Neuronal Stress and Its Hormetic Aspects

Chapter 15

Neuronal Stress and Its Hormetic Aspects Sarah Ann Hofbrucker-MacKenzie1, Iswariya Sivaprakasam1, Yuanyuan Ji1,2, Michael Manfred Kessels1 and Britta Qualmann1 1

Institute for Biochemistry I, Jena University Hospital — Friedrich Schiller University Jena, Jena, Germany, 2Hans Berger Department of Neurology,

Jena University Hospital — Friedrich Schiller University Jena, Jena, Germany

15.1

INTRODUCTION

Hormesis is defined by toxicologists to describe a biphasic dose response to an environmental agent with a low-dose stimulation showing beneficial effects and a high-dose stimulation showing inhibitory or toxic effects. While hormesis is most often thought of in the context of exposures to exogenous agents or environmental conditions, it should be recognized that hormesis also is integral to normal physiological functions of cells and organisms. Neuronal cells stand out for their extraordinary degree and variety of adaptive responses that include altering their synaptic strength, called functional plasticity, and/or changing their overall pattern of connectivity leading to different kinds of structural plasticity. Signaling pathways in neurons do not follow a simple, linear dose response. Due to the complex signal integration processes and the multiple layers of crosstalk and fine-tuning of signals in our brain, cellular responses to synaptic transmission are strongly dependent on dose and frequency of stimulation. Important and very basic principles underlying these complex patterns of signal integration and modified output can be described by a hormetic dose response (Fig. 15.1). As will be described in detail in the following, low to medium doses of the major excitatory neurotransmitter glutamate mediate synaptic transmission and plasticity as well as learning and memory. Such doses activate adaptive cellular stress response pathways from which not only information processing in the brain but also the individual nerve cells of the neuronal networks themselves benefit, as their growth and survival is promoted. Importantly, different stimulation paradigms are hereby known to result in either an increase or a decrease of synaptic strength resulting in long-term potentiation (LTP) or depression (LTD) (Figs. 15.1 and 15.2) — two important mechanisms behind learning and memory. However, excessive amounts of the neurotransmitter glutamate can damage and even kill nerve cells. This toxic process is therefore often referred to as excitotoxicity (Figs. 15.1 and 15.2). Similar to the adaptations caused by doses and stimulation patterns corresponding to the beneficial part in the classical hormesis diagram, also excitotoxicity is of high biological relevance, as it is a major pathomechanism in severe epileptic seizures and stroke. Hormetic dose responses in neurosciences can furthermore be observed for a whole variety of additional substances targeting neurons, e.g., toxins, hormones, drugs of abuse, medication to modulate pain, treatment of anxiety, depression, antiepileptic drugs (reviewed in [1]). Additionally, also environmental factors and stressors are modulating neuronal responses and functions in a manner that in part follows hormetic principles [2,3]. Since these topics have been reviewed before, this chapter will exclusively focus on the adaptive responses of neurons to the signaling underlying the general physiology of excitatory neurons, the signaling pathways triggered by the endogenous signaling factor glutamate. Information can be passed between two neurons at specialized connections termed synapses, which can be electrical or chemical. Electrical synapses are fast and bidirectional, allowing the flow of current from one cell to another through gap junctions with less resistance. Unlike electrical synapses, chemical synapses have no contact between the cytoplasm of the cells. By contrast, chemical synapses are separated by a 20 nm wide gap, the synaptic cleft, and the interaction occurs through neurotransmitters. The chemical synapses are compartmentalized into pre- and postsynaptic elements. The presynapse is a terminal of an axon that contains neurotransmitter-filled vesicles ready to be released upon

The Science of Hormesis in Health and Longevity. DOI: https://doi.org/10.1016/B978-0-12-814253-0.00015-2 © 2019 Elsevier Inc. All rights reserved.

171

172

SECTION | III Hormetic Stressors

FIGURE 15.1 Scheme of a hormetic dose response curve in neurons. The curve illustrates that depending on the levels of the endogenous signaling factors glutamate and/or Ca21 either beneficial or toxic effects are seen as a response. Low to moderate levels of glutamate and/or Ca21 lead to beneficial effects corresponding to the green zone of the curve. Low levels of the stressors will lead to long-term depression, while moderate levels will lead to long-term potentiation. High doses of glutamate and/or Ca21 lead to toxic effects corresponding to the red zone of the curve. In neurons, cell injury and death caused by too high glutamate concentrations is termed excitotoxicity.

stimulation. Synaptic vesicles are docked at the presynaptic membrane in a region called the active zone that is enriched with proteins that help in tethering the vesicles and mediating vesicle fusion [4]. Once an action potential is received at the presynaptic terminal, it leads to depolarization followed by an opening of calcium (Ca21) channels. The Ca21 entry causes the neurotransmitter-containing vesicles to fuse at the active zone and a release of neurotransmitter into the synaptic cleft. The neurotransmitter diffuses across the cleft and interacts with the receptors in the postsynaptic membrane causing changes in membrane potential of the postsynaptic terminal. The further down-stream reactions then largely depend on the neurotransmitter. Glutamate, an excitatory neurotransmitter, will cause depolarization of the postsynaptic neuron, thus stimulating the firing of action potentials. Gamma-aminobutyric acid is one example for an inhibitory neurotransmitter that causes hyperpolarization of the postsynaptic neuron thus inhibiting the firing of action potentials. The released neurotransmitter is active only for a very short period of time as it is immediately cleared from synaptic cleft either by reuptake or degradation [4]. This ensures a high reactivity in neuronal communication. Glutamate sensitivity and fast excitatory transmission mark a majority of synapses in the central nervous system. These synapses therefore are an important focus in contemporary research. Fast synaptic transmission is mediated by ionotropic receptors, which are ion channels allowing a quick alteration in membrane potential and thus fast signaling and transmission. In contrast, metabotropic neurotransmitter receptors that are coupled to G-proteins will exert slower effects when a neurotransmitter binds. Glutamate is a nonessential amino acid that is, however, mainly taken from dietary sources. It is the most abundant neurotransmitter in the human brain and can bind to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), N-methyl-D-aspartate receptors (NMDARs), kainate and metabotropic glutamate receptors, of which the first three are ionotropic receptors [5]. AMPARs and NMDARs are the main routes for ion conductance. Both are cation channels being permeable to potassium (K1), sodium (Na1) and in the case of NMDAR also to Ca21 [6]. There are four different types of AMPAR subunits (GluA1 4) that form a heterotetrametric unit. Each subunit is comprised of four membrane-spanning regions. NMDA receptors are tetramers compiled of variations of the three different subunits (GluN1 3) where each subunit has several isoforms [5]. The postsynaptic compartment is often located in protrusions on the dendritic part of the neuron, called dendritic spines. The spine, comprised of a head, neck, and base, allows spatial confinement and compartmentalization of signaling molecules such as Ca21, which can be cytotoxic at high levels. The region important in synaptic transmission is called postsynaptic density (PSD) and can be found in the head of the spine (Fig. 15.2). It contains receptors for interacting with the neurotransmitters released from the presynaptic compartment. Moreover, it has a complex network of proteins involved in anchoring, trafficking and regulating receptors [7]. Examples are postsynaptic density protein 95 (PSD-95), important in stabilizing AMPARs and NMDARs in the PSD, as well as signaling molecules including Ca21/ calmodulin kinase II (CaMKII) and serine/threonine phosphatase 1 (PP1) [8]. Other proteins important in AMPAR scaffolding are glutamate receptor interacting proteins (GRIPs) that specifically interact with the C-termini of GluA2 and GluA3 subunits of AMPARs [5]. In excitatory synaptic transmission, glutamate is released into the synaptic cleft from the presynaptic compartment binding to AMPARs in the postsynaptic membrane. As a result, the AMPAR undergoes a conformational change allowing cation flux through the ion channels; mainly Na1 will flow into the spine, depolarizing it and thus the signal is transmitted [5] (Fig. 15.2A).

Neuronal Stress and Its Hormetic Aspects Chapter | 15

173

FIGURE 15.2 Models depicting the effects of different stimulations with glutamate on the postsynaptic response of neurons. (A) During basal synaptic transmission, glutamate released by vesicle exocytosis in the presynaptic terminal binds to both AMPA and NMDA receptors in the PSD. Binding of glutamate will allow flux of Na1 and K1 ions through AMPARs. As mainly Na1 is entering the postsynaptic cell, it is depolarized and the signal is transmitted from the presynaptic to the postsynaptic cell. Glutamate binding to NMDARs will cause no net change in ion flux due to Mg21 blocking the channel. (B) During LTD, low frequency stimulations (LFS) causing low levels of glutamate released during synaptic transmission result in a slight increase in Ca21 levels in the dendritic spines, as most of the NMDAR will still be blocked by Mg21. The intracellular Ca21 forms complexes with calmodulin and activates a crucial Ca21 sensor protein, calcineurin. Activated calcineurin further activates the phosphatase PP1 that dephosphorylates both AMPARs and stabilizing proteins in the PSD. These pathways involving calcineurin and PP1 lead to weakening of synaptic strength by disassembling actin filaments and enhancing endocytosis and lateral diffusion of AMPARs. Altogether, the efficacy of the synapse is reduced due to effective removal of AMPARs and morphological changes to the spine as a result of LFS and low levels of glutamate and Ca21. (C) During LTP, high frequency stimulations (HFS) and therefore moderate levels of glutamate released during synaptic transmission result in glutamate binding to NMDARs coinciding with depolarization of the postsynapse. As a result, the Mg21 block is released and moderate Ca21 levels will flow into the dendritic spines. The increased intracellular Ca21 forms complexes with calmodulin and this, in turn, activates Ca21 sensors, mainly CaMKII and PKA. The activation of CaMKII and PKA results in phosphorylation of AMPARs and enhances channel conductance. Additionally, actin polymerization is enhanced. This results in the enlargement of dendritic spines followed by increased accumulation of AMPARs at the PSD by either exocytosis or lateral diffusion toward the PSD. Therefore, the moderate increase in glutamate concentration leads to enlargement of dendritic spines and increased accumulation of AMPARs at the PSD of dendritic spines resulting in synaptic strengthening. (D) During excitotoxicity, high levels of glutamate are released leading to high intracellular Ca21 levels in the dendritic spine. This activates another Ca21 sensor protein, the protease calpain, which degrades many cytoskeletal proteins and membrane receptors that are crucial for maintaining the integrity of the synaptic connection. The massive overactivation of downstream signaling pathways due to highly increased intracellular Ca21 level is a potential cause of neuronal damage and can also lead to cell death.

Repetitive stimulations at a synapse can lead to lasting activity-dependent alterations leading to either an increased or a diminished subsequent response (Fig. 15.2). These processes of synaptic plasticity are called LTP and LTD, respectively. A special feature of the NMDAR allows for the differential responses to the same neurotransmitter glutamate: at resting membrane potential, the NMDAR is blocked by magnesium (Mg21), which is only released by depolarization of the cell [9].

174

SECTION | III Hormetic Stressors

Therefore, the NMDAR serves as a coincidence detector that will only allow Ca21 into the cell when the cell is simultaneously depolarized and glutamate is bound [10]. Low-frequency stimulation will cause low levels of Ca21 and a weakening of the synapse, hence LTD, and high-frequency stimulation leads to an accumulation of glutamate and a large influx of Ca21, which will signal to strengthen the synapse, termed LTP (Fig. 15.2). The changes in the response are achieved by alterations in the postsynaptic surface availability of AMPARs in the PSD. The AMPAR number can, for instance, be modulated by taking up the receptor into the cell via endocytosis or by inserting it into the postsynaptic membrane by exocytosis of receptor-carrying vesicles. Also, the morphology of the postsynapse is modulated [11]. Together, these synaptic processes are the basis for memory and learning. By contrast, high levels of glutamate and subsequently Ca21 are cytotoxic and pose a major threat to neuronal homeostasis [12] (see Fig. 15.2). Altogether, glutamate shows a hormetic dose response curve where low levels lead to LTD, higher levels cause LTP and very high levels lead to excitotoxity (Fig. 15.1). In the following, the role of glutamate and Ca21 as stressors at the synapse will be discussed and the differential pathways and adaptive responses at different doses of the stressors will be elucidated.

15.2

LONG-TERM DEPRESSION

LTD is a process, in which activity at a given synapse reduces its efficacy of synaptic transmission lasting hours or longer. Consecutive stimulations of the synapse are then only recognized and transmitted in a suppressed way. Responding to the same cue in a different way after a first experience with this cue is a basic characteristic for learning and memory. Therefore, synaptic plasticity is considered the cellular and molecular correlate of learning and memory [11]. In the following, the depression of an excitatory synapse mediated by NMDARs will be the center of attention as it is also the most studied model of LTD. NMDAR-induced weakening of the synapse is a result of a reduction of AMPARs by lateral diffusion and/or receptor endocytosis and a structural modulation of the dendritic spine harboring the postsynaptic signal reception apparatus (Fig. 15.2B). In the hippocampus, LTD can be achieved experimentally by low-frequency stimulation (LFS), typically below 10 Hz, by L-glutamate uncaging, or by a brief application of NMDA generating the so-called chemical LTD [11]. These stimulations of LTD seem to signal through the same pathways and converge on the same effector proteins modulating receptor availability and postsynaptic organization [11]. Low-frequency stimulation of an excitatory synapse results in persistent low levels of glutamate being released from the presynaptic terminal (Fig. 15.2B). The consequence of these persistent, yet relatively low levels of glutamate is that the threshold for Mg21-block release of NMDARs is not reached. The lack of Mg21-block release and depolarization results in only low concentrations of Ca21 (Fig. 15.2B) [13]. The levels of Ca21 achieved at least locally during LTD are sufficient to activate various enzymes mediating AMPAR endocytosis [14]. This uptake of AMPARs is thought to take place at sites adjacent to the PSD [15]. The removal of AMPARs from the PSD will result in a smaller postsynaptic response upon subsequent stimulations by glutamate. In contrast to enzymes responsible for LTP, such as CaMKII with a Ca21-dependent dissociation constant of about 6 μM [16], the phosphatases important in LTD mediation have a high affinity for Ca21. This explains the differential response to the same stressors, glutamate and sequentially Ca21, at different concentrations, specifically low Ca21 concentrations in LTD opposed to high Ca21 levels during LTP. When Ca21 enters through the NMDARs it binds to the Ca21 sensor protein calmodulin and Ca21/calmodulin as a complex will activate the phosphatase calcineurin. Calcineurin seems to play an important role in AMPAR endocytosis [17]. It forms a complex with dynamin 1, which consecutively binds to amphiphysin 1 [18]. Amphiphysin is a Bin amphiphysin Rvs (BAR) domain protein able to bend membranes [19]. Calcineurin dephosphorylates endocytic proteins, thus, promoting their assembly into a functional endocytic complex [17]. This then is hypothesized to enhance the endocytic removal of AMPARs [17] from the PSD causing a desensitization of the synapse. Besides promoting the assembly of the endocytic machinery, calcineurin also affects neurotransmission more directly. Calcineurin was found to dephosphorylate and thereby inactivate inhibitor-1 (I-1), which inhibits PP1. Calcineurin activity thus activates PP1 [20]. The further actions of PP1 are still mostly unknown and controversial. However, it has been suggested that PP1 dephosphorylates Ser845 on GluA1, an AMPAR subunit and Ser295 of PSD95 [21]. This results in an increase of receptor dynamics in the postsynaptic compartment, as AMPARs are thought to diffuse laterally and to be internalized more effectively upon GluA1 Ser845 dephosphorylation. Dephosphorylation of PSD-95 and of other structural PSD proteins is thought to additionally destabilize the integration of AMPARs into the PSD [21] and thus, encourage lateral diffusion and endocytosis.

Neuronal Stress and Its Hormetic Aspects Chapter | 15

175

Apart from the Ca21/calmodulin-mediated activation of calcineurin, other selective Ca21 sensors might play a role in the divergent response to different levels of Ca21 in synaptic plasticity. One candidate is hippocalcin, which can sense small changes in the Ca21 concentration. Upon translocation to the plasma membrane in response to a Ca21 increase, hippocalcin forms a complex with the adaptor protein 2 (AP2) complex and with GluA2. This promotes clathrin-mediated endocytosis of AMPARs [22] and thus also leads to reduced AMPAR availability at the postsynaptic plasma membrane. Protein interacting with C kinase-1 (PICK1), another BAR-domain protein, was also found to be a Ca21 sensor. PICK1 binds GluA2 with a biphasic-binding profile [23], in which GluA2 is bound at lower but not at high Ca21 concentration. The optimal binding is at 15 μM. Binding of PICK1 was thought to enhance AMPAR endocytosis; however, Citri et al. [24] suggested it to rather be important in retention of the AMPARs intracellularly. Besides AMPAR endocytosis, other structural changes take place due to LTD. The spines, which form the postsynaptic compartment and effectively retain the various signals spatially, are destabilized during LTD [25]. Both the spine head and the size of the PSD are reduced upon LTD (see Fig. 15.2B). This shrinking of the postsynaptic signaling compartment occurs independent of AMPAR endocytosis and additionally promotes the decline in the synaptic efficacy further [26]. Structural reorganization of the spine also consolidates the reduction of synaptic efficacy for longer periods of time. The morphological changes observed upon LTD are driven by alterations in the actin cytoskeleton within spines. Filamentous actin (F-actin) is crucial in maintaining the spine morphology. The dynamics of actin filaments play a major role in alterations of spine size and in structural rearrangements of the postsynaptic compartment and the signaling apparatus embedded into the PSD. Actin filaments are not static scaffolds but steadily turnover with varying rates. Depolymerization of F-actin was shown to be important in LTD [26]. Inhibition of F-actin turnover by Jasplakinolide resulted in impairment of LTD. Here, the Ca21sensors calcineurin and PICK1 also seem to play a role. Calcineurin dephosphorylates the actin-binding protein cofilin thereby activating it so that it can disassemble actin filaments [27]. Moreover, the actin nucleator Cobl may be blocked upon an increase in Ca21 concentrations [28]. However, the function of Cobl in synaptic plasticity remains unknown. More established is the role of another actin nucleator, the actinrelated protein-2/3 (Arp2/3) complex. The Arp2/3 complex is a powerful machine triggering the formation of new actin filaments — preferentially from already existing actin filaments. It is inhibited by PICK1 [29]. Thus, PICK1 association is thought to decrease the pool of F-actin and thereby destabilize the postsynaptic spine compartment. Altogether, low-level stimulation with glutamate causes intermediate Ca21 levels in the postsynapse where specific 21 Ca sensors involved in phosphatase signaling work to destabilize and depress the synapse by decreasing functional, synaptic AMPARs and shrinking the spine. The word depression suggests that LTD is a harmful change and one might assume that a decrease in efficiency at the synapse would be negative. However, LTD is essential for proper functioning of the various brain regions. Several genetic models demonstrate that knocking out vital molecules in LTD signaling disrupts not only LTD but also impairs proper brain function. For instance, mice lacking a certain NMDAR subunit important in LTD showed a decrease in spine density in hippocampal neurons and performed worse than their wild-type counterparts in established memory tests [30]. This highlights the importance of LTD in memory formation. Consistently, dysregulation of LTD has been implicated in several diseases. A study inducing major depression by chronic stress application in mice found elevated LTD. This effect could be counteracted by administration of fluvoxamine, an antidepressant [31]. Altogether, this demonstrates the necessity for tight and controlled regulation of LTD for the physiological function of the brain.

15.3

LONG-TERM POTENTIATION

LTP is defined as the long-lasting strengthening of synaptic connections. This strengthening is accomplished by enlargement of dendritic spines and increased accumulation of AMPARs in the PSD [32]. LTP is a widely studied cellular model for learning and memory and can experimentally be achieved by either repeated high-frequency stimulations (HFS) or theta-burst stimulations (TBS). The HFS is achieved by tetanic stimulation of typically 100 Hz for the duration of 1 second and TBS generally by four pulses at 100 Hz repeated with 200 milliseconds interburst intervals [33]. As mentioned in the introduction, neuronal stressors like glutamate and Ca21, depending on their dose, activate signaling cascades either to strengthen or to weaken synaptic connections [33] (Fig. 15.1 and 15.2). A critical step for LTP to be initiated is the influx of Ca21 over the postsynaptic membrane. This influx is mainly mediated through NMDARs and leads to the activation of intracellular signaling cascades resulting in enhanced rapid accumulation of AMPARs in the PSD to strengthen the synaptic connection [34].

176

SECTION | III Hormetic Stressors

Upon a strong synaptic transmission by HFS, a depolarization state is achieved by Na1 influx through AMPARs. This results in a release of the Mg21 blockade in NMDARs. Once the Mg21 blockade is released, the flow of Na1 and K1, and particularly the influx of Ca21 occurs through NMDARs [35]. This increases the postsynaptic intracellular Ca21 concentration to low micromolar levels [34] and augments the formation of Ca21/calmodulin complexes (Fig. 15.2C). The Ca21/calmodulin complex activates CaMKII by autophosphorylation at Thr286 [36]. Once activated by autophosphorylation, CaMKII switches from a Ca21/calmodulin complex dependent to an independent state by retaining its kinase activity even after the complex dissociates [37]. Immediately after the HFS stimulation, a rapid activation of CaMKII by autophosphorylation is observed. CaMKII’s activity persists for at least an hour until it is dephosphorylated by PP1 [38]. This means that a transient increase in Ca21 levels will lead to a prolonged kinase activity of CaMKII that continues even after the subsequent Ca21 decline until CaMKII is inactivated by PP1 [39] (Fig. 15.2C). Once autophosphorylated, CaMKII gets translocated from the cytoplasm to the postsynaptic compartment and binds to the PSD. Along with CaMKII, another Ca21-dependent kinase, protein kinase C (PKC), can phosphorylate the C-terminus of the AMPAR subunit GluA1 at Ser831 [40]. Additionally, protein kinase A (PKA) is also involved in phosphorylation of GluA1 at Ser845 [41]. Phosphorylated GluA1 has the potential to increase AMPAR-mediated channel conductance to enhance LTP. This occurs by increased influx of Na1 that lowers the resting potential resulting in an amplified response to the incoming impulses [39]. During LTP, Ca21 along with its sensor protein calmodulin does not only activate CaMKII but also directly or indirectly activates other proteins, such as adenylate cyclase (AC), PKA, and Rho family GTPases [42]. Once AC gets activated, it produces cyclic adenosine monophosphate (cAMP) and this results in activation of PKA. Downstream, activated PKA stimulates I-1. PKA thus is a counterplayer of calcineurin, which responds to low Ca21 levels and inactivates I-1 [43]. The PKA-activated I-1 inhibits PP1 and this inhibition of PP1 through a Ca21-mediated cAMP/PKA signaling cascade prolongs CaMKII’s activity by maintaining it in its phosphorylated state [38,44]. This stands in contrast to LTD, where — due to low levels of Ca21 and calcineurin-mediated inactivation of I-1 — PP1 is activated and thereby inhibits CaMKII’s activity by dephosphorylation. Hence, depending on the Ca21 concentration either the CaMKII or the calcineurin pathway gets activated to induce either LTP or LTD, respectively [42]. Additionally, activated CaMKII has an impact on the morphology of dendritic spines during LTP. CaMKII has an influence on members of the Rho family of GTPases like Cdc42, Rac1, and RhoA [45]. Through the Rho/ROCK pathway, LIM domain kinase (LIMK) is activated, which in turn inhibits the activity of actin depolymerizing factor (ADF)/ cofilin by phosphorylating Ser3 [46,47] resulting in an inhibition of its F-actin depolymerizing activity [48]. Other members of the Rho family of GTPases, such as Rac1 and Cdc42, are involved in enhancing actin polymerization through activators of the Arp2/3 complex [49]. This CaMKII-mediated increase in F-actin polymerization leads to an enlargement of spines and such enlarged spines are able to accommodate additional postsynaptic AMPARs enhancing synaptic strengthening [32] (Fig. 15.2C). Although the mechanism behind the insertion of AMPARs in the PSD following spine enlargement remains unclear, one emerging model is the CaMKII-mediated slot hypothesis. Here, rearrangements of the PSD could make slots available, which can be filled by AMPARs that diffuse laterally toward the PSD within the postsynaptic plasma membrane (Fig. 15.2C) and are then trapped by PSD-95. Another suggested model for increased AMPAR insertion in the postsynaptic membrane is exocytosis, where newly generated actin filaments could support transport of AMPAR-containing vesicles to the spine head and/or fusion with the plasma membrane [32]. During LTP, kinases like CaMKII activate synaptic Ras GTPase-activating protein 1 (SynGAP) by phosphorylation and the activated SynGAP is shown to be involved in AMPAR membrane trafficking [50]. Apart from the NMDAR-mediated Ca21 entry, there are other modes of increase in intracellular Ca21 levels in the postsynapse that have an influence on LTP. They are mediated through voltage-dependent Ca21 channels in the plasma membrane and through Ca21 release from internal Ca21 stores — mostly from the endoplasmic reticulum. In any case, the increase in intracellular Ca21 levels upon synaptic stimulation normally is short lived as Ca21 buffering proteins like calbindin, parvalbumin, and calretinin are involved in balancing the transient increase in Ca21 levels. Apart from the involvement of Ca21 buffering proteins in balancing, other mechanisms lowering local synaptic Ca21 concentrations rapidly are diffusion that occurs within B1 millisecond and active ion transport processes through ATP-dependent Ca21 pumps like plasmalemmal Ca21 ATPase (PMCA) pumps and sarcoendoplasmic reticulum Ca21 ATPase (SERCA) pumps [51]. Besides strengthening synaptic connections during LTP, Ca21 is also involved in LTP maintenance and memory consolidation. The induction of LTP stimulates Ca21-mediated pathways like CAMKs and MAPKs/extracellular signalregulated kinases (ERKs) to phosphorylate cAMP response element-binding protein (CREB) at Ser133. Such

Neuronal Stress and Its Hormetic Aspects Chapter | 15

177

phosphorylated CREB upregulates the expression levels of a neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF is a crucial factor for long-term memory formation [34]. Apart from Ca21, additional widely implicated secondary messengers noted for their roles in LTP signal transduction mechanisms are cAMP and cyclic guanosine monophosphate (cGMP) [52]. A recent animal behavioral study shows that cGMP behaves in a hormetic fashion in regulating beneficial levels of Aβ peptide to enhance LTP and memory [53]. Interestingly, also peptides like leptin and angiotensin IV show biphasic dose responses in enhancing learning and memory when tested in behavioral studies by facilitating LTP [54,55]. Furthermore, two notable studies show concentration-dependent biphasic response with glucocorticoids on aspects like working memory [56,57] and emotional memory [58] by increasing glutamate transmission. Overall, depending on the levels of Ca21, either the LTP-inducing kinase pathway or the LTD-inducing phosphatase pathway is activated [44]. During LTP, the synaptic strengthening is achieved by high-frequency stimulation leading to increased intracellular Ca21 that activates many kinases resulting in spine enlargement and increased AMPAR accumulation at the PSD.

15.4

EXCITOTOXICITY

Excitotoxicity is the pathological process in which the neurons are damaged or killed by excessive stimulation of neurotransmitters like glutamate [59]. A lot of factors, such as aging, energy shortage, and genetic factors, can induce the excessive release of glutamate [60], which causes severe neuron damage. In experimental settings, high glutamate concentrations, such as 100 μM, induced B50% neuronal death in cultured cerebellar granule cells [61] and even over 80% neuronal death in cortical neurons [62]. While neurons may undergo excitotoxic death in pathological conditions, it is also clear that the neurons activate hormetic pathways including neurotrophic signaling pathways and the activation of survival proteins, such as protein chaperones, Ca21-binding proteins, and inhibitors of apoptosis proteins to help the neurons to cope with the glutamate stress [59]. For example, pretreatment of neurons with ultra-low dose glutamate (10218 M) can protect them from being damaged by subsequent higher levels of glutamate [63]. As pointed out in the introduction, glutamate at physiological levels exerts its activity by binding to postsynaptic glutamate receptors causing membrane depolarization followed by Ca21 influx. However, during ischemic conditions, the deprivation of oxygen and glucose levels in addition to decreased ATP levels induces an excessive release of glutamate [64]. This increased extracellular glutamate causes overactivation of glutamate receptors including NMDARs, which leads to a strong Ca21 influx into postsynaptic neurons. This excessive Ca21 influx further activates pathways like cysteine proteases and induces oxidative stress and triggers programmed cell death (apoptosis) (Fig. 15.2D) [59]. In the following, the Ca21-activated protease pathway will be discussed, as it most directly has an impact on the synaptic signal transmission machinery. As described before, excess of Ca21 leads to the overactivation of cysteine proteases. The so-called calpain proteases are ubiquitously expressed in the nervous system. There are two highly expressed calpain isoforms — μ-calpain and m-calpain (also called calpain I and calpain II, respectively). They are similar in substrate specificity but differ in their sensitivity to Ca21: m-calpain requires 0.4 0.8 mM Ca21 for half-maximal proteolytic activity in vitro, whereas μ-calpain requires 3 50 μM Ca21 [65]. Calpains play a crucial role in excitotoxicity by degrading a variety of substrates including cytoskeletal proteins, membrane receptors, and metabolic enzymes [59]. Interestingly, the subunits of the NMDARs, GluN1, GluN2A, and GluN2B, are substrates for calpain cleavage following excitotoxicity [66]. Recent studies using cortical neurons exposed to a toxic insult with NMDA showed cleavage of both GluN2A and GluN2B by calpain [67]. Moreover, the same experimental conditions induced a calpain-mediated cleavage of PSD-95, a protein highly expressed in the PSD that plays an important role in anchoring NMDARs [67]. Together, these results suggest that in addition to the degradation of NMDAR subunits, the lack of the scaffold protein PSD-95 could alter the NMDAR levels and thus modify the efficacy of synaptic transmission. However, the consequences of the NMDARs cleavage are not clear, and the role of NMDARs also remains elusive. There are evidences that GluN2A and GluN2B mediate prosurvival signaling but also contribute to the prodeath signaling [68]. Another family of ionotropic glutamate receptors targeted by calpains under excitotoxic conditions is the AMPA receptors. The subunits GluA1, GluA2, and GluA3 all are targets of calpain [65]. Biochemical analysis further confirmed that excitotoxic conditions (glutamate or NMDA stimulation) induce a calpain-mediated truncation of the 106 kDa GluA1 subunit to a 98 kDa cleavage product in pyramidal cortical neurons, which decreases the total and surface expression of GluA1 [69]. In addition to the subunits, AMPAR scaffolding and interacting proteins, such as GRIP, were also degraded by calpains [70] under excitotoxic conditions. GRIP1 contributes to the stabilization of receptor subunits, and another study showed that interference with GluA2 GRIP1 binding decreased the number of dendritic

178

SECTION | III Hormetic Stressors

protrusions and induced a loss of synaptic contacts [71]. In addition, lack of GluA2 AMPAR contributes to ischemiainduced neuronal death. It is thought that this effect is caused by enhancing the vulnerability of neurons to neuronal insults and mediating the rise of toxic ions [72]. In addition to ionotropic glutamate receptors, the roles of metabotropic glutamate receptors, especially of group I metabotropic glutamate receptors, have been studied in excitotoxicity. It was shown that calpain-mediated mGluR1alpha truncation in the intracellular C-tail of the receptor plays a critical role in excitotoxicity by both disrupting neuroprotective signaling and enhancing neurotoxic signaling [73]. Besides the neurotransmitter receptors and scaffold proteins, proteins involved in establishing and controlling neuronal morphology might get cleaved in excitotoxic conditions. A recent study showed that calpain mediates the cleavage of drebrin in cultured hippocampal neurons subjected to excitotoxic stimulation with NMDA [74]. Drebrin is an Factin-binding protein [75], and is localized preferentially to dendritic spines of mature neurons [76]. Downregulation of drebrin A, the isoform expressed in mature neurons, was shown to decrease the density and width of dendritic spines. Reduction of drebrin levels furthermore inhibits the synaptic clustering of NMDARs [77]. However, the role of drebrin in neuronal death in the ischemic brain remains to be determined. Not only postsynaptic proteins, but additionally also presynaptic proteins are targets of calpain. Growth-associated protein 43 (GAP43) is enriched in axonal growth cones. The phosphorylation status of GAP43 plays an important role in the regulation of the actin cytoskeleton. Phosphorylated GAP43 stabilizes long actin filaments, whereas the dephosphorylated form of the protein reduces actin filament length [78]. GAP43 is truncated by calpain [79], and the cleavage of GAP43 participates in repulsion of axonal growth cones and induction of neuronal death. Although the molecular mechanism behind this is not fully understood, it was suggested that the cleavage products may be involved in apoptotic signaling. Together, excessive stimulation by glutamate and Ca21 induces the overactivation of downstream proteases, which will go on to damage cell structures, such as components of the cytoskeleton and membrane receptors, and will ultimately result in cell death. However, the mechanisms of how the calpain-targeted proteins are involved in neuronal death are not fully understood. Therefore, understanding the substrates of calpain in excitotoxicity will be crucial for our understanding of how detrimental conditions causing the irreversible loss of neurons in our nervous system could be prevented or improved by therapeutic interventions. Altogether, this chapter on neuronal stress briefly highlighted the mechanisms that neurons apply to adapt in order to ensure their survival and protection as a response to different doses of stressors—specifically, glutamate and Ca21. The emerging evidence for hormetic dose responses in neurons will set the ground for the application of this concept in clinical studies and therapeutics.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

Calabrese EJ. Neuroscience and hormesis: overview and general findings. Crit Rev Toxicol 2008;38(4):249 52. Mattson MP. Hormesis defined. Ageing Res Rev 2008;7(1):1 7. Kagias K, Nehammer C, Pocock R. Neuronal responses to physiological stress. Front Genet 2012;3:222. Di Maio V. Regulation of information passing by synaptic transmission: a short review. Brain Res 2008;1225:26 38. Willard SS, Koochekpour S. Glutamate, glutamate receptors, and downstream signaling pathways. Int J Biol Sci 2013;9(9):948 59. Citri A, Malenka RC. Synaptic plasticity: multiple forms, functions, and mechanisms. Neuropsychopharmacology 2008;33(1):18 41. Ryan TJ, Grant SG. The origin and evolution of synapses. Nat Rev Neurosci 2009;10(10):701 12. Ziff EB. Enlightening the postsynaptic density. Neuron 1997;19(6):1163 74. Vargas-Caballero M, Robinson HP. Fast and slow voltage-dependent dynamics of magnesium block in the NMDA receptor: the asymmetric trapping block model. J Neurosci 2004;24(27):6171 80. Blanke ML, VanDongen AMJ. Activation mechanisms of the NMDA receptor. In: Van Dongen AM, editor. Biology of the NMDA receptor. Boca Raton, FL: Frontiers in Neuroscience; 2009. Collingridge GL, Peineau S, Howland JG, Wang YT. Long-term depression in the CNS. Nat Rev Neurosci 2010;11(7):459 73. Dong XX, Wang Y, Qin ZH. Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin 2009;30(4):379 87. Luscher C, Malenka RC. NMDA receptor-dependent long-term potentiation and long-term depression (LTP/LTD). Cold Spring Harb Perspect Biol 2012;4 a005710. Cummings JA, Mulkey RM, Nicoll RA, Malenka RC. Ca21 signaling requirements for long-term depression in the hippocampus. Neuron 1996;16(4):825 33. Lu J, Helton TD, Blanpied TA, Racz B, Newpher TM, Weinberg RJ, et al. Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer. Neuron 2007;55(6):874 89. Tse JK, Giannetti AM, Bradshaw JM. Thermodynamics of calmodulin trapping by Ca21/calmodulin-dependent protein kinase II: subpicomolar Kd determined using competition titration calorimetry. Biochemistry 2007;46(13):4017 27.

Neuronal Stress and Its Hormetic Aspects Chapter | 15

179

[17] Beattie EC, Carroll RC, Yu X, Morishita W, Yasuda H, von Zastrow M, et al. Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD. Nat Neurosci 2000;3(12):1291 300. [18] Lai MM, Hong JJ, Ruggiero AM, Burnett PE, Slepnev VI, De Camilli P, et al. The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis. J Biol Chem 1999;274(37):25963 6. [19] Qualmann B, Koch D, Kessels MM. Let’s go bananas: revisiting the endocytic BAR code. EMBO J 2011;30(17):3501 15. [20] Mulkey RM, Herron CE, Malenka RC. An essential role for protein phosphatases in hippocampal long-term depression. Science 1993;261 (5124):1051 5. [21] Kim MJ, Futai K, Jo J, Hayashi Y, Cho K, Sheng M. Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95. Neuron 2007;56(3):488 502. [22] Palmer CL, Lim W, Hastie PG, Toward M, Korolchuk VI, Burbidge SA, et al. Hippocalcin functions as a calcium sensor in hippocampal LTD. Neuron 2005;47(4):487 94. [23] Hanley JG, Henley JM. PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking. EMBO J 2005;24(18):3266 78. [24] Citri A, Bhattacharyya S, Ma C, Morishita W, Fang S, Rizo J, et al. Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression. J Neurosci 2010;30(49):16437 52. [25] Zhou Q, Homma KJ, Poo MM. Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses. Neuron 2004;44 (5):749 57. [26] Wang XB, Yang Y, Zhou Q. Independent expression of synaptic and morphological plasticity associated with long-term depression. J Neurosci 2007;27(45):12419 29. [27] Okamoto K, Nagai T, Miyawaki A, Hayashi Y. Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat Neurosci 2004;7(10):1104 12. [28] Hou W, Izadi M, Nemitz S, Haag N, Kessels MM, Qualmann B. The actin nucleator Cobl is controlled by calcium and calmodulin. PLOS Biol 2015;13(9):e1002233. [29] Rocca DL, Martin S, Jenkins EL, Hanley JG. Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis. Nat Cell Biol 2008;10(3):259 71. [30] Brigman JL, Wright T, Talani G, Prasad-Mulcare S, Jinde S, Seabold GK, et al. Loss of GluN2B-containing NMDA receptors in CA1 hippocampus and cortex impairs long-term depression, reduces dendritic spine density, and disrupts learning. J Neurosci 2010;30(13):4590 600. [31] Holderbach R, Clark K, Moreau JL, Bischofberger J, Normann C. Enhanced long-term synaptic depression in an animal model of depression. Biol Psychiatr 2007;62(1):92 100. [32] Herring BE, Nicoll RA. Long-term potentiation: from CaMKII to AMPA receptor trafficking. Annu Rev Physiol 2016;78:351 65. [33] Hernandez RV, Navarro MM, Rodriguez WA, Martinez Jr JL, LeBaron RG. Differences in the magnitude of long-term potentiation produced by theta burst and high frequency stimulation protocols matched in stimulus number. Brain Res Brain Res Protoc 2005;15(1):6 13. [34] Marambaud P, Dreses-Werringloer U, Vingtdeux V. Calcium signaling in neurodegeneration. Mol Neurodegener 2009;4:20. [35] Nicoll RA. A brief history of long-term potentiation. Neuron 2017;93(2):281 90. [36] Giese KP, Fedorov NB, Filipkowski RK, Silva AJ. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science 1998;279(5352):870 3. [37] Braun AP, Schulman H. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 1995;57:417 45. [38] Barria A, Muller D, Derkach V, Griffith LC, Soderling TR. Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation. Science 1997;276(5321):2042 5. [39] Soderling TR, Derkach VA. Postsynaptic protein phosphorylation and LTP. Trends Neurosci 2000;23(2):75 80. [40] Benke TA, Luthi A, Isaac JT, Collingridge GL. Modulation of AMPA receptor unitary conductance by synaptic activity. Nature 1998;393 (6687):793 7. [41] Banke TG, Bowie D, Lee H, Huganir RL, Schousboe A, Traynelis SF. Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J Neurosci 2000;20(1):89 102. [42] Xia Z, Storm DR. The role of calmodulin as a signal integrator for synaptic plasticity. Nat Rev Neurosci 2005;6(4):267 76. [43] Blitzer RD, Connor JH, Brown GP, Wong T, Shenolikar S, Iyengar R, et al. Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP. Science 1998;280(5371):1940 2. [44] Kang-Park MH, Sarda MA, Jones KH, Moore SD, Shenolikar S, Clark S, et al. Protein phosphatases mediate depotentiation induced by highintensity theta-burst stimulation. J Neurophysiol 2003;89(2):684 90. [45] Nakayama AY, Harms MB, Luo L. Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons. J Neurosci 2000;20(14):5329 38. [46] Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 1998;393(6687):805 9. [47] Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 1998;393(6687):809 12. [48] Fukazawa Y, Saitoh Y, Ozawa F, Ohta Y, Mizuno K, Inokuchi K. Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo. Neuron 2003;38(3):447 60. [49] Lamprecht R, LeDoux J. Structural plasticity and memory. Nat Rev Neurosci 2004;5(1):45 54.

180

SECTION | III Hormetic Stressors

[50] Araki Y, Zeng M, Zhang M, Huganir RL. Rapid dispersion of SynGAP from synaptic spines triggers AMPA receptor insertion and spine enlargement during LTP. Neuron 2015;85(1):173 89. [51] Higley MJ, Sabatini BL. Calcium signaling in dendritic spines. Cold Spring Harb Perspect Biol 2012;4(4):a005686. [52] Bernabeu R, Schmitz P, Faillace MP, Izquierdo I, Medina JH. Hippocampal cGMP and cAMP are differentially involved in memory processing of inhibitory avoidance learning. Neuroreport 1996;7(2):585 8. [53] Palmeri A, Ricciarelli R, Gulisano W, Rivera D, Rebosio C, Calcagno E, et al. Amyloid-beta peptide is needed for cGMP-induced long-term potentiation and memory. J Neurosci 2017;37(29):6926 37. [54] Wayner MJ, Armstrong DL, Phelix CF, Wright JW, Harding JW. Angiotensin IV enhances LTP in rat dentate gyrus in vivo. Peptides 2001;22 (9):1403 14. [55] Farr SA, Banks WA, Morley JE. Effects of leptin on memory processing. Peptides 2006;27(6):1420 5. [56] Lupien SJ, Gillin CJ, Hauger RL. Working memory is more sensitive than declarative memory to the acute effects of corticosteroids: a doseresponse study in humans. Behav Neurosci 1999;113(3):420 30. [57] Lupien SJ, Wilkinson CW, Briere S, Menard C, Ng Ying Kin NM, Nair NP. The modulatory effects of corticosteroids on cognition: studies in young human populations. Psychoneuroendocrinology 2002;27(3):401 16. [58] Abercrombie HC, Kalin NH, Thurow ME, Rosenkranz MA, Davidson RJ. Cortisol variation in humans affects memory for emotionally laden and neutral information. Behav Neurosci 2003;117(3):505 16. [59] Mattson MP. Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromol Med 2003;3(2):65 94. [60] Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: bridge to various triggers in neurodegenerative disorders. Eur J Pharmacol 2013;698(1 3):6 18. [61] Marini AM, Rabin RS, Lipsky RH, Mocchetti I. Activity-dependent release of brain-derived neurotrophic factor underlies the neuroprotective effect of N-methyl-D-aspartate. J Biol Chem 1998;273(45):29394 9. [62] Vaarmann A, Kovac S, Holmstrom KM, Gandhi S, Abramov AY. Dopamine protects neurons against glutamate-induced excitotoxicity. Cell Death Dis 2013;4:e455. [63] Jonas W, Lin Y, Tortella F. Neuroprotection from glutamate toxicity with ultra-low dose glutamate. Neuroreport 2001;12(2):335 9. [64] Cross JL, Meloni BP, Bakker AJ, Lee S, Knuckey NW. Modes of neuronal calcium entry and homeostasis following cerebral ischemia. Stroke Res Treat 2010;2010:316862. [65] Wu HY, Lynch DR. Calpain and synaptic function. Mol Neurobiol 2006;33(3):215 36. [66] Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 2014;115:157 88. [67] Gascon S, Sobrado M, Roda JM, Rodriguez-Pena A, Diaz-Guerra M. Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95. Mol Psychiatr 2008;13(1):99 114. [68] Curcio M, Salazar IL, Mele M, Canzoniero LM, Duarte CB. Calpains and neuronal damage in the ischemic brain: the swiss knife in synaptic injury. Prog Neurobiol 2016;143:1 35. [69] Yuen EY, Gu Z, Yan Z. Calpain regulation of AMPA receptor channels in cortical pyramidal neurons. J Physiol 2007;580(Pt 1):241 54. [70] Lu X, Wyszynski M, Sheng M, Baudry M. Proteolysis of glutamate receptor-interacting protein by calpain in rat brain: implications for synaptic plasticity. J Neurochem 2001;77(6):1553 60. [71] Heisler FF, Lee HK, Gromova KV, Pechmann Y, Schurek B, Ruschkies L, et al. GRIP1 interlinks N-cadherin and AMPA receptors at vesicles to promote combined cargo transport into dendrites. Proc Natl Acad Sci USA 2014;111(13):5030 5. [72] Liu SJ, Zukin RS. Ca21-permeable AMPA receptors in synaptic plasticity and neuronal death. Trends Neurosci 2007;30(3):126 34. [73] Xu W, Wong TP, Chery N, Gaertner T, Wang YT, Baudry M. Calpain-mediated mGluR1alpha truncation: a key step in excitotoxicity. Neuron 2007;53(3):399 412. [74] Chimura T, Launey T, Yoshida N. Calpain-mediated degradation of Drebrin by excitotoxicity in vitro and in vivo. PLOS One 2015;10(4):e0125119. [75] Asada H, Uyemura K, Shirao T. Actin-binding protein, drebrin, accumulates in submembranous regions in parallel with neuronal differentiation. J Neurosci Res 1994;38(2):149 59. [76] Aoki C, Sekino Y, Hanamura K, Fujisawa S, Mahadomrongkul V, Ren Y, et al. Drebrin A is a postsynaptic protein that localizes in vivo to the submembranous surface of dendritic sites forming excitatory synapses. J Comp Neurol 2005;483(4):383 402. [77] Takahashi H, Mizui T, Shirao T. Down-regulation of drebrin A expression suppresses synaptic targeting of NMDA receptors in developing hippocampal neurones. J Neurochem 2006;97(Suppl 1):110 15. [78] He Q, Dent EW, Meiri KF. Modulation of actin filament behavior by GAP-43 (neuromodulin) is dependent on the phosphorylation status of serine 41, the protein kinase C site. J Neurosci 1997;17(10):3515 24. [79] Zakharov VV, Mosevitsky MI. M-calpain-mediated cleavage of GAP-43 near Ser41 is negatively regulated by protein kinase C, calmodulin and calpain-inhibiting fragment GAP-43-3. J Neurochem 2007;101(6):1539 51.