Calcium Dysregulation and Homeostasis of Neural Calcium in the Molecular Mechanisms of Neurodegenerative Diseases Provide Multiple Targets for Neuroprotection

Abstract

The intracellular free calcium concentration subserves complex signaling roles in brain. Calcium cations (Ca²⁺) regulate neuronal plasticity underlying learning and memory and neuronal survival. Homo- and heterocellular control of Ca²⁺ homeostasis supports brain physiology maintaining neural integrity. Ca²⁺ fluxes across the plasma membrane and between intracellular organelles and compartments integrate diverse cellular functions. A vast array of checkpoints controls Ca²⁺, like G protein-coupled receptors, ion channels, Ca²⁺ binding proteins, transcriptional networks, and ion exchangers, in both the plasma membrane and the membranes of mitochondria and endoplasmic reticulum. Interactions between Ca²⁺ and reactive oxygen species signaling coordinate signaling, which can be either beneficial or detrimental. In neurodegenerative disorders, cellular Ca²⁺-regulating systems are compromised. Oxidative stress, perturbed energy metabolism, and alterations of disease-related proteins result in Ca²⁺-dependent synaptic dysfunction, impaired plasticity, and neuronal demise. We review Ca²⁺ control processes relevant for physiological and pathophysiological conditions in brain tissue. Dysregulation of Ca²⁺ is decisive for brain cell death and degeneration after ischemic stroke, long-term neurodegeneration in Alzheimer's disease, Parkinson's disease, Huntington's disease, inflammatory processes, such as in multiple sclerosis, epileptic sclerosis, and leucodystrophies. Understanding the underlying molecular processes is of critical importance for the development of novel therapeutic strategies to prevent neurodegeneration and confer neuroprotection. Antioxid. Redox Signal. 14, 1275–1288.

Introduction: General Principles of Calcium Cations Signaling

Cellular signaling requires harnessing protein functions by charge distribution in response to the changing environment (17). Besides phosphate anions, calcium cations (Ca²⁺) are predestined to alter protein conformation, since Ca²⁺ can accommodate 4–12 oxygen atoms in its primary coordination sphere. Under physiological conditions, electrical pulses or receptor-mediated stimuli in the neuron generate different Ca²⁺ signals with distinct spatial dimensions, temporal extension, amplitude, subcellular localization, and, in some cases, oscillations. Spatial variability ranges from nanodomains in cellular organelles up to gradients covering the whole cell body.

The versatility of Ca²⁺ as an intracellular messenger is derived from varying cytosolic Ca²⁺ concentrations, most of which are generated by regulated openings of Ca²⁺-permeable channels expressed in the plasma membrane and in different organelles.

High signal speed and effectiveness is achieved by a 20,000-fold gradient between the low intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and high extracellular Ca²⁺ concentration during the resting state (100 nM vs. mM). This gradient is maintained by Ca²⁺-buffering proteins as well as by membrane-intrinsic Ca²⁺ transport systems capable of removing Ca²⁺ from the cytosol into the extracellular milieu. The Ca²⁺ extrusion is possible even uphill this very large concentration gradient.

To trigger cellular processes, myriads of cellular proteins, acting both locally and globally, have been adapted to bind Ca²⁺. Their affinities range from nanomolar to millimolar. In Ca²⁺ binding proteins, six to seven carboxyl and carbonyl groups are coordinated to surround Ca²⁺ in a pentagonal bipyramid (37). The helix-turn-helix motifs of the archetypical EF hand domain, the professional protein chelator of Ca²⁺, have negatively charged oxygen atoms to encompass Ca²⁺ within a 12 amino acid loop between two orthogonal α-helices. The affinities of EF hand domains for Ca²⁺ vary, depending on a variety of factors, ranging from critical amino acids in the Ca²⁺ binding loop to side chain packing in the protein core. The subsequent readout of Ca²⁺ signals employs downstream signaling proteins, which transmit the Ca²⁺ message to cellular effectors.

Cellular Factors That Determine Ca²⁺ Signaling in the Brain

Role of mitochondria and endoplasmic reticulum

In intracellular Ca²⁺ signaling, mitochondria accumulate Ca²⁺ from the cytosol. Mitochondrial Ca²⁺ accumulation is a tightly controlled process. It regulates three Ca²⁺-sensitive dehydrogenases of the citric acid cycle and thus controls the synthesis of ATP. Specifically, pyruvate dehydrogenase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, the ATP synthase (complex V), and the adenine nucleotide translocase are activated by Ca²⁺. Further, Ca²⁺ from mitochondria shapes the amplitude and spatio-temporal patterns of [Ca²⁺]_i signals, and is instrumental to determining cell death or survival. Mitochondria can store large amounts of Ca²⁺ in the matrix without dangerously increasing its osmotic concentration, since they accumulate inorganic phosphate alongside with Ca²⁺, and thus precipitate in the matrix deposits of amorphous hydroxyapatite. When mitochondria accumulate excessive Ca²⁺, however, they trigger a vicious cycle that will exacerbate the cellular Ca²⁺ overload. Further, mitochondria produce nitric oxide (NO), and in stress situations, mitochondria release apoptogenic factors into the cytosol. Moreover, mitochondrial dynamics (fission, fusion, and migration) is important for neurotransmission, synaptic maintenance, and neuronal survival.

Regulated Ca²⁺ release from the endoplasmic reticulum (ER) controls many neuronal functions, from plasmalemmal excitability to synaptic plasticity. Enzymatic cascades that are localized in the ER, dependent on the Ca²⁺ concentration in the ER lumen, integrate rapid Ca²⁺ signaling with long-lasting adaptive responses through modifications in protein synthesis and processing (14). Mitochondria form junctions with the ER that support signal transduction and biosynthetic pathways and affect distribution of the organelles. These junctions have a pivotal role in mediating Ca²⁺ signal propagation to the mitochondria. In the ER membrane, Ca²⁺ release channels and Ca²⁺ pumps provide for excitability (38).

In neurodegenerative diseases and aging, mitochondrial dysfunction is associated with compromised cellular energy production, the induction of apoptosis, and the generation of oxidative stress (33, 76).

Cross-talk between calcium and reactive oxygen species signaling

It is now well established that cross-talk between Ca²⁺ and redox signals plays a key role in physiological functions in the brain. However, conditions that promote oxidative/nitrosative stress result in excessive Ca²⁺ release that can provoke pathological responses and neuronal death (55, 101).

Reactive oxygen species (ROS) are generated by tightly regulated NADPH oxidases, which were found recently even in neurons (82, 93). Further, main sources of ROS are mitochondria, where free oxygen radicals are generated as by-products from the electron transport chain and from enzymes of the tricarbonic acid cycle (4, 25). Excessive stimulation of NADPH oxidases, disturbed electron transport chain, or tricarbonic acid cycle results in oxidative stress, a process that damages cell structures (5). This is particularly important in the nervous system, since this relatively small tissue accounts for over 20% of the oxygen consumption of the body and, as a result, produces large quantities of ROS. Additionally, the nervous system is particularly sensitive to oxidative stress because of enrichment of polyunsaturated fatty acids in many of the nerve cell membranes. Finally, oxygen radicals generated by xanthine oxidase are shown to play a significant role in neuronal cell death after ischemia (3).

Components of ROS homeostasis are regulated by Ca²⁺-dependent pathways. Thus, a positive correlation between the Ca²⁺-dependent mitochondrial metabolic rate and ROS generation was shown (107). Further, Ca²⁺ stimulates NO synthase, which has been shown to inhibit complex IV and in turn leads to ROS production at the complex III (41). Similarly, increased [Ca²⁺]_i during reoxygenation after anoxia correlates with ROS generation from NADPH oxidase in neurons (3). Free radical generation in response to β-amyloid (Aβ) was shown to be dependent on extracellular Ca²⁺ and to be prevented by inhibition of NADPH oxidase (2).

Conversely, ROS modify Ca²⁺ signaling proteins and reshape local and global Ca²⁺ amplitudes and kinetics. Many of the Ca²⁺ transporters and signaling proteins in the ER-mitochondrial web are sensitive to redox regulation and are directly exposed to the ROS produced in the mitochondria and ER (38). ROS have been shown to modulate directly ryanodine receptors (RyR). Moreover, ROS induce oxidation of NO to nitrosium ions (NO⁺) and the subsequent reaction with free thiols in RyR to form S-nitrosothiol, which stimulates RyR-mediated Ca²⁺ release from the ER (48). Similarly, ROS has been shown to stimulate inositol(1,4,5)trisphosphate receptors (InsP₃R). Further, sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCA) and plasma membrane Ca²⁺ ATPases (PMCAs) were shown to be inhibited by ROS in the heart, pancreas, and brain, and Na⁺/Ca²⁺ exchangers (NCX) were shown to be stimulated or inhibited during oxidative stress (41).

Homo- and hetereocellular interactions in Ca²⁺ regulation of neural cells

The major cellular constituents of the central nervous system (CNS) are three cell types of neural descent, neurons, astrocytes, and oligodendrocytes, as well as one cell type of myeloid descent, namely, microglia. Whereas neurons comprise not more than 10% of the cells in the brain, astrocytes comprise approximately half of the volume of the adult mammalian brain. They represent the primary neuronal trophic element since they were shown to supplement neurons with nutrients, to modulate the extracellular milieu mainly by buffering the glutamate level and by eliminating ROS (52). Astrocytes, which are organized into distinct nonoverlapping domains, interact intimately with synapses and the cerebrovasculature (7). Astrocytes express a large variety of G protein-coupled receptors (GPCR) that affect a diverse set of signaling cascades, including many different GPCR coupled to G_q, which are activated by neurotransmitters released from presynaptic terminals to produce astrocytic increase of Ca²⁺ level. Prevailing evidence indicates that spontaneous astrocytic Ca²⁺ rise generally remains localized and is unlikely to activate distant astrocytes via intercellular Ca²⁺ waves (15).

Neuroactive substances, primarily glutamate and ATP, are secreted from astrocytes. They act back onto synapses in a process known as gliotransmission (7). Very recently, it has been reported that neither increasing nor obliterating astrocytic Ca²⁺ fluxes affects spontaneous and evoked excitatory synaptic transmission or synaptic plasticity (6). These findings suggest that the mechanism of gliotransmission in the three-partite synapse might be Ca²⁺ independent and thus needs to be reconsidered.

Ca²⁺ Toolkit in Neural Signaling and in Control of Neurodegenerative Processes

Ca²⁺ fluxes across the plasma membrane and between intracellular compartments play critical roles in fundamental functions of neurons. Ca²⁺ signals are produced in response to stimuli, like membrane depolarization, mechanical stretch, noxious insults, extracellular agonists, intracellular messengers, and the depletion of intracellular Ca²⁺ stores. Neurite outgrowth, synaptogenesis, synaptic transmission, plasticity, and cell survival in degeneration processes are regulated by Ca²⁺ signals.

The diversity of events controlled by Ca²⁺ is a consequence of distinct types of signals that differ spatially, temporally, and in magnitude. A given change of the Ca²⁺ concentration modifies functions in the same type of neurons in a multitude of ways and, thus, produces distinct outcomes over short, medium, or long distances and times.

The following sections summarize important components of the Ca²⁺ signaling system in neural cells that are able to create a diverse array of signaling units that can deliver Ca²⁺ signals with very different spatial and temporal properties (17). Figure 1 schematically depicts the Ca²⁺ regulating proteins, which are involved in normal neurophysiological processes, firstly, in the plasma membrane and, secondly, in intracellular organelles, ER, mitochondria, and the nucleus. These proteins, in a coordinate manner, induce [Ca²⁺]_i changes, from where all Ca²⁺ signals emanate.

FIG. 1.

Ca²⁺ homeostasis in brain under normal physiological conditions. Stimuli induce the entry of external Ca²⁺ via Ca_V, TRP channels, and ROC. Activation of GPCR and other signals enable release of internal Ca²⁺ from the ER by formation of second messengers that open channels of receptors for InsP₃R and RyR. The latter pathway is also activated by Ca²⁺ through calcium influx. Ca²⁺ depletion of intracellular ER Ca²⁺ stores further signals to the activation of capacitative Ca²⁺ entry from the Ca²⁺ sensor STIM to Orai/TRP channels. Mitochondria sequester Ca²⁺ through the uniporter MCU, and Ca²⁺ is released back into the cytosol by the NCX_mito. In conditions of acute or lasting damage, Ca²⁺ is spilled out by the mPTP, which is shown in Figures 2 and 3; this pore might also have some physiological function, replenishing [Ca²⁺]_i. K_Ca contribute to reducing overexcitation by hyperpolarizing the plasma membrane. Ca²⁺ is removed from the cell by extrusion of Ca²⁺ to the outside, mediated by the NCX and the PMCA. The SERCA pumps Ca²⁺ back into the ER. Intracellular Ca²⁺ spatiotemporally binds to buffers and effectors and, thus, activates a plethora of cellular processes. Ca²⁺, calcium cations; [Ca²⁺]_i, intracellular free Ca²⁺ concentration; Ca_V, voltage-gated Ca²⁺ channels; TRP, transient receptor potential; ROC, receptor-operated channels; GPCR, G protein-coupled receptors; ER, endoplasmic reticulum; InsP₃R, inositol(1,4,5)-trisphosphate receptor; RyR, ryanodine receptor; MCU, mitochondrial Ca²⁺ uniporter; NCX, Na⁺/Ca²⁺ exchanger; NCX_mito, mitochondrial exchanger; mPTP, mitochondrial permeability transition pore; K_Ca, Ca²⁺-activated K⁺ channels; PMCA, plasma-membrane Ca²⁺-ATPase; SERCA sarco(endo)plasmic reticulum Ca²⁺-ATPase; STIM, stromal interacting molecule. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

G protein-coupled receptors

Over 90% of nonsensory GPCR are expressed in the brain. In the CNS, GPCR function primarily, but not exclusively, as mediators of slow neuromodulators rather than of fast neurotransmitters, and their role is critical for normal brain function. After activation, GPCR can be desensitized by being phosphorylated by members of the family of GPCR kinases (44). Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways. Under- or overactivity of many individual GPCR systems in the brain may contribute to pathological conditions, ranging from hypo-dopaminergic movement disorders to mania and depression. Thus, these receptors are primary or downstream targets for a variety of useful therapeutic agents. Decreased levels of extracellular Ca²⁺ are sensed by G protein-coupled Ca²⁺ sensing receptors that initiate the phospholipase C pathway. Expression of these receptors in glial cells indicates potential roles in the maintenance of local ionic homeostasis (21). In gonadotropin-releasing hormone neurons the activation of these Ca²⁺ receptors promotes cell migration (35).

Ion channels

Ion channels provide for an aqueous bridge across the hydrophobic environment of the plasmalemma allowing for selective transition of ions across these membranes. The channel proteins are constantly flickering between an activated state (open), a deactivated state (closed), and an inactivated state (closed)—often in a voltage- and time dependent fashion. For any condition, there exists an equilibrium, which can be shifted by binding of particular ions or molecules.

Voltage-gated Ca²⁺ channels

The family of voltage-gated Ca²⁺ channels (Ca_V) transduces cell surface membrane potential changes into local rise of [Ca²⁺]_i. These ion channels play a role in the generation and propagation of the nerve impulse and in cell homeostasis. Ca_V are formed as a complex of several subunits, such as α₁, α₂δ, β_1–4, and γ. The α₁ subunit forms the ion-conducting pore, whereas the associated subunits have several auxiliary functions, including control of channel expression and modulation of current kinetics. The voltage sensor in the pore-containing subunit comprises a series of positively charged amino acid residues, which are physically displaced by the change in the membrane electric field: the physical displacement in turn causes a conformational change in the ion channel protein, leading to channel opening.

Molecular biology studies have identified 10 different genes in mammals, each coding for a different α₁ subunit. According to sequence similarities, they are divided into three subfamilies, Ca_v1, Ca_v2, and Ca_v3. In the laboratory, it is possible to distinguish them by studying their physiological roles and/or inhibition by specific toxins. The Ca_v1 isoforms correspond to the dihydropyridine-sensitive L-type channels, which are responsible for excitation–contraction coupling of skeletal, smooth and cardiac muscle and for hormone secretion in endocrine cells. The Ca_v2 isoforms represent the neuronal currents (N-, R-, P/Q-), sensitive to spider and cone snail toxins. The N-type channel is blocked by ω-conotoxin, and the R-type channel (R stands for resistant to the other blockers and toxins) is involved in so far poorly defined processes in the brain. The closely related P/Q-type channel is blocked by ω-agatoxins. The Ca_v3 isoforms form the so-called T-type channels, which are insensitive to either the typical L-type blockers or the organic non-L-type inhibitors (32, 40). The modulation of voltage-gated ion channels by signaling pathways activated by GPCR constitutes widespread mechanisms for regulating neuronal excitability, neurotransmitter release, and neuronal plasticity.

Receptor-operated Ca²⁺ channels

Receptor-operated channels (ionotropic receptors) open in response to specific ligand molecules binding to the extracellular domain of the receptor. Ligand binding causes a conformational change in the structure of the channel protein that ultimately leads to the opening of the channel gate and subsequent ion flux across the plasma membrane. Examples of such channels include the monovalent cation-permeable nicotinic acetycholine receptor, ionotropic glutamate-gated receptors and ATP-gated P2X receptors, and the anion-permeable γ-aminobutyric acid-gated receptor.

Seven genes code for P2X receptor subunits, which are 40%–50% identical in amino acid sequence. All P2X receptors are permeable to small monovalent cations; some have significant Ca²⁺ permeability. Functional responses of P2X receptors are seen in neurons and glia cells. On sensory nerves, P2X receptors are involved in the initiation of afferent signals in several viscera (e.g., bladder, intestine) and play a key role in sensing tissue damage, pain, and inflammatory stimuli. Paracrine roles for ATP signaling through P2X receptors are likely in the neurohypophysis (78).

N-methyl-D-aspartate receptors (NMDAR) are a subtype of ionotropic glutamate receptors with an important role in the physiology and pathophysiology in the mammalian CNS. Physiological levels of NMDAR activity play important roles in neurotransmission and synaptic plasticity and can promote neuronal survival and resistance to trauma.

Inappropriate levels of Ca²⁺ influx through the NMDAR, however, can contribute to neuronal loss under pathophysiological conditions. This is called excitotoxicity. Increased understanding of the molecular mechanisms underlying both the neuroprotective and neurodestructive effects of NMDAR activation will lead to the discovery of new therapeutic targets and strategies for excitotoxic disorders (47, 80).

Mitochondrial channels

An electrophoretic uniporter mediates the charge-uncompensated entry of Ca²⁺ into mitochondria in response to the high negative membrane potential generated by the asymmetric topography and operation of the respiratory chain components (54). This Ca²⁺ uptake is mediated by the mitochondrial Ca²⁺ uniporter located in the organelle's inner membrane. The uniporter passes Ca²⁺ down the electrochemical gradient maintained across this membrane without direct coupling to ATP hydrolysis or transport of other ions.

Additionally, Ca²⁺-activated potassium channels (K_Ca; see the section Ca²⁺-sensitive potassium channels) were found also in mitochondrial inner membranes. They might function to stabilize the mitochondrial membrane and thereby have a neuroprotective function (57).

Transient receptor potential ion channel

The family of transient receptor potential (TRP) channels now comprises >30 members subdivided into seven families. TRP channels are widely expressed in the CNS where they contribute to changes in [Ca²⁺]_i by providing Ca²⁺ entry pathways, by modulating the driving force for the Ca²⁺ entry, and very likely also by providing intracellular pathways for Ca²⁺ release from cellular organelles. TRP channels are activated by a wide range of stimuli, including intra- and extracellular messengers, chemical, mechanical, and osmotic stress, and some by the filling state of intracellular Ca²⁺ stores (77, 83).

Receptor-operated Ca²⁺ channels of the ER and acidic stores

RyR and InsP₃R are localized in the ER and secondarily in the internal nuclear membrane (66) and predominantly operated by second messengers. Cyclic ADP ribose is synthesized from NAD⁺ by the bifunctional ectoenzymes of the CD38 family (49). InsP₃ originates from hydrolysis from membrane bound phosphatidylinositol (4,5)P₂, as induced by G_q-activated phospholipase Cβ (42). The depletion of Ca²⁺ from the ER is sensed by stromal interacting molecule (STIM) proteins. They oligomerize, translocate to junctions adjacent to the plasma membrane, organize Orai or TRP channel proteins into clusters, and open these channels to induce store-operated Ca²⁺ entry (27).

There are at least three different isoforms of RyR, but only RyR3 is expressed in the brain. Redox modification of RyR channels has physiological and pathological consequences for neuronal function (41, 48).

InsP₃R constitute a family of three paralogs, from which InsP₃R1 is expressed in neurons. InsP₃R mediate cell physiological processes ranging from gene transcription to forming learning traces for memory (42). Synaptic activity regulates the level of expression of InsP₃R1 in neurons (28). Metabotropic and ionotropic glutamate receptors collaborate to generate InsP₃ signals (50). Phosphorylation dynamics of InsP₃R1 modulate intracellular Ca²⁺ release (84). InsP₃R were demonstrated to be activated furthermore by calmodulin-like neuronal Ca²⁺-binding proteins (53).

Store-operated Ca²⁺ entry has been shown to be regulated by both Ca²⁺-selective RyR or InsP₃R and Ca²⁺-independent phospholipase A₂ as described before (89, 91).

Ca²⁺ from intracellular acidic stores has been shown to be mobilized by nicotinic acid-adenine dinucleotide phosphate. Cellular levels change in response to a variety of agonists, confirming its role as a Ca²⁺-mobilizing messenger also in the nervous system (18). Nicotinic acid-adenine dinucleotide phosphate interacts with InsP₃ and cyclic ADP-ribose in shaping cytosolic Ca²⁺ signals in the cytosol as well as in the nucleus (18).

Ca²⁺-sensitive potassium channels

K_Ca share the basic architecture with voltage-gated potassium channels. On the basis of the size of ionic conductance, being big, intermediate, or small, K_Ca are divided into three categories, called BK channels (K_Ca1.1), IK channels (K_Ca5.1), and SK channels (K_Ca2.1, K_Ca2.2, K_Ca2.3, K_Ca3.1, K_Ca4.1, and K_Ca4.2) (104). They are present in a wide range of excitable and nonexcitable cells and are tightly associated with calmodulin. On activation by low concentrations of Ca²⁺, K_Ca channel opening results in hyperpolarization of the membrane and changes in cellular excitability. Their activation limits the firing frequency of action potentials and is important for regulation of after-hyperpolarization in central neurons. K_Ca are involved in induction of synaptic plasticity and therefore play important roles in memory and learning (94).

Calcium binding proteins and transcriptional networks

Interaction of Ca²⁺ with specific sensors transduces the specificities of the [Ca²⁺]_i signal in terms of amplitude, duration, frequency, and spatial distributions into changes in the transcription rate of specific genes and cellular functions (86). Further, Ca²⁺-mediated posttranslational modifications on appropriate molecules in the transcriptional machinery result in modified expression of specific single genes or in readjustments of the activity of entire transcriptional networks (70).

A number of Ca²⁺ binding proteins are expressed in the nervous system. Their affinity for Ca²⁺, their localization in relation to the Ca²⁺ signal, and their interactions with other proteins determine their specific role. Functionally, they may be distinguished into two groups. Being chelators, they can act as Ca²⁺ buffers, which modify the spatiotemporal aspects of Ca²⁺ transients. As Ca²⁺ sensors, these proteins have a higher affinity and lower capacity towards Ca²⁺ and translate [Ca²⁺]_i changes into signaling cascades.

Three basic mechanisms that follow the recognition of changes in [Ca²⁺]_i by a Ca²⁺ sensor were differentiated (69). First, there is an activation of signaling cascades of phosphorylation and dephosphorylation that either modify the transactivating properties of transcription factors or affect the nucleosome structure. This changes the accessibility of the RNA polymerase II to specific genes. In some occasions, phosphorylation and dephosphorylation are coupled events that act on a particular protein or even on a particular phosphorylated amino acid residue. In those cases, the specificities of the Ca²⁺ signal to activate the two events of phosphorylation and dephosphorylation alternatively require a delicate fine-tuning. Two major phosphatases in this context are calcineurin, also termed protein phosphatase 2B (PP2B), and PP1 (70). Second, there is an induction of Ca²⁺-dependent protein–protein interactions between the Ca²⁺ sensor and transcription factors. Because of this interaction, binding to DNA or recruitment of certain cofactors is modified. Third, Ca²⁺-induced changes in the binding properties of the calcium sensor to specific sites in the DNA can be found.

The best studied example among the Ca²⁺ sensors is the ubiquitously occurring calmodulin with four EF-hand binding motifs (69). Neuronal Ca²⁺ sensor-1 (NCS-1) is a member of five NCS proteins and interacts with different other proteins in a Ca²⁺-dependent or Ca²⁺-independent manner (26). Recently, it has been shown that NCS-1 has a role in learning and memory (87). Calretinin, calbindin D-28, and parvalbumin belong to a family of Ca²⁺-binding proteins, which in man comprises >200 members. They are particularly enriched in specific cerebellar neurons. Several studies suggest that these proteins have evolved as functionally distinct, physiologically relevant modulators of intracellular Ca²⁺ transients. They are involved in regulating the Ca²⁺ pools, which are critical for synaptic plasticity. Whether they play a major role as endogenous neuroprotectants is not clear.

The cAMP-dependent transcription factor CREB (cAMP response element binding) is thought in neurons to be involved in the formation of long-term memory and to mediate neuronal survival (29). Distinct protein kinase signaling pathways mediate converging effects of Ca²⁺ and cAMP on CREB activation. Binding of a neurotransmitter to its receptor may be coupled to activation of the cAMP-dependent protein kinase. Alternatively, elevation of [Ca²⁺]_i leads to activation of a Ca²⁺/calmodulin-dependent kinase (CaMK) (90). CaMKs and the protein kinase C isoenzymes represent two major groups of Ca²⁺ sensitive enzymes. CaMKII represents between 1% and 2% of the protein in the brain. Besides transcription factor regulation, CaMKII plays a role in neurotransmitter secretion, glycogen metabolism, and synaptic plasticity (61).

Calcium pumps and exchangers

ATP-driven Ca²⁺ pumps and ion gradient-dependent Ca²⁺ exchangers are the major systems responsible for uphill transport of Ca²⁺ across membranes. Both types of transporters are simultaneously expressed in both astrocytes and neurons. This implies that the two transporters with different kinetic properties have different functions in these cells.

Ca²⁺/cation antiporter

Ca²⁺/cation antiporters are a superfamily containing three members, which subserve a variety of roles, including neuronal signaling (92). The NCX, a 9-transmembrane domain protein able to couple the efflux/influx of Ca²⁺ to the influx/efflux of Na⁺ ions, operates in a bidirectional way. It is the major regulator of Na⁺ and Ca²⁺ homeostasis. In the brain, unlike in other tissues, this exchanger is present as three different gene products, NCX1, NCX2, and NCX3, with a distinct distribution pattern in different brain regions. Under physiological conditions, its primary role is to extrude Ca²⁺ through a forward mode of operation. NCX responds to a depolarization or to an increase in intracellular Ca²⁺ concentration coupled to receptor stimulation.

The Na⁺/Ca²⁺–K⁺ exchanger (NCKX) transports K⁺ and Ca²⁺ in exchange for Na⁺, and comprises five members, four of which (NCKX2-5) are expressed in the brain. The NCKX proteins play a role in regulating Ca²⁺ flux in environments, which experience wide and frequent fluctuations in Na⁺ concentration. Evidence emerges for multiple roles for these exchangers, also in processes such as synaptic plasticity.

The Ca²⁺/cation exchanger branch has only one mammalian member, Na⁺/Ca²⁺–Li⁺ exchanger (NCKX6 or NCLX), whose physiological function remains unclear, despite a broad pattern of expression (63).

Mitochondrial exchangers

While in nonexcitable cells the Ca²⁺ efflux from mitochondria is essentially mediated by the H⁺/Ca²⁺ exchanger, in neurons and other excitable cells, the mitochondrial NCX (NCX_mito) is the main system extruding Ca²⁺ ions from the mitochondrial matrix. NCX_mito was shown to have an impact on mitochondrial physiology by controlling the activity of the Ca²⁺-sensitive enzymes pyruvate-, α-ketoglutarate-, and isocitrate-dehydrogenase, and by consuming energy because of its indirect effect on the H⁺ gradient (30). NCX_mito determines a shoulder in neuronal [Ca²⁺]_c responses to neurotransmitters and depolarizing stimuli. This shoulder may outlast the stimulus duration. The persistent NCX_mito-dependent Ca²⁺ release has a role in post-tetanic potentiation, a form of short-term synaptic plasticity. Published data suggest that the three isoforms of plasma membrane NCX contribute to NCX_mito in neurons and astrocytes (30).

Ca²⁺ shuttling between the ER and mitochondria was detected as the pacemaker of Ca²⁺ oscillations. Ca²⁺ release from the ER induces the initial Ca²⁺ oscillation and loads mitochondria with Ca²⁺. The following Ca²⁺ peak is induced by means of NCX_mito. Increased [(Ca²⁺]_i provokes further peaks by regenerative Ca²⁺ release from ER, which generates partial reloading of the mitochondria. This sequence is repeated until mitochondrial Ca²⁺ is depleted (51).

Plasma membrane Ca²⁺ ATPases

PMCAs are quickly activated by a rise in [Ca²⁺]_i and affected by binding of Ca²⁺-calmodulin. In the brain, four separate genes code for the major PMCA isoforms 1–4. In particular, PMCA2 is enriched primarily in neurons in some CNS regions. The identification of a growing number of further specific interacting proteins with regulatory, targeting, and signaling functions supports the paradigm that PMCAs are not only responsible for global Ca²⁺ homeostasis but are highly dynamic participants in spatially defined Ca²⁺ signaling (22).

Endoplasmic reticulum Ca²⁺ATPase

The ER serves as a dynamic Ca²⁺ pool being thus involved in rapid signaling events in nerve cells associated with cell stimulation by either electrical (action potential) or chemical (neurotransmitters) signals. This function is supported by Ca²⁺ release channels (InsP₃R and RyR) and SERCA Ca²⁺-ATPases residing in the endoplasmic membrane (102). In addition, the ER provides a specific environment for the posttranslational protein processing and transport of various molecules toward their final destination. In parallel, the ER acts as a calcium tunnel, which facilitates Ca²⁺ movements within the cell by avoiding cytoplasmic routes. Finally, the ER appears as a source of numerous signals aimed at the nucleus and involved in long-lasting adaptive cellular responses. All these important functions are controlled by intra-ER free Ca²⁺, which integrates various signaling events and establishes a link between fast signaling, associated with ER Ca²⁺ release/Ca²⁺ uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis (14, 22). Recently, SERCA has to been shown to be functionally coupled to the Ca²⁺ sensor in the ER, called STIM, and Orai, a member of the TRP family, to allow efficient refilling of the ER (65).

Disruption of ER Ca²⁺ homeostasis triggers several forms of cellular stress responses and is intimately involved in neurodegeneration and neuronal cell death (105).

Dysregulation of Ca²⁺ Signaling in Brain and Neural Degeneration

The central role of Ca²⁺ in signaling functions in brain underlines its potential relevance for neurodegenerative diseases, a heterogeneous group of disorders characterized by gradual and progressive, selective loss of anatomically or physiologically related neuronal systems. Despite the wide spectrum of appearances of these diseases, there are striking similarities in the molecular pathogenesis of these diseases as they all involve dysregulation of Ca²⁺ homeostasis and signaling. This is associated with alterations of Ca²⁺ buffering capacities, deregulation of Ca²⁺ channel activities, and alterations of other Ca²⁺-regulating proteins due to excitotoxicity, perturbed energy metabolism, and oxidative stress. Abnormal cellular Ca²⁺ load can trigger cell death by activating proteases, by reinforcing signals leading to caspase activation or by triggering other catabolic processes mediated by lipases and nucleases.

Involvement of ER and mitochondria in Ca²⁺ homeostasis is an important common theme in neurodegenerative diseases, since these organelles are intimately involved in neuronal cell death (102). The link between Ca²⁺ dysregulation, mitochondria, and cellular derangement became particularly evident, when usage of animals as genetic disease models and analysis of the impact of environmental factors allowed to identify common traits in the pathogenic routes for these diseases (33, 60).

Neurological diseases should be also considered as gliopathologies, which determine the progression and final outcome of the neuropathological process (75). Glial function is closely regulated by cellular Ca²⁺ signaling that underlies the so-called glial calcium excitability. Glial Ca²⁺ signals triggered by activation of multiple GPCR are primarily driven by Ca²⁺ release from the ER (52).

The identification of precise neurotoxic molecular events in neurodegenerative disorders is a challenging task. A recent collection of overviews covered the role of imbalanced Ca²⁺ homeostasis and pathological Ca²⁺ signaling in various neurological diseases (74). We will focus in the following section on the question of how perturbation of Ca²⁺ homeostasis contributes to the pathophysiology of specified neurodegenerative disorders (12, 36, 60, 67).

Aging

Oxidative and metabolic stress, and impaired cellular stress adaptation are mechanisms of aging that render neurons vulnerable to degeneration. Many Ca²⁺-dependent processes, such as susceptibility to neurotoxicity, the after-hyperpolarization amplitude, induction of synaptic plasticity, long-term potentiation, long-term depression, and cell excitability are altered with age (43). Indeed, multiple lines of evidence have implicated Ca²⁺ dysregulation in brain aging and dementia. These changes have been associated with age-related deficits in learning and memory.

Aging-related increases in the Ca²⁺ spikes and currents result from changes in local Ca²⁺ levels. There is evidence for an increased L-type Ca²⁺ channel activity functionally linked to RyR. In turn, the increased Ca²⁺ transients result in dysregulation of multiple Ca²⁺-dependent processes and, through different pathways, in accelerated functional decline during aging (43, 99).

Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related dementia in which patients show neurodegeneration and loss of cognitive abilities. Cognitive impairment and emotional disturbances in AD result from the degeneration of synapses and neuronal death in the limbic system and some regions of the cerebral cortex. Biochemically, AD is characterized by the perturbed expression and subsequent accumulation of several proteins, such as reelin and presenilins (PS). Reelin is involved in regulation of neuronal migration, synaptic plasticity, and dendritic spine development. Presenilins are part of the γ-secretase protease complex and play a role in presynaptic neurotransmitter release, induction of long-term potentiation and in the modulation of [Ca²⁺]_i. γ-Secretase in coordination with β-secretase induces overproduction of Aβ, which forms oligomers. Amyloid plaques and neurofibrillary tangles are found as protein aggregates in the brain cells in AD.

The Aβ hypothesis postulates that Aβ deposits are the fundamental cause of the disease. Figure 2 summarizes the Aβ-associated Ca²⁺ dysregulation combined with the Ca²⁺-dependent cell death pathways that are prominent in AD cellular pathology. Under normal conditions, a sequential cleavage of the Aβ precursor protein (APP) by β-secretase and γ-secretase generates Aβ. AD-causing mutations in APP and PS compromise normal processing of these proteins in the plasma membrane.

FIG. 2.

Perturbed brain Ca²⁺ homeostasis in Alzheimer's disease due to mutations of several proteins and formation of protein aggregate deposits. Several pathways lead to [Ca²⁺]_i overload. Thus, Aβ oligomers form pores in the plasma membrane through which Ca²⁺ passes into the cytoplasm. Aβ can also interact with Fe²⁺ and Cu⁺ to generate HOO^• and OH^•, resulting in membrane lipid peroxidation that impairs the function of PMCA. As a result, the plasma membrane becomes depolarized; NMDAR and Ca_V open and cause flux of Ca²⁺ into the cytoplasm. Interaction of reelin with the ApoER2 enhances Ca²⁺ influx through NMDAR by a mechanism involving a SFk. Increased [Ca²⁺]_i further increases [Ca²⁺]_i by calpain-mediated inhibition of PMCA and nNOS-induced activation of TRPM7. Aβ acts on mitochondria, to cause Ca²⁺ overload-increased ROS production, depolarization, and decreased ATP production. Mutations of PS result in excessive accumulation of Ca²⁺ in the ER via SERCA and, thus, enhance Ca²⁺ release through RyR and InsP₃R channels. Further, the intracellular APP domain AICD, which is generated by proteolysis from amyloid precursor protein translocates to the nucleus and alters Ca²⁺-dependent gene transcription. AICD, amyloid intracellular C-terminal domain; Aβ, β-amyloid; NMDAR, N-methyl-D-aspartate receptor; ApoER2, apolipoprotein E receptor; SFk, src family tyrosine kinsase; NO, nitric oxide; nNOS, neuronal NO synthase; ROS, reactive oxygen species; PS, presenilins; APP, Aβ precursor protein; ΔΨ_c, cytosolic membrane potential; ΔΨ_m, mitochondrial membrane potential. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Central to the neurodegenerative process is the inability of neurons to properly regulate intracellular Ca²⁺ levels (67, 99). Thus, toxic forms of Aβ may induce Ca²⁺ influx into neurons by formation of an oligomeric pore in the plasma membrane, thereby rendering neurons vulnerable to excitotoxicity and apoptosis (19). Aβ interacts with Fe²⁺ and Cu⁺ to generate hydrogen peroxide and hydroxyl radical (HO^•), resulting in membrane lipid peroxidation, which generates aldehydes that impair the function of PMCA. As a result, the membrane becomes depolarized. NMDAR channels and Ca_V open, and a flux of toxic amounts of Ca²⁺ into the cytoplasm follows (16).

In addition, Aβ accumulates in mitochondria and thereby impairs the activity of complexes III and IV of the respiratory chain (9). Further, evidence for an involvement of NCX_mito in pathological mechanisms of AD was reported (30, 100). Aβ affects mitochondria, whereby it causes elevated cytoplasmic Ca²⁺ levels and oxidative stress, and it reduces ATP synthesis and further increases the Ca²⁺ overload and oxidative stress (60).

There is also evidence that PS modulate SERCA to alter Ca²⁺ uptake and release in the ER (19). This effect of PS results in excessive accumulation of Ca²⁺ in the ER and, as a consequence, enhances Ca²⁺ release through RyR, whose expression levels are increased in mutant PS1-expressing AD mouse models (96). In the cortex of AD patients, reelin levels were significantly increased compared with control brain. Interaction of reelin with the apolipoprotein E receptor 2 was shown to enhance Ca²⁺ influx through NMDAR channels by a mechanism involving a src family tyrosine kinsase. Amyloidogenic APP processing prevents the α-secretase from α-cleavage of APP, which would otherwise generate a secreted form of APP. The latter is normally produced in response to synaptic activity and is known to be involved in neuroprotective pathways. During AD, amyloidogenic processing also generates by proteolysis a free intracellular APP domain, designated amyloid intracellular C-terminal domain, which translocates to the nucleus and perturbs gene transcription that regulates Ca²⁺ homeostasis (19). Finally, Ca²⁺ homeostasis disruption mediated by TRP channels has been suggested in AD (106). High [Ca²⁺]_i induces stimulation of neuronal NO synthase, which leads to production of reactive oxygen and nitrogen species and to the feedforward activation of TRPM7 and TRPM2-like currents.

Ischemia and brain energy deprivation

A few minutes after uncompensated brain ischemia, cell death pathways overcome survival-promoting pathways, leading to neuronal death through three interacting mechanisms: excitotoxicity attributable to excess glutamate, oxidative stress, and/or stimulation of apoptotic-like pathways (12, 62). Figure 3 highlights the mechanisms connected to Ca²⁺ dysregulation in ischemia/stroke-induced cell death. These mechanisms were shown above in Figure 1 within the context of normal physiology.

FIG. 3.

Perturbation of neural Ca²⁺ homeostasis under ischemic conditions. Neuronal and glial cell death is primarily mediated by oxidative stress and excess glutamate. Ca²⁺ entry through NMDAR mainly triggers the excitotoxic Ca²⁺ overload. As a result, calpains are activated, which inactivate by cleavage both NCX3 and PMCA. This precludes the capability to remove accumulated Ca²⁺. High [Ca²⁺]_i induces stimulation of nNOS, which leads to production of ONOO^•− and to the feedforward activation of TRPM7 currents. The decrease in [Ca²⁺] in the extracellular space disinhibits Ca²⁺-sensing channels, such as K_Ca3.1, leading to a further membrane depolarization. As a result of high [Ca²⁺]_i, excessive rise of intramitochondrial Ca²⁺ triggers the opening of the mPTP. Mitochondria also release Ca²⁺ via NCX_mito. This induces a depletion of ATP. Under ischemic conditions, the mode of operation of NCX_mito is reverted. Then, the NCX_mito acts as an influx pathway for Ca²⁺ into the mitochondrial matrix. ROS induce a high activity response of RyR2 and thus amplify the increase of [Ca²⁺]_i. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Glutamate activates NMDAR at synaptic and extra-synaptic sites, causing prolonged neuronal depolarization and triggering deregulation of cellular ion homeostasis, mainly [Ca²⁺]_i and [Na⁺]_i. The entry of Ca²⁺ through NMDAR is accepted as the major pathway leading to the excitotoxic, delayed cell death associated with the ischemic periods of stroke. As a consequence of the excitotoxic stimulus in neurons, calpains cleave NCX and PMCA (Fig. 3) and thus inhibit its capability to remove accumulated Ca²⁺ (13). On the other hand, during hypoxic conditions, a nuclear factor-κB-dependent increase of NCX1 activity was shown enabling Na⁺ extrusion and Ca²⁺ influx. This may help to refill the ER Ca²⁺ stores and to prevent ER stress during anoxic conditions, thus contributing to cell rescue (92).

The decrease in [Ca²⁺] in the extracellular space, resulting from NMDAR stimulation, disinhibits Ca²⁺-sensing channel currents, leading to a further decrease in extracellular [Ca²⁺] and also membrane depolarization, which enhances removal of the Mg²⁺ block from NMDAR. Similarly, the decrease in extracellular [Ca²⁺] disinhibits some ion channels, thus resulting in a more pronounced depolarization and further relief of the block of NMDAR (64).

During ischemia, mitochondria release Ca²⁺ via NCX_mito inducing a depletion of ATP due to the decrease in mitochondrial membrane potential (ΔΨ_m) (73). In this condition, the operation of the NCX_mito is shifted to the Ca²⁺ accumulation mode. It has been shown that NCX_mito can go into its reverse mode of operation and then acts as an influx pathway for Ca²⁺ into the mitochondrial matrix (Fig. 3). Excessive rise of intramitochondrial Ca²⁺ is a trigger for mitochondrial permeability transition pore (mPTP) opening and apoptosis (30). The opening of the mPTP is a critical step in neurodegenerative processes (11, 45). This pore structure with its control elements provides a very promising target for neuroprotective strategies (45). Nevertheless, the biochemical composition of this pore is still a mystery despite many years of intense investigations.

Ischemia-induced local production of ROS increases high activity response of RyR2 by S-glutathionylation and thus amplifies the increase of [Ca²⁺]_i (8, 24).

Members of the melastatin subfamily of TRP proteins, particularly TRPM7 and TRPM2, may play key roles in neuronal death that is activated by oxidative stress downstream from excitotoxic signaling pathways (1, 64). Moreover, spreading depression-like hypoxic depolarization after vascular stroke is associated with an increase in [Ca²⁺]_i. Intriguingly, TRPM4 and 5 could be candidates for triggering this dramatic event, although at present there is no direct experimental evidence in support of this conjecture (10).

Parkinson's disease

Parkinson's disease (PD), the most frequent movement disorder, is caused by the progressive loss of the dopamine neurons within the substantia nigra pars compacta (SNc) and the associated deficiency of the neurotransmitter dopamine in the striatum. Most cases of PD occur sporadically with unknown cause, but mutations in several genes, such as α-synuclein, Parkin, DJ-1, PINK1, and LRRK2, have been linked to genetic forms of PD.

In humans and animal models, mitochondrial dysfunction is a defect occurring early in the pathogenesis of both sporadic and familial PD. Both PINK1 and Parkin play crucial roles in the regulation of mitochondrial dynamics and function. Mutations in DJ-1 and Parkin render animals more susceptible to oxidative stress, and mitochondrial toxins were implicated in sporadic PD. Mitochondrial association of α-synuclein in cells was also linked to impairment of respiratory complex I activity, oxidative modification of mitochondrial proteins, and increased levels of Ca²⁺ and NO (81). In autoptic samples of substantia nigra from patients affected with the disease the activity of complex I was shown to be reduced (23, 30).

L-type Ca²⁺ channels are engaged during autonomous pacemaking rendering SNc dopaminergic neurons susceptible to mitochondrial toxins. This indicates that homeostatic Ca²⁺ stress could determine their selective vulnerability (34, 97). Further, reduction in the cell surface expression of the TRP channel isoform 1 may be involved in dopaminergic neurodegeneration, and TRP channel isoform 1 may provide neuroprotection against PD-inducing agents (20).

Multiple sclerosis and brain inflammatory processes

Multiple sclerosis is characterized by inflammation, demyelination, oligodendrocyte death, gliosis, axonal damage, and neurodegeneration. The clinical symptoms include paresthesia, optic neuritis, diplopia, fatigue, paralysis, and cognitive dysfunction. NCX, PMCA2, several ion channels, and SERCA play critical roles in this type of neuronal pathology. Increase of Na⁺ concentration induces a reversal in the activity of NCX and the subsequent increase of [Ca²⁺]_i. Reduction in PMCA2 expression causes injury due to activation of Ca²⁺-dependent proteases, including calpains and PPs. One consequence is the dephosphorylation of cytoskeletal proteins, such as neurofilaments. This affects neuronal stability and increases the vulnerability of cytoskeletal proteins (58, 71).

Huntington's disease

Huntington's disease (HD) is characterized by motor, cognitive, and psychiatric symptoms, including depression, personality changes, weight loss, and movement irregularity (chorea) developing in the terminal stage into severe akinesia (33). The disease is inherited in an autosomal dominant fashion. The mutant huntingtin gene was identified to be reponsible for the disease. This protein is considered to play a role in axonal transport, regulation of transcription, exocytosis, bioenergetic metabolism, prevention of apoptosis, and Ca²⁺ homeostasis (31).

Mitochondrial Ca²⁺ overload is a decisive commitment step for the disease. Thus, a facilitated opening of the mPTP in permeabilized mutant huntingtin-expressing cells was demonstrated (59). Mutant but not wild-type huntingtin directly impairs the mitochondrial function (98). Further, huntingtin forms a ternary complex with huntingtin-associated protein-1A and InsP₃R1. In this complex, mutant huntingtin facilitates Ca²⁺ release from the ER and renders neurons more sensitive to Ca²⁺-mediated cellular dysfunction (98).

Epilepsy

Up to 50% of all cases of epilepsy are initiated by neurological insults. Status epilepticus, stroke, and traumatic brain injury are three major causes that can lead to the development of acquired epilepsy. The neurons that survive the increase in extracellular glutamate concentration and the perturbed Ca²⁺ homeostasis sustain long-term changes in Ca²⁺ signaling that are prominent features of the epileptic phenotype (39, 73).

Genes of Ca²⁺-dependent ion channels and several other genes contribute to the genetic background of the pathogenesis of idiopathic epilepsy syndromes (72). Idiopathic generalized epilepsies are inherited with additive effects of genetic variation. Ca_V are involved in the release of neurotransmitters and in the sustained depolarization phase of paroxysomal depolarisation shifts; they are also the genetic substrate of generalized tonic-clonic convulsions. Various Ca²⁺-regulated enzymes found in spines have been implicated in epileptogenesis, including the nonreceptor protein tyrosine kinases Src and Fyn, CaMKII, and calcineurin (68).

A broad variety of mutations in mitochondrial DNA or nuclear genes have been associated with epileptic phenotypes. Ca²⁺ entrance into mitochondria through the uniporter induces mitochondrial depolarization. Impairment of the mitochondrial respiratory chain causes an increased metabolic demand in epileptic neurons (30). Mitochondrial dysfunction has been identified as a cause of epileptic seizures. The cross-talk between astrocytes and neurons promotes increased dendritic Ca²⁺ levels and synchronous firing of neurons, a hallmark of epileptiform activity (56).

Refsum disease and accumulation of branched-chain phytanic acid

In a inherited peroxisomal disorder, Refsum disease, mutations in the PHYH gene cause a buildup of the saturated branched-chain fatty acid phytanic acid in the plasma and tissues. Clinical features of this and other leukosdystrophies result from the malformation of myelin sheaths and comprise neurologic damage, cerebellar degeneration, and peripheral neuropathy. Phytanic acid and its peroxisomal α-oxidation product pristanic acid were recently shown to induce mitochondrial depolarization, Ca²⁺ deregulation, ROS production, and cell death in brain cells (85).

Conclusions

Outlook: search for novel targets of protection against neurodegeneration related to Ca²⁺ dysregulation

The mechanisms of neural diseases reported in the Dysregulation of Ca²⁺ Signaling in Brain and Neural Degeneration section involve a broad spectrum of Ca²⁺ dysregulation. This proves that stabilization of neuronal Ca²⁺ homeostasis may retard cell degeneration in acute and chronic neurodegenerative conditions. Causal mutations of genes coding for Ca²⁺-regulating proteins are genetic risk factors. These act together with environmental factors to determine whether a neurodegenerative disease process develops. If we were able to identify the mechanisms that determine whether the nervous system adapts positively and copes with the stress, or shows a disease phenotype during development, then the disease processes could be averted. For that purpose, drugs targeting the Ca²⁺ homeostasis might be helpful.

In line with this concept, it can be positively noted that drugs that suppress Ca²⁺ influx support neuronal survival in models of neurodegenerative disorders. Indeed, beneficial effects of the Ca_V1 (L-type Ca²⁺ channel) blocker nimodipine and the NMDAR channel blocker memantine have been shown in AD patients. On the other hand, clinical trials of NMDAR antagonists have failed to prevent ischemia-induced cell death since these antagonists may be protective only in the early phase of the insult, but not at later stages (67).

Recent exciting developments in therapeutics for neurodegenerative disorders have been the use of immunization. If antibodies were able to remove Aβ from the brain, immunization would be expected to prevent or reverse also the Aβ-induced neuronal Ca²⁺ dysregulation. For individuals at high risk for AD, prophylactic treatment may be prescribed, including anti-inflammatory drugs and immunization (19). Administration of the Ca_V blockers bepridil and nitrendipine significantly ameliorated experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Similar results were obtained by inhibition of receptor-operated and other Ca²⁺-regulating channels (71). For HD, promising results have been published using a specific animal model of HD (79). In mice expressing the full-length mutant huntingtin, histone deacetylase inhibitors were shown to ameliorate the deficits in mitochondrion-dependent Ca²⁺ handling. Thus, the inhibitors induced neuroprotection by improving the neuronal ability to cope with excitotoxic stimuli. For PD, no proven neuroprotective therapy with regulation of the Ca²⁺ balance has been described up to now. The search for such treatments is vigorously pursued (97). For neuroprotection against damage after status epilepticus, early NMDAR antagonism has been proposed to block the fatal Ca²⁺ accumulation in neurons (103).

Open questions and further perspectives

To stimulate further the development of promising therapeutic agents, which target neuronal Ca²⁺ signaling, several open questions have to be elucidated. These questions concern (i) the impact of risk factors of disease on neuronal Ca²⁺ homeostasis, both at the molecular and the cellular level, (ii) the identification of the subset of neurons affected in a particular type of neuronal degeneration, (iii) further elucidation of interactions of different neural cell types in brain Ca²⁺ homeostasis, (iv) the role played by mitochondria in pathogenesis, and (v) the potential help of antioxidants and cellular energy-promoting agents in the context of Ca²⁺ homeostasis.

Development of neuroprotective strategies comprises the sophisticated analysis of the pathways to control Ca²⁺ homeostasis and the understanding of the sequences of downstream events of Ca²⁺ signaling. Distinct Ca²⁺ signals may define whether protection is initiated or not. For example, the protease thrombin at low concentrations mediates hippocampal neuroprotection against ischemia via activation of protease-activated receptors, but thrombin causes neuronal degeneration at high concentrations (95). Further, the involvement of phospholipase A₂ activation in the up- or downregulation of [Ca²⁺]_i in astrocytes promises neuroprotection (88). The development of better-focused antiexcitotoxic strategies requires considerations of pro-survival NMDAR signaling. The selective targeting of pro-death pathways downstream of the NMDAR may represent an effective therapeutic strategy (47). Cell death in ischemia depends on the neuronal subtype, the severity, and duration of the episode, and the position of the neuron within the lesion (infarct core or penumbra). The use of NMDAR antagonists that are activated only in the pathological state is a promising strategy to consider this fact. This should minimize negative side effects (46).

Although in animal models of neurodegenerative disorders many Ca²⁺ targeting drugs have been demonstrated to protect neurons, only few have been successful in preclinical trials. To speed up the advancement of promising drugs to therapeutic intervention, rigorous scientific evidence for the suggested mechanism of action of the compound in question is needed; additionally, critical clinical evaluation of neuroprotectants in combination with other therapies is necessary. Further, growing attention has to be directed to the stimulation of neuroregeneration.

Abbreviations Used

Aβ

β-amyloid

AD

Alzheimer's disease

AICD

amyloid intracellular C-terminal domain

APP

Aβ precursor protein

Ca²⁺

calcium cations

[Ca²⁺]_i

intracellular free Ca²⁺ concentration

CaMK

Ca²⁺/calmodulin-dependent kinase

Ca_V

voltage-gated Ca²⁺ channels

CNS

central nervous system

CREB

cAMP response element binding

ER

endoplasmic reticulum

GPCR

G protein-coupled receptor

HD

Huntington's disease

InsP₃R

inositol(1,4,5)trisphosphate receptor

K_Ca

Ca²⁺-activated potassium channels

MCU

mitochondrial Ca²⁺ uniporter

mPTP

mitochondrial permeability transition pore

NCKX

Na⁺/Ca²⁺–K⁺ exchanger

NCS

neuronal Ca²⁺ sensor

NCX

Na⁺/Ca²⁺ exchanger

NCX_mito

mitochondrial NCX

NMDAR

N-methyl-D-aspartate receptor

nNOS

neuronal NO synthase

NO

nitric oxide

PD

Parkinson's disease

PS

presenilins

PMCA

plasma membrane Ca²⁺ ATPases

PP

protein phosphatase

ROC

receptor-operated channels

ROS

reactive oxygen species

RyR

ryanodine receptor

SERCA

sarco(endo)plasmic reticulum Ca²⁺-ATPases

SNc

substantia nigra pars compacta

STIM

stromal interacting molecule

TRP

transient receptor potential

Acknowledgments

The work was supported by grants from Bundesministerium für Bildung und Forschung (BMBF), Europäischer Fonds für Regionalentwicklung (EFRE), and from Land Sachsen-Anhalt.

We apologize to many authors who have published substantial scientific contributions in the field of this specific calcium research whose work could not or not sufficiently be cited here. This is due to space restrictions.