IUBMB

Life, 59(4 5): 280 285, April May 2007

Critical Review
Calcium, Iron and Neuronal Function
Cecilia Hidalgo1 and Marco T. Nunez2
Centro FONDAP de Estudios Moleculares de la Celula and Programa de Biologia Celular y Molecular, Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile 2 Department of Biology and Millennium Institute for Cell Dynamic and Biotechnology, Faculty of Sciences, Universidad de Chile, Santiago, Chile
1

Summary Calcium and iron play dual roles in neuronal function: they are both essential but when present in excess they cause neuronal damage and may even induce neuronal death. Calcium signals are required for synaptic plasticity, a neuronal process that entails gene expression and which is presumably the cellular counterpart of cognitive brain functions such as learning and memory. Neuronal activity generates cytoplasmic and nuclear calcium signals that in turn stimulate pathways that promote the transcription of genes known to participate in synaptic plasticity. In addition, evidence discussed in this article shows that iron deciency causes learning and memory impairments that persist following iron repletion, indicating that iron is necessary for normal development of cognitive functions. Recent results from our group indicate that iron is required for long-term potentiation in hippocampal CA1 neurons and that iron stimulates ryanodine receptor-mediated calcium release through ROS produced via the Fenton reaction leading to stimulation of the ERK signaling pathway. These combined results support a coordinated action between iron and calcium in synaptic plasticity and raise the possibility that elevated iron levels may contribute to neuronal degeneration through excessive intracellular calcium increase caused by iron-induced oxidative stress. IUBMB Life, 59: 280285, 2007 Keywords
ERK signaling; oxidative stress; ryanodine receptor; Fenton reaction; synaptic plasticity; gene expression; long-term potentiation. calcium release; CREB, cAMP/calcium response element binding protein; ERK, extracellular signalregulated kinases; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptors; IRE, ironresponsive element; IRP, iron regulatory protein; LTP, long-term potentiation; NMDA, N-methyl D-aspartate; NFAT, nuclear factor of activated T cells; ROS, reactive oxygen species; RyR, ryanodine receptor. Received 15 January 2007; accepted 16 January 2007 Address correspondence to: Prof. Cecilia Hidalgo, ICBM, F. Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile. Tel: 56 2 978 6510. Fax: 56 2 777 6916. E-mail: chidalgo@med.uchile.cl
ISSN 1521-6543 print/ISSN 1521-6551 online 2007 IUBMB DOI: 10.1080/15216540701222906

INTRODUCTION Calcium and iron share the common feature of being both essential for normal neuronal function and toxic at high concentrations. Iron is necessary for the normal development of cognitive functions whereas calcium is an important mediator of activity-dependent gene expression and of other key neuronal functions. In particular, specic neuronal stimuli produce calcium signals that through the sequential activation of signaling cascades and transcription factors promote the expression of genes involved in synaptic plasticity, a neuronal response seemingly correlated with learning and memory. In this article we will review existing evidence indicating that both calcium and iron have central roles in neuronal function, including our recent ndings showing that changes in iron levels aect synaptic plasticity. We will also address briey how calcium and iron via calcium release elicited by reactive oxygen species (ROS)1 produced by the Fenton reaction may jointly inuence normal and pathological neuronal responses.

Abbreviations CaM, Calcium/calmodulin; CICR, calcium-induced

CALCIUM AND NEURONAL FUNCTION Neuronal calcium signals produce diverse physiological responses that occur in dierent temporal domains and which include, among others, secretion of neurotransmitters, synaptic plasticity, gene expression and axonal growth (1 6). Among these dierent calcium dependent responses, we will focus in this article on how activity-dependent calcium signals promote neuronal gene expression. Calcium signals can arise in cells via calcium entry through plasma membrane calcium channels activated by voltage, neurotransmitters or store depletion (1). Additionally, activation of calcium release channels present in intracellular stores, such as the ryanodine receptors (RyR) or the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), also gives rise to

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calcium signals (7, 8). Through calcium-induced calcium release (CICR), which is based on the activation of RyR or IP3R by calcium, small and localized calcium signals can be amplied and propagated to the mitochondria and the nucleus (1, 8). The calcium levels in these organelles are bound to receive increasing attention because the functional importance of both nuclear and mitochondrial calcium signals is becoming increasingly apparent. Nuclear calcium signals inuence the expression of a variety of genes whereas the mitochondrial calcium concentration, which results from the balance between calcium uptake and release, has a central role in mitochondrial function and if too high may prompt cell death. Mitochondria can take up calcium released from neighboring endoplasmic reticulum stores and release it later, giving rise to delayed calcium signals (9). Calcium signals can arise at the nucleus from propagated cytoplasmic signals or through other mechanisms including calcium release directly at the nucleus. Neuronal activity generates calcium signals that result in the expression of genes that are essential for dendritic development, synaptic plasticity and neuronal survival. Activity-dependent gene expression involves nuclear and cytoplasmic factors that can stimulate or inhibit specic transcription factors by decoding the spatial and temporal properties of particular calcium signals (1, 6, 10 13). A case in point is the activation of cytoplasmic or nuclear calciumdependent kinases and phosphatases, which through phosphorylation or dephosphorylation modify the transactivating properties of transcription factors such as the nuclear transcription factor cAMP/calcium response element binding protein (CREB). It is now widely accepted that activitydependent CREB phosphorylation is crucial for synaptic plasticity (14, 15), a neuronal response considered essential for learning and memory (16, 17) which entails the expression of new gene products that cause long-term structural and functional changes (16, 18). Activity-dependent calcium signals produced via calcium entry through L-type calcium channels and/or N-methyl D-aspartate (NMDA) receptors stimulate cytoplasmic signaling cascades, which via stimulation of CREB phosphorylation (19 21) prompt the transcription of genes containing cAMP/ calcium response element promoter sequences, such as c-fos and brain derived nerve factor (10 12, 19, 22 25). In hippocampal neurons cytoplasmic and nuclear calcium signals can stimulate calcium/calmodulin-dependent (CaM) kinases or extracellular signal-regulated kinases (ERK). Once activated, both kinases promote CREB phosphorylation at the nucleus albeit with dierent kinetics; in addition, nuclear calcium is required to stimulate CaM kinases and phosphorylate a coactivator protein of CRE-mediated transcription (20, 26 29). Several reports indicate that CREB-dependent transcription of genes involved in synaptic plasticity entails long-term CREB phosphorylation by the ERK pathway (16, 26, 30 32). Enhanced ERK phosphorylation occurs downstream of Ras

activation by calcium, which seems to be due primarily to calcium activation of Ras-specic exchange factors (33). Dierent Ras-specic exchange factors can act as calcium sensors for dierent NMDA receptor types and through activation of particular MAP kinase cascades, they can induce opposing forms of synaptic plasticity (34). The above description highlights the relevant association between neuronal activity and calcium-dependent gene expression. In addition, the source of calcium seems to convey specic signals to the nucleus. For example, the association between L-type calcium channels and signaling protein complexes that promote CREB phosphorylation and synaptic plasticity varies according to L channel subtype and neuronal origin (6, 19, 35, 36). Likewise, calcium inux via P/ Q-type voltage-dependent calcium channels does not promote gene expression if calcium release from intracellular stores is suppressed (37). Additionally, calcium entry via NMDA receptors generates just below the postsynaptic membrane calcium signals that can eectively activate ERK and CREB (21); in contrast, stimulation of extrasynaptic hippocampal NMDA receptors induces a CREB shut-o pathway (38). Postsynaptic sub-membranous calcium signals also stimulate the transcription factor NF-kB (39). Yet, as recently reviewed elsewhere (5) neuronal gene expression via calcium activation of other transcription factors, including NF-kB and the nuclear factor of activated T cells (NFAT), has been less studied in comparison to CREB.

IRON AND NEURONAL FUNCTION Iron is a necessary element for the normal development of cognitive functions. Late fetal and early postnatal iron deciency is a condition that causes learning and memory impairments in humans that persist following iron repletion (40 44). Enzymes involved in neurotransmitter synthesis that possess iron as a prosthetic group are recognized targets of iron deciency (45, 46). Tyrosine hydroxylase, required for dopamine and norepinephrine synthesis (47), tryptophan hydroxylase, required for serotonin synthesis (48), glutamate decarboxylase and glutamate transaminase, involved in GABA and L-glutamate synthesis (49, 50) and the monoamine oxidases A and B involved in dopamine catabolism (51, 52), all belong to this group. One of the major symptoms of iron deciency in humans is a decline in cognitive capacity (42, 44), a characteristic replicated in rat iron-deciency models (51). Research groups around the world have studied the cognitive behavior of iron decient children (44, 53 57). Despite variations in methods, race and cultural peculiarities they have all reached essentially the same conclusion: the two more important factors in determining the degree of the cognitive decit are the magnitude and the duration of the period of iron deciency (58). Similarly, iron decient rats are slow learners, as evidenced by their behavior in the water maze and the

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Morris Water Maze tests (59, 60); in these animals, the degree of learning decit correlates with the extent of iron deciency. In vertebrates, cellular iron levels are post-transcriptionally controlled by the activity of the iron regulatory proteins (IRP1 and IRP2), cytosolic proteins that bind to structural elements called iron-responsive elements (IREs). Three of the fundamental proteins that regulate cellular iron homeostasis the transferrin receptor and DMT1, both involved in cell iron uptake, and the cytosolic iron-storage protein ferritin possess IREs in the untranslated regions of their respective mRNAs (reviewed in (61). The activities of IRP1 and IRP2 respond to changes in cellular iron through dierent mechanisms. In iron-replete conditions IRP1 has a 4S-4Fe cubane structure that renders the protein active as a cytosolic aconitase but inactive for IRE-binding. Unexpectedly, eectors such as nitric oxide (62, 63), hydrogen peroxide (64) and iron-induced oxidative stress (65, 66) activate IRP1 by disruption of the 4Fe-4S cluster. In contrast, IRP2 is activated by hypoxia (67) and is down-regulated by iron-induced oxidative damage followed by ubiquitination and proteasome degradation (68). Genetically modied IRP17/7 mice are normal and exhibit only slight defects in iron metabolism in the kidney and brown fat (69), whereas IRP27/7 mice develop a neuronal disorder in adulthood characterized by ataxia, bradykinesia and tremor (69, 70). Thus, IRP2 seems to dominate the physiological regulation of iron metabolism whereas IRP1 seems to be associated basically with pathophysiological conditions. The ux of iron into or out of cells is determined by the respective activities of the iron uptake transporter DMT1 and the iron removal transporter ferroportin. In agreement with the presence of an IRE element in its 30 anking region, DMT1 expression is down-regulated by high cell iron contents, albeit there is no general agreement about the specic mechanisms responsible for this regulation. Ferroportin, the only member of the SLC40 family of transporters, is the rst reported protein to promote iron exit from cells (71). This protein is expressed mainly in enterocytes and macrophages, but the presence of both DMT1 and ferroportin has also been described in neurons, neuroblastomas and astrocytes (72, 73). The mechanisms responsible for the regulation of Ireg1 expression are currently unknown. Iron deciency induces Ireg1 expression in enterocytes (71) but has the opposite eect in macrophages (74). In a recent study we reported that SHSY5Y neuroblastoma cells and hippocampal neurons in primary culture which survive an iron accumulation protocol evoke an adaptive response characterized by decreased DMT1 and increased Ireg1 synthesis (73). It follows that the concerted regulation of iron transporters is cell-specic and adjusts to the particular functions of cells: DMT1 expression is activated by low iron levels in all cell types whereas ferroportin is activated by low iron in intestinal cells, thus allowing for higher intestinal iron absorption, and by high iron in neuronal

cells and lung macrophages, thus allowing for diminished cell iron levels. Most electrophysiology studies on the role of iron in brain function have centered in the hippocampus, due to its unique role in learning and memory processes and spatial learning tasks. Hippocampal slices taken from iron decient rats exhibit immature long-term potentiation (LTP) patterns 30 days after birth (P30) and lower LTP responses at P65 (75). Similarly, immunohistochemical analyses of dendritic growth indicate that iron-decient P30 pups have an immature dendritic morphology in CA1 neurons, a trait that is conserved at P65 even after iron supplementation (76). Additionally, rats subjected to perinatal nutritional iron deciency show impaired hippocampus-dependent learning when exposed to a fear-conditioning protocol (77). These results indicate that iron plays a key role in the development and function of the nervous system, and suggest that distinctive hippocampal regions (e.g., CA1) may be particularly compromised by iron deciency. In spite of the signicant knowledge gained in studies of iron-decient conditions, the molecular mechanisms behind the requirement for iron for the normal development of cognitive functions remain largely unknown. Current studies, however, have started to uncover the participation of iron in signal transduction mechanisms related to synaptic plasticity (see below). In addition, we recently reported that the iron chelator desferrioxamine inhibits tetanic LTP induction in the CA1 area of hippocampal slices obtained from late fetal and early postnatal iron-sucient rats; in contrast, basal synaptic transmission and paired-pulse facilitation were not aected by desferrioxamine (78). These ndings strongly suggest that iron is needed for LTP, and provide a potential model to account for the learning and memory decits exhibited by humans and animals exposed to iron deciency.

THE CALCIUM-IRON CONNECTION As already mentioned, calcium released from neuronal intracellular stores stimulates the ERK pathway and CaM kinases, promotes CREB-dependent gene transcription and is likely to reach the nucleus via CICR (1, 8, 20, 79). In particular, RyR-mediated CICR signicantly amplies the calcium signals produced by synaptic stimulation of hippocampal NMDA receptors (80). Likewise, RyR-mediated calcium release is required in hippocampal neurons for tetanically induced LTP and CREB phosphorylation (81) and presumably underlies the stimulation of CREB and c-fos expression caused by stimulation of ionotropic acetyl choline receptors with nicotine (82). Many studies have shown that RyR activity, which directly aects RyR-mediated CICR, is markedly inuenced by the redox state of a few cysteine residues of the RyR protein (83 85). This remarkable redox sensitivity, which has led to the proposal that RyR are cellular redox sensors (84), acquires

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special relevance in hippocampal neurons. Thus, tetanic stimulation causes a signicant enhancement in the generation of ROS that are required for synaptic plasticity (78, 86) and which could readily stimulate RyR-mediated calcium release. In fact, we have recently reported that hydrogen peroxide induces RyR redox modications that trigger a cascade of sequential events in hippocampal neurons, starting with the generation of RyR-mediated calcium signals and followed by ERK and CREB phosphorylation (87) and the enhanced expression of the immediate early genes c-fos and egr-1 (78). Furthermore, our recent work points toward novel correlations among neuronal iron levels, ROS generation and RyR-mediated calcium signals. Thus, in PC12 cells an increase of cellular iron levels led to increased ROS generation and activated the ERK pathway through stimulation of RyR-mediated calcium release (88). Mannitol (an hydroxyl radical trapping agent) and desferrioxamine suppressed these eects (88) implying RyR stimulation by the hydroxyl radical generated through the Fenton reaction (1): Fe2 H2 O2 ! Fe3 OH OH 1

increases beyond physiological limits, as happens during aging and in certain neuronal degenerative states, iron-generated calcium signals may become too large to be controlled, leading to neuronal cell death.

ACKNOWLEDGEMENTS This work was supported by Fondo de Investigacion Avanzada en Areas Prioritarias (FONDAP) Center for Molecular Studies of the Cell, Fondo Nacional de Investigacion Cient ca y Tecnologica (FONDECYT) grant 15010006, and by FONDECYT grant 1040448. The experimental work related to the iron-calcium interaction reviewed here was part of the PhD thesis of Dr Pablo Munoz.

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We have also found that addition of NMDA or iron to PC12 cells and rat hippocampal neurons generates ryanodinesensitive calcium signals and stimulates ERK phosphorylation; once again hydroxyl radical scavengers and desferrioxamine signicantly reduced both eects (78, 88). Thus, iron generates calcium signals through ROS-mediated RyR stimulation that lead to ERK activation. Since ERK activation is a requisite step for CREB-dependent transcription of genes involved in synaptic plasticity, iron-induced calcium release may contribute to this activity-dependent neuronal response.

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