ], also located at the plasma membrane, promoted calcium release from intracellular stores displaying calcium spikes of smaller intensity and a more widespread increase of calcium concentration within the cytosol. Taking into consideration the large number of different chemical molecules that promote neuronal stimulation within the brain and the high frequency of the stimulation events, it is Figure 1 . Diagrammatic image illustrating the major calcium transport systems controlling the concentration of cytosolic calcium in the neuronal soma. Yellow and white arrows indicate cytosolic calcium entry and extrusion transport systems, respectively. The thickness of the arrow indicates the relative relevance. Abbreviations: Endopl.Ret., endoplasmic reticulum (green space); NMDAr, NMDA receptor; AMPAr, AMPA receptor; L-VOCC, L-type voltage-operated calcium channel; PMCA, plasma membrane calcium pump; NCX, sodium-calcium exchanger; MR, metabotropic receptor; RyR, ryanodine receptor; IP 3 R, IP 3 receptor; SERCA, endoplasmic reticulum calcium pump; PTP, mitochondrial permeability transition pore; CaUP, mitochondrial calcium uniporter. Neurochemistry 62 wonderful for its simplicity that the concerted action of only a few calcium transport systems can maintain neurons functional and alive for so many years during human lifetime. Why such a simple design? "s the evolution selects the living structures and organisms that optimize the use of metabolic energy [ ] and the bioenergetics costs for building a complex structural design is always higher than the costs for building simpler structures, probably the answer is to optimize the use of the metabolic energy in neurons. Let us recall here that the large needs of metabolic energy to continuously restore the electric potential of the plasma membrane of active brain neurons, which is essential for their proper biological functions, would not allow them to develop a safe system for the control of cytosolic calcium homeostasis of a highbioenergetics cost of maintenance and repair. Noteworthy, maximal energy optimization within the cells can be attained when the coupling between molecules involved in energy transduction makes use of the information or entropic energy stored in subcellular structures, i.e. minimizing stochastic collisional events that dissipate a large amount of energy, and this seems to be the case. For example, the subcellular distribution of the calcium transport systems in neurons enables them to use cytosolic calcium for highly polarized, rapid and specific synaptic responses, and also for more slowly developing adaptative responses, like long term post-synaptic potentiation or depression [ , ]. Furthermore, the different levels of expression of ionotropic and metabotropic receptors in distinct types of neurons allows for differential selectivity and sensitivity in calcium modulation of neuronal threshold excitability, thereby linking regionalization of neuronal responses within the brain structures with the major neurotransmitter pathways. . . The calcium entry systems of the neuronal plasma membrane "ll neurons express different types of functional VOCC. On the basis of their unitary conductance, on their rate of inactivation and their subcellular location the most relevant for neuronal calcium homeostasis are the L-VOCC. The L-VOCC unitary conductance has been reported to be in the range of -pS, while reported unitary conductances for N-, P/Q-and R-type range between and pS, and L-VOCC inactivation kinetics is slower than that of the other VOCC types [ -]. In addition, L-VOCC are polarised in the neuronal soma and at the conical neck leading to neurite extensions [ ], whereas N-, P/Q and R-types of VOCC are largely enriched in the presynaptic plasma membranes and its activation serves largely to elicit neurotransmitter release at the synapses [ , -]. On these grounds, taking also into consideration the rate of kinetics inactivation of the P/Q-VOCC, these channels should afford a contribution to the cytosolic calcium homeostasis of the neuronal soma much lower than that of L-VOCC but higher than that of N-, R-and T-types of VOCC. Indeed, using specific channels blockers we have experimentally assessed that the sum of the contributions of non-L-VOCC calcium channels to the cytosolic calcium homeostasis of the neuronal soma of primary cultures of cerebellar granule neurons in a standard Locke s medium with mM K + is lower than %, while the L-VOCC contribution is % or higher unpublished results . L-VOCC, which are expressed in all neurons, are by far the most relevant calcium channels not only for the tuning of steady-state cytosolic calcium homeostasis in neurons see above , but also for the overall threshold neuronal excitability, see [ -]. The L-VOCC family, also Cytosolic Calcium Homeostasis in Neurons -Control Systems, Modulation by Reactive Oxygen and... http://dx.doi.org/10.5772/57576 63 known as Ca V , has four subtypes Ca V . , Ca V . , Ca V . and Ca V . [ ]. Ca V . and Ca V . are expressed in neurons, cardiac and endocrine cells, while Ca V . and Ca V . are specific of skeletal muscle and retina, respectively [ ]. In brain, near % of L-VOCC belongs to the Ca V . subtype and -% to the subtype Ca V . [ ]. It has been reported that inactivation of the gene encoding for Ca V . in the hippocampus and neocortex of mouse Ca V . HKO leads to a selective loss of N-methyl-D-aspartate NMD" receptors-independent long-term potentiation [ ]. The activity of these calcium channels is modulated not only by the plasma membrane potential but it is also dependent upon their phosphorylation by protein kinases. Meanwhile the activation of different isoforms of protein kinase C PKC has been reported to produce stimulation or inhibition of L-VOCC activity in different cellular types [ ], the activation of protein kinase " PK" and of calcium/calmodulin-dependent protein kinase II CaMKII have been shown to increase the activity of L-VOCC. Moreover, both PK" and CaMKII have been shown to form complexes with L-VOCC subunits. In brain, PK" associates with L-VOCC subunit c [ ]. L-VOCC subunits c and are phosphorylated by PK" [ -], and this produces an increase of L-VOCC activity. It has been demonstrated that this increase of L-VOCC activity is mediated by phosphorylation of Ser and Ser of thesubunit and also by phosphorylation of Ser of the c-subunit, as their mutations led to complete elimination of the PK"-induced increase of calcium currents catalyzed by L-VOCC [ , ]. Regarding CaMKII, the amino acids sequence near Thr of the L-VOCC subunit a shows a high homology with the self-inhibitory domain of the CaMKII and with the binding domain of this kinase in the NR " subunit of NMD" receptors [ ]. Indeed, it has been shown the co-localization within neurons of the L-VOCC Ca V . type and CaMKII [ ] and also of the L-VOCC subunit a with CaMKII, and this has led to the suggestion that the L-VOCC subunit a can act as an associated protein of CaMKII in vivo [ ]. Phosphorylation of L-VOCC by CaMKII takes place not only in Thr of the a subunit but also in Ser and Ser of the subunit and leads to an increase of the intensity of calcium currents through these channels [ , -]. It has been proposed that the modulation of L-VOCC by CaMKII can be relevant to potentiate the raise of cytosolic calcium concentration in response to hormones and growth factors [ , ]. In contrast, the excessive activation of the L-VOCC Ca V . type by CaMKII over-stimulation has been correlated with the loss of dendritic spines in the striatum observed after dopamine depletion in animal models of parkinsonism [ ]. The most potent calcium ionotropic receptors present in the neurons of the mammalian brain are L-glutamate receptors of the NMD" and -amino--hydroxy--methylisoxazole-propionic acid "MP" subtypes, except those "MP" receptors formed only with GluR subunits [ , ], and P X -purinergic receptors [ ]. NMD" and "MP" receptors are present in most fast excitatory synapses in the brain, allowing for neuronal responses in the milliseconds time scale range, and P X -purinergic receptors display also a widespread distribution in the brain. The more limited distribution in brain of L-glutamate receptors of the kainate subtype, its low ionic selectivity for calcium and the slight calcium currents generated upon its activation compared to those observed upon activation of "MP" and NMD" receptors [ ], suggest that they can play at most a secondary role in the tuning of cytosolic calcium homeostasis of a very limited number of brain neurons. Neurochemistry 64 The L-glutamate receptors are expressed in the vast majority of glutamatergic neurons, and they are present in the major structures of mammalian brain neocortex, striatum, hippocampus and cerebellum . NMD", "MP" and kainate receptors are oligomeric integral membrane proteins, being their calcium channel structure predominantly formed by a combination of different, though highly homologous, subunits [ ]. "mong these receptors, NMD" receptors play an outstanding role in neurosciences, as supported by many experimental evidences in studies of brain development [ ], long term post-synaptic potentiation [ ] or brain damage after ischemia-reperfusion [ , ]. Three major reasons allows to explain the dominant role of NMD" receptors over "MP" and kainate receptors in the brain the NMD" single channel conductance is higher than "MP" single channel conductance, -pS versus ~ pS [ , ] -synaptic potentiation LTP , and the latter kinase also enhances the channel conductance of this receptor [ -]. On the other hand, it has been shown that facilitation of L-glutamate release by P x activation can lead to a stronger NMD" receptor activation. The calcium channel in the NMD"-receptor structure can be formed by different combinations of subunit NR and one of the isoforms of subunit NR ", NR ", NR C and NR D [ ]. The expression of functional NMD" receptors is a relatively slow process during the maturation of neurons [ ]. Therefore, in molecular terms there are different isoforms of functional NMD" receptors whose level of expression varies from one type of neurons to another, and also during neuronal maturation. In addition, NMD" receptors are found in synaptic and in extra-synaptic locations [ -]. "s activation of extra-synaptic NMD" receptors can lead to a less focalized increase of cytosolic calcium, the extra-synaptic NMD" receptors are likely to play a role more relevant than synaptic NMD" receptors in the control of cytosolic calcium homeostasis in the neuronal soma. Phosphorylation of NMD" receptors in vitro by PK" and by some PKC isoforms increases their activity [ ]. The costimulation of PK" and PKC elicits the phosphorylation of Ser and Ser leading to activation of NMD" receptors, while phosphorylation of Ser by only PKC leads to a subcellular re-localization of the NR subunit of NMD" receptors, which is reverted upon dephosphorylation [ ]. . . Transport systems that release calcium from intracellular stores The long-term control of neuronal calcium homeostasis also involves several major calcium transport systems of the subcellular organelles that behave as relevant neuronal intracellular stores, namely, endoplasmic reticulum and mitochondria. Cytosolic Calcium Homeostasis in Neurons -Control Systems, Modulation by Reactive Oxygen and... http://dx.doi.org/10.5772/57576 65 The endoplasmic reticulum Ca + -"TPase SERC" helps to pump calcium out from the cytosol to the endoplasmic reticulum internal space [ ], while calcium release from the endoplasmic reticulum involves the activation of IP receptors and/or ryanodine receptors in different neuronal responses and in synaptic plasticity [ , -]. "lthough the neuronal endoplasmic reticulum can accumulate much lower amounts of calcium than the sarcoplasmic reticulum of muscle cells, it is still significant for hippocampal neurons as shown in several studies, see e.g. [ , ]. In these cases, calcium release from the endoplasmic reticulum can sustain a moderate increase of cytosolic calcium, which has been shown to play a significant role in the process of LTP in hippocampal neurons [ , ]. "y itself, calcium filling of the endoplasmic reticulum is relevant for neuronal survival to ensure the correct protein folding of many proteins, particularly proteins of the plasma membrane or to be secreted to the extracellular space, as the activity of several endoplasmic reticulum protein-chaperones is dependent on the calcium concentration in the internal space of the endoplasmic reticulum [ ]. Depletion of calcium in the endoplasmic reticulum has been shown to elicit the opening of specific calcium channels of the plasma membrane, the store-operated calcium entry SOCE [ , -]. The presence of SOCE in neurons has been documented during last years [ , ], and its opening elicits a transient increase of cytosolic calcium under neuronal stress conditions to restore the calcium levels of intracellular stores. The inhibition of SERC" by selective inhibitors, thapsigargin or cyclopiazonic acid, is needed to induce the large calcium depletion in the endoplasmic reticulum required for SOCE in experiments with cells in culture. Thus, this process can be seen as a 'rescue call' at the cellular level and operates under conditions of severe energetic depletion of the neurons. Indeed, it is to be recalled here that these channels and in particular the isoforms TRPC-and -have been also involved in neuronal survival of CGN [ ]. More recently, Selvaraj et al. [ ] have demonstrated that in a mouse neurotoxin-based model of Parkinson s disease, reduced Ca + influx through transient receptor potential C TRPC channels in the plasma membrane of dopaminergic neurons triggers a cell death-inducing endoplasmic reticulum-stress response. These latter results highlighted for the first time the relevance of calcium homeostasis in Parkinson s disease. In contrast, the large population of neuronal mitochondria can store relatively large amounts of calcium, high enough to elicit a large increase of cytosolic calcium as shown by several studies, see e.g. [ , ]. Nevertheless, the rate of calcium fluxes across the mitochondrial membrane transporters in normal cells is much slower than that measured for the major endoplasmic reticulum calcium transport systems listed above. However, calcium release from mitochondria high enough to promote a large and sustained rise of cytosolic calcium in neurons has been observed only during the development of neuronal cell death, as a consequence of the steady opening of the high permeability mitochondrial transition pore [ ]. On these grounds, large calcium release from mitochondria has been proposed to be part of the molecular mechanism that triggers irreversible events in neuronal cell death through calpains activation. On the other hand, the uptake of calcium by mitochondria takes place through a calcium uniporter [ , ], with a rate of uptake in the submicromolar calcium range much lower than the major cytosolic calcium extrusion pathways, namely, PMC" and SERC" in neurons [ , ]. Neurochemistry 66 . . The transport systems involved in calcium extrusion from the cytosol The major plasma membrane calcium extrusion systems, PMC" and Na + /Ca + -exchanger NCX are expressed in all neuronal types. PMC" provides the major extrusion pathway operating in neurons for the maintenance of cytosolic calcium concentrations below the neurotoxic calcium range, i.e. < . μM cytosolic calcium [ , , ]. "s PMC" is active at cytosolic calcium concentrations below . μM [ , ], neurons must spend a significant amount of metabolic energy "TP to maintain cytosolic calcium within the short concentration range which is required for neuronal survival. Therefore, the cytosolic calcium concentration can be considered a key bioenergetics marker of neuronal activity and survival. In contrast, NCX is more potent than the PMC" at cytosolic calcium concentrations ≥ . μM [ , ]. On these grounds, NCX can be seen as a safety system to minimise neuronal damage associated with cytosolic calcium ≥ . μM, as its activation when cytosolic calcium reaches this range allows neurons to rapidly reset cytosolic calcium to the concentration window that allows neuronal survival, see above. The expression levels of different neuronal isoforms of PMC" undergo significant changes during neuronal maturation [ ], and a similar observation has been reported for NCX isoforms [ ]. This has been seen as a neuronal adaptative response to the fine set of free cytosolic calcium concentration and control of cytosolic calcium homeostasis, since it has been demonstrated that different PMC" isoforms show different affinity for calcium [ ]. On the other hand, although both PMC" and NCX are found in the plasma membrane of the neuronal soma and neuronal dendrites, recent data cast doubt on the current assumption that both PMC" and NCX are homogeneously distributed in the plasma membrane. For example, regulatory effects of actin cytoskeleton have been recently reported on the NCX activity [ ], and actin filaments are components of caveolin-rich structures associated with 'lipid rafts' [ ]. SERC", which catalyzes the "TP-dependent calcium uptake by this subcellular organelle, plays only a secondary role as a system for calcium extrusion from the cytosol because in neurons PMC" is a calcium pump more potent than SERC" [ ]. The calcium uptake by mitochondria is performed mainly via the Ca + uniporter driven by the large mitochondrial inner membrane potential [ ], although the contribution of an alternate transport system yet ill-defined in molecular terms cannot be excluded under conditions of high frequency of cytosolic calcium peaks [ ]. Nevertheless, in neurons the rate of calcium uptake by mitochondria is much slower than the rate of calcium extrusion from the cytosol via the plasma membrane systems, i.e. the PMC" and NCX, and via the SERC". . Compartmentation of calcium transport systems relevant for the control of cytosolic calcium homeostasis in nanodomains of the neuronal plasma membrane and functional implications Many recent experimental evidences have demonstrated that the calcium transport systems of the neuronal plasma membrane more relevant for the control of cytosolic calcium homeostasis are clustered within focalized nanodomains of a diameter size lower or equal to few Cytosolic Calcium Homeostasis in Neurons -Control Systems, Modulation by Reactive Oxygen and... http://dx.doi.org/10.5772/57576 67 hundreds of nanometers. Lipid rafts of the plasma membrane are dynamic nanodomains of a dimension between and nm [ ], which define cellular sub-microdomains of the plasma membrane anchoring caveolins, see e.g. [ ], and it has been suggested that caveolin-rich nanodomains associated with neuronal plasma membrane lacking the morphological appearance of caveola invaginations can serve to focalize signal transduction in neurons [ ]. Indeed, the putative implication of lipid rafts in the regulation of intracellular calcium homeostasis and calcium signalling pathways was already suggested in the ´s [ , ], but only during the last decade this hypothesis has been experimentally demonstrated, see [ , ]. Lipid rafts are enriched in cholesterol and sphingolipids [ ], including a lipid family particularly enriched in the plasma membrane of neurons the gangliosides [ ], and define nanodomains of the plasma membrane for the anchoring of caveolins, flotillin, actin microfilaments and also an increasingly higher number of palmitoylated or farnesylated proteins, see [ ]. The isoform caveolin-binds to cholesterol and sphingolipids [ -], and also promotes the transport of cholesterol from the endoplasmic reticulum to the plasma membrane [ ]. These nanodomains are merging as unique platforms for intracellular signalling in neurons, as pointed out in [ , , ], and their stability is currently rationalized in terms of specific protein/ protein or protein/lipid interactions. Noteworthy, as caveolins can act as scaffolding proteins in protein/protein interactions within these nanodomains [ , ], these interactions also bear functional relevance for the protein partners and, therefore, these nanodomains cannot be solely seen as structural elements of the plasma membrane. In this regard, it has been reported that cholesterol depletion with methyl--cyclodextrin, a chemical widely used to solubilise lipid rafts, alters the basal current of L-VOCC in foetal mouse skeletal muscle cells and cardiomyocytes [ , ]. "lso the calcium-dependent exocytosis in synaptosomes is sensible to the cholesterol content of the plasma membrane [ ], and probably one of the best documented functions of caveolins is their implication in the maintenance of intracellular cholesterol homeostasis [ ]. Noteworthy, using hippocampal neurons in culture it has been demonstrated the regulation of caveolins expression by L-glutamate [ ], and an increased level of caveolins expression has been reported in "lzheimer s disease which has been correlated with the increased level of cellular cholesterol observed in these patients [ ]. On the other hand, knockout mice in caveolin-have impaired nitric oxide and calcium signalling pathways, displaying severe vascular and pulmonary anomalies and uncontrolled cellular proliferation [ ], and caveolins mutations has been associated with muscle disorders and cancer [ ]. Moreover, lipid rafts alterations have been reported in a significant number of pathologies [ , ]. The association of the muscle type of L-VOCC with lipid rafts sub-microdomains in cardiomyocytes was established nearly years ago [ , ]. Later, we have demonstrated L-VOCC association with lipid rafts nanodomains in mature primary cultures of cerebellar granule neurons using FRET microscopy imaging [ ]. This association of L-VOCC with lipid rafts nanodomains has a major functional relevance for the regulation by protein kinases of the calcium influx through these channels in neurons. First, as noted previously in this chapter within the brain the c subunit of L-VOCC forms a complex with PK" [ ] and Razani et al. [ ] have demonstrated the co-localization and direct interaction between the scaffolding Neurochemistry 68 domain of caveolin-and the catalytic subunit of PK" in vivo and in vitro, respectively. Second, some experimental data have suggested the possibility of direct association of CaMKII with lipid rafts [ ], which is consistent with the reported co-localization of Ca V . , the predominant L-VOCC subtype in the brain, and CaMKII [ ]. Functional regulation of L-VOCC by lipid rafts is also supported by the modulation of the level of phosphorylation of L-VOCC by cholesterol depletion in cardiomyocytes [ ]. Since the two major subtypes of L-VOCC present in the brain, namely Ca V . and Ca V . , directly interact with many proteins having the PDZ binding domain [ , ], proteins that also bind to the NMD" receptor [ ], the association of these receptors with lipid rafts nanodomains is not an unexpected finding. The presence of NMD" receptors in isolated lipid rafts has been shown by different investigators [ -], and using fluorescence resonance energy transfer FRET microscopy imaging their association with lipid rafts nanodomains in mature primary cultures of cerebellar granule neurons has been demonstrated in a recent work of our laboratory [ ]. The critical role of proteins with PDZ domains in the association of NMD" receptors with neuronal lipid rafts has been experimentally demonstrated using genetically modified mice, as mutations in the NR " and NR " subunits which impair their interaction with PDZ domains led to a reduction of NMD" receptors association with lipid rafts [ ]. It has been suggested that the clustering of NMD" receptors in lipid rafts-associated sub-microdomains can potentiate the activation of these receptors, thereby serving as a molecular mechanism for potentiation of the synaptic efficiency in neuronal connections [ , ]. "ecause "MP" receptor clustering near NMD" receptors plays a key role for NMD" receptor activation and LTP induction, it is of special neurophysiological relevance to note here that the association of "MP" receptors with molecular components of the lipid rafts of neuronal plasma membranes has also been experimentally demonstrated [ , , ]. The association with lipid rafts of the major systems of the neuronal plasma membrane for extrusion of calcium from the cytosol, PMC" and NCX, has also been experimentally assessed, although to the best of our knowledge only in the case of PMC" this has been reported with neuronal plasma membranes at the time this chapter was written. PMC" association with lipid rafts has been shown using preparations of synaptic plasma membranes [ ] and also in primary cultures of rat cortical and hippocampal neurons [ ]. Earlier, it was shown that the C-terminal domain of the PMC" interacts with proteins with PDZ domains [ ]. Moreover, Jiang et al. [ ] showed that disruption of lipid rafts domains by chronic depletion of cholesterol elicited a marked decrease of PMC" activity, suggesting that PMC" associated with lipid rafts is more active than PMC" bound to non-raft domains. NCX has been shown to be associated with lipid rafts in the smooth muscle of coronary arteries [ ], it has also been shown to be present in membrane fractions of vascular endothelial cells enriched in the lipid rafts markers caveolin-and e-NOS [ ] and the direct interaction of cardiac NCX with caveolin-has been demonstrated by co-precipitation [ ]. On these grounds, lipid rafts nanodomains of the neuronal plasma membrane can be seen as microchip-like structures for the fine coupling and control of systems playing a major role in the maintenance of a cytosolic calcium homeostasis within the range that allows for survival and normal functionality of neurons. "ecause of the relevance of oxidative stress in neurode-Cytosolic Calcium Homeostasis in Neurons -Control Systems, Modulation by Reactive Oxygen and... http://dx.doi.org/10.5772/57576 69 generation it is of utmost importance to note that two enzymatic sources of ROS/RNS have been shown to be also associated with these lipid rafts nanodomains in the neuronal plasma membrane, namely, neuronal nitric oxide synthase nNOS and cytochrome b 5 reductase Cb 5 R . Sato et al. [ ] showed that two domains of the nNOS, the oxygenase and the reductase domains, interact with the scaffolding domain of caveolin-. More recently, using FRET microscopy imaging our group has shown that nNOS is associated with lipid rafts nanodomains enriched in NMD" receptors and L-VOCC in mature cultures of primary cerebellar granule neurons [ ]. Since nitric oxide play a very important role in neuromodulation, this association bears a special relevance as protein/protein interactions regulate the enzyme activity of nNOS as well as define anchoring points for the subcellular location of this protein [ , ]. Indeed, it has been shown that the interaction of nNOS with caveolin-in skeletal muscle modulates the catalytic activity of NOS [ ]. In addition, previous works of our laboratory have shown that the Cb 5 R, whose deregulation at the onset of neuronal apoptosis generates a burst of superoxide anion that stimulates the entry in the irreversible phase characterized by caspases activation [ , -], is also associated with lipid rafts nanodomains enriched in L-VOCC and NMD" receptors in mature cultures of primary cerebellar granule neurons [ , , ]. Moreover, the association with these lipid rafts nanodomains of a source of nitric oxide nNOS and of a source of superoxide anion Cb 5 R point out that these nanodomains may play also a major role in the focalized generation of the harmful oxidant peroxynitrite in the plasma membrane when the neurons are exposed to sustained cellular stress conditions. Let us recall here also that some mitochondria, a widely accepted major ROS-producing subcellular compartment, are also close to the plasma membrane in many neuronal types, because the cell nucleus occupies a large volume of the neuronal soma. This protein clustering associated with lipid rafts nanodomains of the neuronal plasma membrane is summarized in the Table , where proteins of the cytoskeleton typically associated with lipid rafts are also included. Noteworthy, ROS significantly alter the actin polymerisation/depolymerisation dynamics, reviewed in [ ]. "ecause actin microfilaments are part of the structural protein network of proteins associated with lipid rafts nanodomains, ROS are expected to produce a significant distortion of this protein network, like nNOS which has been shown to associate with the neuronal cytoskeleton in synaptic terminals [ ]. Indeed, regulatory effects of actin cytoskeleton have been reported on NMD" receptors activation [ ], on the distribution of L-type calcium channels in myocytes [ ], and on the activity of NCX [ ]. Structural elements Calcium transport systems ROS/RNS sources Regulatory kinases Cholesterol, Caveolins, L-VOCC, nNOS and Cb 5 R PKA and CaMKII Sphingolipids, Flotillin, NMDA and AMPA receptors, Actin microfilaments, PMCA and NCX PDZ-binding proteins Table 1 Molecules associated with lipid rafts in the neuronal plasma membrane of special relevance for cytosolic calcium homeostasis and ROS/RNS-calcium signalling cross-modulation. Neurochemistry 70 In spite of the well known relevance of L-glutamate "MP" and NMD" receptors clustering in LTP, the regulation of incorporation and dissociation of proteins in nanodomains or submicrodomains associated with lipid rafts is still poorly understood and, thus, it is a pending issue. It is to be noted also that knowledge of the time scale range of the clustering dynamics of proteins within these nanodomains is a basic need to properly understand their formation and plasticity, and this is particularly relevant to reach firm conclusions regarding their role as structural or adaptive elements in rapid and slow neuronal responses. In conclusion, a close spatial location of these calcium transport proteins in the neuronal plasma membrane can also afford a fast and fine tuning of cytosolic calcium concentrations. Moreover, as major redox centers producing ROS are also tighly associated with lipid rafts nanodomains, this compartmentation allows also to rationalize on simple grounds the intimate cross-talk between ROS and calcium signalling in neurons, as well as between oxidative stress and sustained cytosolic calcium deregulation, reviewed in [ , ]. . Sustained alteration of cytosolic calcium homeostasis in neuronal death Neuronal survival is extremely dependent of the fine tuning of cytosolic calcium homeostasis, because cytosolic calcium concentration has to be maintained within a relatively narrow window for neuronal survival [ ], for example, between and nM for cerebellar granule neurons in culture [ , ]. "n overwhelming amount of experimental data reported by many investigators from different countries show that sustained deviations of cytosolic calcium concentration out of this narrow window lead to neuronal cell death. "esides rapid necrotic neuronal death induced by sustained cytosolic calcium concentration higher than . μM for periods in the minutes time scale range [ , , , , , ], it has also been shown that apoptotic neuronal death can be induced when cytosolic calcium concentration remains very low for longer periods of time, in the hours time scale range [ , ]. "s the extracellular free calcium concentration is approximately mM, this implies that neurons need to sustain a large calcium gradient across their plasma membranes. Owing to the large number of synaptic connections established by neurons in the brain, these cells need to spend a large amount of metabolic energy to maintain their cytosolic calcium homeostasis, because during synaptic activity calcium entry is activated through VOCC and some ionotropic receptors, mainly NMD" receptors. In addition, many neuronal processes are extremely dependent upon cytosolic calcium concentration, such as neurotransmitter secretion and synaptic plasticity [ ], neurite growth and sprouting [ ] and signalling pathways which mediate the metabolic neuronal responses to a large number of relevant extracellular stimuli [ , ]. Therefore, the cytosolic calcium concentration should be considered a major bioenergetic marker for neuronal activity and survival. The increase of oxidative stress in brain is a biochemical marker associated with neurodegenerative insults, like ischemia-reperfusion or inflammation, or neurodegenerative diseases of high prevalence and relevance to humans, for example, "lzheimer s, Parkinson s, amyotrophic lateral sclerosis and Huntington s diseases. Many studies have shown that cellular oxidative stress is caused by an imbalance between endogenous antioxidant defences and ROS produc-Cytosolic Calcium Homeostasis in Neurons -Control Systems, Modulation by Reactive Oxygen and... http://dx.doi.org/10.5772/57576 71 tion in favour of the latter, which results in an excessive exposure of cells to harmful ROS/RNS. On the other hand, it is well established now that the calcium transport systems most relevant for the cytosolic calcium homeostasis in neurons are molecular targets for ROS/RNS and that their chemical modification by these reactive species lead to their functional impairment. Indeed, oxidative chemical modifications of these calcium transport systems have been reported to take place in vivo. Moreover, many experimental studies reported during last years led to the conclusion that ROS produce a sustained deregulation of cytosolic calcium homeostasis in neurons. For example, neuronal death mediated by calpains activation can be taken as a biological marker of a sustained rise of cytosolic calcium concentration [ -]. "nother examples are provided by the central role of L-VOCC and ROS in the apoptosis induced by low extracellular potassium concentration [ , , , , , ], and also by Lglutamate excitoxicity-induced neuronal death [ , , ]. Thus, sustained alterations of neuronal cytosolic calcium are expected to be a convergent cellular mechanism in brain neurodegeneration. Consistent with this hypothesis, alterations of neuronal calcium homeostasis and brain oxidative stress have been reported in the case for the brain neurodegenerative diseases of higher incidence to humans, like "lzheimer s [ , ] and Parkinson s [ , ], or in the acute neurodegeneration observed in amyotrophic lateral sclerosis [ , ], and also for major brain insults, such as excitotoxicity in trauma and ischemia-reperfusion [ , ], inflammation [ , ] and neurotoxicity by drugs and environmental chemicals [ , ]. Most ROS/RNS that are produced in cellular oxidative stress in mammalian tissues have been demonstrated to be strongly neurotoxic to neurons in vitro. This is a relatively large list of ROS/ RNS, and we shall concentrate in this chapter in those most studied as agents in brain neurodegeneration, namely, superoxide anion, H O , hydroxyl radicals, lipid hydroperoxides, and nitric oxide-derived ROS, mainly peroxynitrite and nitrogen dioxide. "ecause of the calcium dependence of the activity of nNOS, the main enzymatic system responsible for the production of nitric oxide in neurons [ ], RNS should be expected to play a particularly relevant role as intracellular biomarkers of the level of coordination or deregulation of calcium and ROS signalling pathways in neurons. However, it is still a matter of debate whether in vivo all of these ROS/RNS can reach concentrations high enough to act as causal agents or merely as agents that potentiate or accelerate the rate of an ongoing neuronal death process in the brain. Moreover, the analysis and dissection of the chemical reaction pathways of each one of this ROS/RNS is further complicated by the fact that in vivo they generate radicalic chain chemical reactions. Therefore, it is critical to identify the major subcellular primary sources of these ROS/ RNS in different neurons and in different degenerative processes in the brain, and this is an issue yet to be settled in many cases, as during lasts years the experimental evidences have pointed out that the relative relevance of different ROS/RNS seems to be largely dependent on the neurodegenerative disease or brain insult. . Modulation by ROS/RNS of calcium transport systems relevant for the control of neuronal cytosolic calcium homeostasis ROS and RNS producing oxidative stress to neurons can be generated by neuronal and also by non-neuronal cells, like microglia or endothelial cells of the brain blood vessels. It is to be Neurochemistry 72 noted that oxidative stress-induced brain degeneration is a relatively slow process, in most neurodegenerative diseases developing in periods of time of years and in acute brain ischemiareperfusion in a time range from minutes to several days, depending upon the intensity of the oxidative stress insult. Thus, in a brain suffering oxidative stress neurons are exposed for relatively large time periods to either extracellularly and/or intracellularly generated ROS/ RNS. "ecause the extracellular liquid bathing the brain and stem neurons is poorer in antioxidants than the blood, due to the low permeability and high selectivity of the blood-brain barrier, the extracellular antioxidant protection in the brain is notably lower than that of other organs and tissues in mammals. Under these environmental conditions the plasma membrane of neurons, where major calcium transport systems controlling the cytosolic calcium homeostais are located, is particularly sensitive to the oxidative stress generated in the brain by vicinal neuronal and non-neuronal cells. The major ROS/RNS reported to play a significant role in the enhanced brain oxidative stress associated with neurodegenerative diseases and insults like ischemia-reperfusion and inflammation can be split into three major groups i primary biochemical ROS/RNS, i.e. chemical species directly generated by some enzymes or proteins during brain activity in normal or pathophysiological conditions, ii secondary biochemical ROS/RNS, chemical species derived by rapid reaction between the primary biochemical ROS/ RNS or by systems involved in their detoxification, and iii radicalic chain ROS/RNS, chemical radicals involved in the initiation of radical reaction chains or that are largely generated within radical reaction chains. Superoxide anion is a primary biochemical ROS that plays a key role in the generation of many of the more harmful ROS and RNS detected in the oxidative stress-induced degeneration of the brain. Superoxide anion can be produced by neuronal and non-neuronal cells within the brain.