Preventing Ca²⁺-mediated nitrosative stress in neurodegenerative diseases: Possible pharmacological strategies

Abstract

Overactivation of the NMDA-subtype of glutamate receptor is known to trigger excessive calcium influx, contributing to neurodegenerative conditions. Such dysregulation of calcium signaling results in generation of excessive free radicals, including reactive oxygen and nitrogen species (ROS/RNS), including nitric oxide (NO). In turn, we and our colleagues have shown that these free radicals trigger pathological production of misfolded proteins, mitochondrial dysfunction, and apoptotic pathways in neuronal cells. Here, we discuss emerging evidence that excessive calcium-induced NO production can contribute to the accumulation of misfolded proteins, specifically by S-nitrosylation of the ubiquitin E3 ligase, parkin, and the chaperone enzyme for nascent protein folding, protein-disulfide isomerase. Additionally, excessive calcium-induced NO generation leads to the formation of S-nitrosylated dynamin-related protein 1, which causes abnormal mitochondrial fragmentation and resultant synaptic damage. In this review, we also discuss how two novel classes of pharmacological agents hold promise to interrupt these pathological processes. Firstly, the NMDA receptor antagonists, Memantine and NitroMemantine, block excessive extrasynaptic glutamate excitation while maintaining synaptic transmission, thereby limiting excessive calcium influx and production of ROS/RNS. Secondly, therapeutic pro-electrophiles are activated in the face of oxidative insult, thus protecting cells from calcium-induced oxidative stress via the Keap1/Nrf2 transcriptional pathway.

1. Introduction

A prominent feature in many neurodegenerative diseases is excessive generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including nitric oxide (NO), which contribute to neuronal cell injury and death [1–3]. N-Methyl-d-aspartate (NMDA)-type glutamate receptors have been linked to ROS and NO production in the nervous system. Overactivation of NMDA receptors causes excessive influx of Ca²⁺ ions, which generates ROS and activates neuronal NO synthase (nNOS) [4]. Reaction of the NO group with critical cysteine thiols of target proteins results in the formation of S-nitrosoproteins (SNO-Ps) and can thus regulate protein function [5]. Lipton and Stamler initially discovered and characterized this biochemical process on the NMDA receptor itself although the activity of potentially hundreds or thousands of other proteins are modulated in this fashion, and coined the term “S-nitrosylation.” S-Nitrosylation can mediate either protective or neurotoxic effects depending on the action of the target proteins. Additionally, mitochondrial respiration produces free radicals, principally ROS, in response to excessive calcium influx, and one species of ROS, superoxide anion (O₂^.−), reacts rapidly with free radical NO to form the very toxic product peroxynitrite (ONOO⁻) [5] (Fig. 1).

Fig. 1.

Possible mechanisms whereby Ca²⁺ signaling contributes to NO generation in neurodegenerative conditions. Hyperactivation of NMDA receptors (NMDARs) by glutamate (Glu) and glycine (Gly) induces excessive Ca²⁺ influx and activation of neuronal NO synthase (nNOS). nNOS produces NO from l-arginine. In Alzheimer’s disease (AD), soluble oligomers of Aβ peptide, thought to be a key mediator in AD pathogenesis, can facilitate neuronal NO production in both NMDAR-dependent and -independent manners. In Parkinson’s disease (PD), mitochondrial dysfunction caused by pesticides or other environmental toxins can trigger NO production possibly via mitochondrial and NMDAR/Ca²⁺ influx pathways. Note that in addition to RNS, ROS are also produced in response to Aβ and pesticides

In recent work, we have shown that S-nitrosylation and further oxidation of critical cysteine residues can lead to protein misfolding. Misfolded proteins form aggregates in many neurodegenerative diseases, and soluble oligomers of these aberrantly folded proteins are thought to adversely affect cell function by interfering with normal cellular processes or initiating cell death signaling pathways [6]. As examples, α-synuclein and synphilin-1 in Parkinson’s disease (PD), and amyloid-β (Aβ) and tau in Alzheimer's disease (AD) form toxic oligomers of non-native secondary structures. The formation of larger aggregates may be an attempt of the cell to wall off these toxic proteins. Protein aggregation is also a signature of Huntington’s disease (a polyQ disorder), amyotrophic lateral sclerosis (ALS), and prion disease [7].

Sporadic forms of neurodegenerative diseases, rather than single gene mutation, constitute the vast majority of cases, and pathologic protein misfolding in these diseases may be the result of posttranslational changes to the protein engendered by nitrosative and/or oxidative stress, which can thus mimic the more rare genetic variants of the disease [8]. Here we focus on specific examples that show the critical roles of S-nitrosylation of ubiquitin E3 ligases, e.g., parkin, and endoplasmic reticulum (ER) chaperones, such as protein-disulfide isomerase (PDI), in accumulation of misfolded proteins in neurodegenerative diseases [9–11]. We also review recent findings on S-nitrosylation of dynamin-related protein 1 (Drp1), which can contribute to the pathological fragmentation of mitochondria. We further discuss therapeutic applications of the NMDA receptor open-channel blockers memantine and its newer NO-derivatives for preventing excessive production of ROS and NO. Finally, we discuss the use of a class of pro-electrophiles for neuroprotection from neurodegenerative disorders [3, 12].

2. Generation of ROS/RNS by Ca²⁺ influx through NMDA receptor channels in response to glutamatergic signaling

The amino-acid glutamate functions as the major excitatory neurotransmitter and is present at millimolar concentrations in the adult central nervous system (CNS). Ca²⁺ stimulates release of glutamate from the presynaptic nerve terminal into the synaptic cleft where it diffuses to postsynaptic receptors on an adjacent neuron. Normal excitatory neurotransmission is essential for synaptic development and plasticity as well as learning and memory. In contrast, excessive glutamate excitation plays a role in a variety of neurological disorders ranging from acute hypoxic-ischemic brain injury to chronic neurodegenerative diseases. Survival pathways appear to be mediated via NMDA receptor synaptic activity, whereas neuronal damage may be mediated by excessive extrasynaptic activity [13, 14]. Severe overstimulation of excitatory receptors can cause necrotic cell death, while less fulminant or chronic overstimulation can cause apoptotic or other forms of cell death [15–17].

Glutamate receptors in the nervous system are divided into two groups, ionotropic (representing ligand-gated ion channels) and metabotropic (coupled to G-proteins). Ionotropic glutamate receptors are represented by three separate classes, NMDA, α-amino–3-hydroxy-5 methyl-4-isoxazole propionic acid (AMPA), and kainate; each receptor type is named for the synthetic ligands that selectively activate them. NMDA receptors, unlike most other types, are highly permeable to Ca²⁺. Depolarization relieves blockade of NMDA receptor-coupled ion channels by Mg²⁺ [18]. Ca²⁺ influx promotes many normal intracellular signaling pathways, but excessive influx promotes pathological signaling, contributing to cell injury and death via production of free radicals such as ROS and NO as well as other enzymatic processes [2, 5, 16, 17, 19] (Fig. 1 and Fig. 2). “Excitotoxicity” is defined as neuronal damage caused by excessive activation of glutamate receptors [20] and is at least partly mediated by excessive Ca²⁺ influx through NMDA receptor-associated ion channel [2, 3, 21]. Increased levels of neuronal Ca²⁺, in conjunction with the Ca²⁺-binding protein CaM, trigger the activation of nNOS and subsequent generation of NO from the amino acid l-arginine [22, 23] (Fig. 2).

Fig. 2.

Overstimulation of NMDA receptors (NMDARs) by glutamate (Glu) and glycine (Gly) induces excessive Ca²⁺ influx, activation of neuronal NO synthase (nNOS), and subsequent formation of SNO proteins. nNOS produces NO from l-arginine, and NO reacts with sulfhydryl groups to form S-nitrosylated proteins. Physiological levels of NO mediate neuroprotective effects, at least in part, by S-nitrosylating the NMDAR and caspases, thus inhibiting their activity. In contrast, we postulate that overproduction of NO can be neurotoxic via S-nitrosylation of Parkin (forming SNO-PARK), PDI (forming SNO-PDI), GAPDH, MMP-2/9, PrxII, and COX-2. S-Nitrosylated parkin and PDI contribute to neuronal cell injury by triggering accumulation of misfolded proteins. S-Nitrosylation of Drp1 (forming SNO-Drp1) causes excessive mitochondrial fragmentation in neurodegenerative conditions.

A connection between ROS/RNS and mitochondrial dysfunction in neurodegenerative diseases, especially in PD, has recently been postulated [1, 24]. Pesticides and other environmental toxins specifically inhibit mitochondrial complex I, generating excessive ROS/RNS, thus contributing to aberrant protein accumulation [9–11]. In animal models, administration of complex I inhibitors, such as MPTP, 6-hydroxydopamine, rotenone, paraquat and maneb, recapitulates many features of sporadic PD, including degeneration of dopaminergic neurons, overproduction and aggregation of α-synuclein, accumulation of Lewy body-like intraneuronal inclusions, and impairment of behavioral functions [1, 24]. Studies such as these, strongly suggest a relationship between ROS/RNS and protein misfolding. Each may be the pathogenic trigger for the other in neurodegenerative diseases, but the mechanism has not yet been determined.

Alternatively, accumulation of ROS can trigger caspase activation resulting in synaptic damage and apoptosis [22]. This process can be exacerbated by the action of endogenous “neurotoxic” electrophiles such as the prostaglandin derivative 15d–PGJ2 and catecholamine metabolites (including dopamine) [22]. Such electrophiles compromise the reductive capacity of the cell by binding reduced cysteine residues, such as in glutathione (GSH), through a reaction called S-alkylation.

3. Nitrosative stress, protein misfolding, synaptic injury, and neuronal cell death

NO participates in cellular signaling pathways that regulate broad aspects of brain function, including synaptic plasticity, normal development, and neuronal cell death [19]. These effects were thought to be largely achieved by activation of guanylate cyclase to form cyclic guanosine-3’,5’-monophosphate (cGMP), but emerging evidence suggests that a more prominent reaction of NO is S-nitros(yl)ation of regulatory protein thiol groups [4, 5, 25]. S-Nitrosylation is the covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH or, more properly, thiolate anion, RS⁻) to form an S-nitrosothiol derivative (R-SNO). Such regulatory modifications are broadly found in mammalian, plant, and microbial proteins. We and our colleagues have found that a consensus motif of nucleophilic residues (generally an acid and a base) surround a critical cysteine, increasing the susceptibility of the sulfhydryl to S-nitrosylation [26]. This process is counterbalanced by denitrosylation by means of thioredoxin/thioredoxin reductase, class III alcohol dehydrogenase, protein-disulfide isomerase (PDI), intracellular GSH, and other mechanisms. We first identified the physiological relevance of the redox-based mechanisms by which NO and related RNS exert seemingly paradoxical effects in the CNS [5]. NO is neuroprotective through S-nitrosylation of NMDA receptors and caspases, yet is neurodestructive through formation of peroxynitrite or S-nitrosylation of matrix metalloproteinase (MMP)-9, GAPDH, and other targets, as discussed below [5, 27, 28] (Fig. 2).

Accumulating evidence suggests that S-nitrosylation is analogous to phosphorylation in regulating the biological activity of many proteins [5, 9–11, 26–29]. However, the chemistry of NO is much more complex. NO is often a good “leaving group,” resulting in further oxidation of the thiol to a disulfide bond between neighboring (vicinal) cysteine residues. Alternatively, as NO “leaves” for another reaction partner, the cysteine residue can react with ROS to yield sulfenic (−SOH), sulfinic (−SO₂H) or sulfonic (−SO₃H) acid derivatives of the protein [10, 11, 27]. S-Nitrosylation may possibly produce a nitroxyl disulfide, in which the NO group is shared by proximate cysteine thiols [30].

What is the consequence of these many oxidative/nitrosative reactions? Recent evidence suggests that NO-related species may play a significant role in the process of protein misfolding. Increased nitrosative and oxidative stress are associated with chaperone and proteasomal dysfunction, resulting in accumulation of misfolded aggregates [8, 25]. However, until recently little was known regarding the molecular and pathogenic mechanisms underlying contributions of NO to the formation of aggregates such as amyloid plaques in AD or Lewy bodies in PD. We and others recently presented physiological and chemical evidence that S-nitrosylation modulates the ubiquitin E3 ligase activity of parkin [9, 10, 31]. Additionally, we found that S-nitrosylation regulates the chaperone and isomerase activities of PDI [11], contributing to protein misfolding and neurotoxicity in models of neurodegenerative disorders.

Nitrosative stress can also result in defects in mitochondrial function. For example, NO affects mitochondrial respiration by reversibly inhibiting complexes I and IV [32, 33]. Mitochondria thus compromised will release ROS, and this in turn could contribute to brain aging and/or pathological conditions associated with neurodegenerative diseases. Additionally, increased nitrosative and oxidative stress can elicit dysfunction of mitochondrial dynamics (fission and fusion events) [34–36]. Although the exact mechanism whereby NO contributes to excessive fragmentation of mitochondria remains enigmatic, our recent findings have shed light on the molecular events underlying this relationship, particularly in AD. Specifically, we have recently discovered (patho)physiological and chemical evidence that S-nitrosylation modulates the GTPase activity of the mitochondrial fission protein, Drp1 (dynamin related protein 1), thus contributing to mitochondrial fragmentation. We found that excessive mitochondrial fragmentation results in bioenergetic impairment, synaptic damage, and eventually frank neuronal loss in models of AD [37].

4. Protein misfolding and aggregation in neurodegenerative diseases

Healthy neurons generally show no accumulation of protein aggregates, indicating that the appearance of such structures is a response to pathological stresses. Considerable evidence suggests that misfolded or otherwise abnormal proteins are produced even in healthy cells. The difference can largely be accounted for by cellular mechanisms for quality control, such as molecular chaperones, the ubiquitin-proteasome system (UPS), and autophagy/lysosomal degradation. A reduction in molecular chaperone or proteasome activities can result in deposition and accumulation of aberrant proteins either within or outside of cells in the brain under pathological conditions. Several mutations in molecular chaperones or UPS-associated enzymes are known to contribute to neurodegeneration [6, 38]. For example, a reduction in proteasome activity was found in the substantia nigra of PD patients [39], and overexpression of the molecular chaperone HSP70 prevented neurodegeneration in vivo in models of PD [40]. Aggregated proteins were first considered to be pathogenic. However, recent evidence suggests that macroscopic aggregates are an attempt by the cell to sequester aberrant proteins, while soluble (micro-) oligomers of such proteins are the most toxic forms [23].

5. S-Nitrosylation of Parkin and the UPS

Studies of rare mutations have revealed key components of the mechanism for protein aggregation and pathology in PD, including sporadic forms of the disease. Such studies revealed that mutated α-synuclein is a major constituent of Lewy bodies in PD patient brains, and that mutant forms of the ubiquitin E3 ligase parkin or the ubiquitin carboxy-terminal hydrolase UCH-L1 (a deubiquinating enzyme) may result in UPS dysfunction and also result in hereditary forms of PD. Formation of polyubiquitin chains on a peptide constitutes the signal for proteasomal degradation. The cascade of activation (E1), conjugation (E2), and ubiquitin-ligase (E3)-type enzymes catalyzes the conjugation of the ubiquitin chain to the proteins marked for degradation. Individual E3 ubiquitin ligases play a key role in the recognition of specific peptide substrates [41].

Parkin is a member of a large family of E3 ubiquitin ligases. Parkin contains a total of 35 cysteine residues, many of which coordinate structurally important zinc atoms, which are often involved in catalysis [42]. Parkin recruits substrate proteins as well as an E2 enzyme (e.g., UbcH7, UbcH8, or UbcH13). Interestingly, mutations in the gene encoding parkin have been associated with Autosomal Recessive Juvenile Parkinson’s disease. In this case, mutations underlying this disorder usually do not produce Lewy bodies. However, other mutations in parkin resulting in adult onset PD have been associated with Lewy body formation. Mutations in both alleles of the parkin gene will cause dysfunction in its activity, although not all mutations result in loss of parkin E3 ligase activity [38]. Additionally, wild-type parkin can mediate the formation of non-classical and “non-degradative” lysine 63-linked polyubiquitin chains [43, 44]. Parkin can also mono-ubiquitinate Eps15, HSP70, and itself, possibly at multiple sites. These activities may explain why some parkin mutations result in the formation of Lewy bodies while others do not. Synphilin-1 (α-synuclein interacting protein) is a well-characterized substrate for parkin ubiquitination, and is found in Lewy body-like inclusions in cultured cells when co-expressed with α-synuclein. Accumulation of these proteins portends a poor prognosis for the survival of dopaminergic neurons in familial PD and possibly also in sporadic PD.

PD is the second most prevalent neurodegenerative disease and is characterized by the progressive loss of dopamine neurons in the substantia nigra pars compacta. Aberrant protein accumulation is observed in patients with genetically-encoded mutant proteins, and recent evidence from our and other laboratories suggests that nitrosative/oxidative stress acts as a potential causal factor for protein misfolding in the much more common sporadic form of PD. Nitrosative/oxidative stress can mimic hereditary PD by promoting protein misfolding in the absence of a genetic mutation [9, 10, 31]. In fact, S-nitrosylation and further oxidation of parkin result in a dysfunctional enzyme and disruption of UPS function [9, 10]. We found that nitrosative stress produces S-nitrosylation of parkin (forming SNO-parkin) in rodent models of PD and in brains of human patients with PD and the related α-synucleinopathy, DLBD (diffuse Lewy body disease). Initially, S-nitrosylation of parkin stimulates its ubiquitin E3 ligase activity, which may contribute to Lewy body formation. Subsequently, with time we found that the E3 ligase activity of SNO-parkin decreases, resulting in UPS dysfunction [10, 31]. Importantly, S-nitrosylation of parkin on critical cysteine residues also compromises its neuroprotective activity [9]. It is likely that S-nitrosylation influences the enzymatic functions of similar ubiquitin E3 ligases, suggesting that this process may be involved in a number of degenerative disorders.

6. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD and AD

In the endoplasmic reticulum (ER), PDI facilitates proper protein folding by introducing disulfide bonds into proteins (oxidation), breaking disulfide bonds (reduction), and catalyzing thiol/disulfide exchange (isomerization), thus facilitating disulfide bond formation, rearrangement reactions, and structural stability [45]. During oxidation of a target protein, oxidized PDI catalyzes disulfide formation in the substrate protein, resulting in the reduction of PDI. In contrast, the reduced form of the active-site cysteines can initiate isomerization by attacking the disulfide of a substrate protein and forming a transient intermolecular disulfide bond. As a consequence, an intramolecular disulfide rearrangement occurs within the substrate itself, resulting in the generation of reduced PDI. Several mammalian PDI homologues, such as ERp57 and PDIp, also localize to the ER and may manifest similar functions [46]. Increased expression of PDIp in neuronal cells under conditions mimicking PD suggests the possible contribution of PDIp to neuronal survival [46]. In many neurodegenerative disorders and cerebral ischemia, the accumulation of immature and denatured proteins results in ER dysfunction [46], but up-regulation of PDI represents an adaptive response promoting protein refolding and may offer neuronal cell protection [46]. We recently reported that S-nitrosylation of PDI (to form SNO-PDI) disrupts normal protein folding and this neuroprotective role [11].

The ER normally manifests a relatively positive redox potential in contrast to the highly reducing environment of the cytosol and mitochondria. This redox environment in the ER can influence the stability of protein S-nitrosylation and oxidation reactions [47]. Excessive NO is known to create ER stress by disruption of Ca²⁺ homeostasis. One possible mechanism is the increased activity of the ER Ca²⁺ channel-ryanodine receptor through S-nitrosylation [48]. Interestingly, we have recently reported that excessive NO, as well as rotenone exposure, which is known to lead to PD, can lead to S-nitrosylation of the active-site thiols of PDI, inhibiting its isomerase and chaperone activities [11]. S-Nitrosylation of PDI prevented its attenuation of neuronal cell death triggered by ER stress, misfolded proteins, or proteasome inhibition. Also, PDI was S-nitrosylated in the brains of virtually all cases we examined of sporadic AD and PD. These results suggest that SNO-PDI can mediate protein misfolding and consequent neuronal cell death or injury.

The activity of the UPS and molecular chaperones normally decline with age [49]. Since we have not found detectable levels of SNO-parkin and SNO-PDI in normal aged brain but only in disease states [9–11], it is likely that S-nitrosylation of these and similar proteins is a key mechanism contributing to neurodegenerative conditions.

7. Mitochondrial fission/fusion machinery in nerve cells

Neurons are particularly vulnerable to mitochondrial defects because they require high levels of energy for their survival and specialized function. Mitochondrial biogenesis, an event that produces new mitochondria, is required in regions that demand high concentrations of ATP, especially the synapse. The distribution of mitochondria at the nerve terminal can control synaptic transmission and structure [50–52].

In healthy neurons, the fission/fusion machinery proteins maintain mitochondrial integrity and insure their presence at critical locations. These proteins includes Drp1 and Fis1, acting as fission proteins, and Mitofusin (Mfn) and Opa1, operating as fusion proteins [53]. In both familial and sporadic neurodegenerative conditions, abnormal mitochondria regularly appear in the brain as a result of dysfunction in the fission/fusion machinery. Genetic mutations in Mfn2 can cause Charcot-Marie-Tooth (CMT) disease, a hereditary peripheral neuropathy that affects both motor and sensory neurons [54, 55]. Mutations in Opa1 cause Autosomal Dominant Optic Atrophy (ADOA), characterized by the loss of retinal ganglion cells and the optic nerve, representing their axons [56]. Recently, Waterham et al. described a heterozygous, dominant-negative mutation of Drp1 in a patient whose symptoms were broadly similar to those of CMT neuropathy and ADOA [57]. Drp1 includes four distinct structural domains: an N-terminal GTPase domain, a dynamin-like middle domain, an insert B domain, and a C-terminal GED domain. The mutation (Ala 395 to Asp) was found in the middle domain of Drp1. This case study further suggested that a defect in mitochondrial fission may have more severe consequences than those of fusion defects, since the Drp1 mutation caused a much earlier onset (prenatal) and fatal outcome. Additionally, it is apparent that the balance between fission and fusion is critical for normal function of mitochondria and determination of phenotype in disease. These fission/fusion proteins are widely expressed in human tissues, clearly supporting the notion that neurons are particularly sensitive to mitochondrial dysfunction.

8. S-Nitrosylation of Drp1 mediates mitochondrial fission and neurotoxicity in cell models of AD

Recent studies have demonstrated that posttranslational modification of mitochondrial fission or fusion proteins can contribute to altered mitochondria dynamics. For example, phosphorylation, ubiquitination, sumoylation, and proteolytic cleavage of Drp1 regulate mitochondrial fission by affecting Drp1 activity, at least in cell culture systems [58–65]. Excessive activation of mitochondrial fission or fusion proteins by posttranslational modification was posited to contribute to neurodegeneration by compromising mitochondrial function. Interestingly, along these lines, we recently reported that excessive NO can lead to S-nitrosylation of Drp1 at Cys644 [37]. Cys644 resides within the GTPase effector domain (GED) of Drp1, which influences both GTPase activity and oligomer formation of Drp1 [66–69]. S-Nitrosylation of Drp1 (forming SNO-Drp1) induces formation of Drp1 dimers, which function as building blocks for tetramers and higher order structures of Drp1, and activates Drp1 GTPase activity; however, substitution of Cys644 to Ala (C644A) abrogated these effects of NO.

We further demonstrated that exposure to oligomeric Aβ peptide results in formation of SNO-Drp1 in cell culture models. Moreover, we and our colleagues have observed that Drp1 is S-nitrosylated in the brains of virtually all cases of sporadic AD [37, 70]. In order to determine the consequences of S-nitrosylated Drp1 in neurons, we exposed cultured cerebrocortical neurons to the physiological NO donor, S-nitrosocysteine (SNOC), or to Aβ oligomers and found that both induced SNO-Drp1 formation and led to the accumulation of excessively fragmented mitochondria. Moreover, mutation of a specific cysteine residue in Drp1 (C644A) prevented these effects of SNOC or Aβ on mitochondrial fragmentation, consistent with the notion that SNO-Drp1 triggered excessive mitochondria fission or fragmentation. Finally, in response to Aβ, SNO-Drp1—induced mitochondrial fragmentation caused synaptic damage, an early characteristic feature of AD, and eventually apoptotic neuronal cell death. Importantly, blockade of Drp1 nitrosylation (using the Drp1(C644A) mutant) prevented Aβ-mediated synaptic loss and neuronal cell death, suggesting that SNO-Drp1 may represent a potential therapeutic target to protect neurons and their synapses in AD. Thus, the posttranslational modification of S-nitrosylation can mimic the effect of rare genetic mutations in contributing to the AD phenotype.

9. Potential protection of neurons from NMDAR-induced excessive Ca²⁺ and oxidative/nitrosative stress

9.1 Memantine and Derivatives

Oxidative/nitrosative stress mediates, at least in part, glutamate-induced neuronal cell injury and death, and several antioxidant molecules have been reported to protect neurons against such assaults [15, 71]. One mechanism that could potentially curtail excessive Ca²⁺ influx and the resultant formation of neurotoxic ROS and RNS is inhibition of NMDA receptors. Until recently, however, drugs in this class (e.g., MK-801) blocked virtually all NMDA receptor activity, including physiological activity, and therefore manifested unacceptable side effects by inhibiting normal functions of the receptor. For this reason, many previous NMDA receptor antagonists have disappointingly failed in advanced clinical trials conducted for a number of neurodegenerative disorders. However, In contrast, we have demonstrated that the adamantane derivative, memantine, preferentially blocks excessive (pathological/extrasynaptic) NMDA receptor activity while relatively sparing normal (physiological/synaptic) activity [14]. Memantine effectively blocks only excessively open channels through an uncompetitive mechanism of action in conjunction with a relatively fast off-rate, resulting in a low affinity for the NMDA receptor. Despite its low affinity for the NMDA receptor, memantine is still relatively selective for this receptor. An uncompetitive antagonist can be distinguished from a noncompetitive antagonist, which acts allosterically at a noncompetitive site, i.e., at a site other than the agonist-binding site. Additionally, the action of an uncompetitive antagonist is contingent upon prior activation of the receptor by the agonist. Hence, a fixed low-dose of memantine will block higher concentrations of agonist to a relatively greater degree than lower concentrations of agonist, providing greater protection when more glutamate is present. Furthermore, the apparent affinity of a drug is determined by the ratio of its “off-rate” divided by its “on-rate” for the target. The on-rate is not only a property of drug diffusion and interaction with the target, but is also influenced by the drug’s concentration. In contrast, the off-rate is an intrinsic property of the drug-receptor complex and diffusion, unaffected by drug concentration. A relatively fast off-rate is a major contributor to memantine’s low affinity for the NMDA receptor as well as its clinical tolerability because this property insures that once excessive activity is normalized, the drug will leave the channel and not disrupt subsequent physiological neurotransmission. Thus, the critical features of memantine’s mode of action are its Uncompetitive mechanism and Fast Off-rate, or what we call a UFO drug – a drug that is present at its site of inhibitory action only when you need it and then quickly disappears (Fig. 3). Many studies in vitro and in animal models of stroke and neurodegenerative disease showed that memantine protects neurons from NMDA receptor-mediated excitotoxic damage [72]. In fact, memantine has been approved for human use by the FDA for moderate-to-severe AD and is currently being studied for other neurodegenerative disorders, including HIV-associated dementia and Huntington’s disease.

Fig. 3.

Memantine and NitroMemantine preferentially block excessive extrasynaptic NMDA receptor activity, while relatively sparing synaptic receptors. (Top) Normal (physiological/synaptic) activity of the NMDAR is required for synaptic function and neuronal survival. Many NMDAR antagonists, such as MK-801, completely block receptor activity, including physiological synaptic activity, and thus result in severe side effects and clinical intolerability. (Bottom) Excessive activation of the NMDAR, predominantly at extrasynaptic sites, is thought to induce neuronal cell injury and death, and is associated with the accumulation of misfolded proteins. Memantine (Mem) and the newer NitroMemantine drugs (NitroMem) preferentially block excessive (pathological) extrasynaptic NMDA receptor activity, while relatively sparing normal (physiological) synaptic activity [14].

Therapeutic results with memantine are meaningful, but as with most first generation drugs, improvements are possible through creation of derivatives. The reaction of NO with the sulfhydryl groups of critical cysteine residues of the NMDA receptor, especially C399 on the external domain of the NR2A subunit, down-regulates (but does not completely shut off) receptor activity [3, 12]. Therefore, we have developed combinatorial drugs called NitroMemantines that use memantine to target NO to the nitrosylation sites of the NMDA receptor in order to avoid the systemic side effects of NO, while still utilizing the uncompetitive channel blocking properties of memantine. Preliminary studies show NitroMemantines to be more effective neuroprotectants in vitro and in animal models than memantine and at lower concentrations [3]. Though much research remains to be done on these second generation NitroMemantine drugs, the combination of memantine with NO has created a new, improved class of UFO drugs that should be both clinically tolerated and neuroprotective.

9.2 Electrophiles

A complementary approach is needed to deal with residual oxidative stress triggered by excessive calcium influx or other mechanisms. One strategy for finding neuroprotective drugs is to search for low-molecular-weight compounds that can regulate the redox state of the cell and thereby block oxidative damage [73]. We and others found electrophilic compounds that transcriptionally induce protective antioxidative enzymes in neurons [74, 75]. The activity of these compounds is attained by inducing the transcription of so-called “phase 2 enzymes,” such as heme oxygenase-1 (HO-1) and sulfiredoxin, that regulate the intracellular redox state of the cell [73, 76, 77]. The Keap1/Nrf2 transcriptional pathway is key in regulating the activity of phase 2 enzymes. Keap1 facilitates ubiquitination of Nrf2, but electrophiles or NO reacting with a critical cysteine on Keap1 can cause the dissociation Nrf2 from Keap1. Nrf2 thus stabilized can translocate to the nucleus where it binds to the antioxidant-responsive element (ARE) in upstream promoters of phase 2 enzymes to activate their transcription [76–78].

The specific nature of the electrophile is important. Chemically, enones, such as curcumin [79] and neurite outgrowth-promoting prostaglandin (NEPP11) [75], are true electrophiles. Some enone-type neuroprotective electrophilic compounds that we have studied, such as NEPP11, accumulate in neurons, exerting a direct protective action through induction of HO-1 via the Keap1/Nrf2 pathway [75]. In contrast, however, we advocate the use of catechols that only become electrophiles upon oxidative conversion to quinones [80]. Thus, catechol-type compounds can function as prodrugs that exert their effects only under oxidative conditions [73, 81]. In this manner, catechols, unlike enones, avoid reaction with other cysteine groups, e.g., those on glutathione, that could paradoxically decrease the antioxidative capacity of the cell. Some catechol-type neuroprotective pro-electrophilic compounds act preferentially in astrocytes, protecting neurons by a paracrine mechanism [74, 82]. Carnosic acid (CA) is a naturally occurring catechol-type poly-phenolic diterpene obtained from Rosmarinus officinalis (the herb rosemary) [83]. Prior work had suggested that CA could exert free radical-scavenging activity [84], but we have recently found that CA, after being converted by oxidative stress from a catechol/pro-electrophile into a true quinone/electrophile, exerts its primary action by activating the Keap1/Nrf2 pathway at sites of oxidative insult [85]. In Parkinson’s disease, oxidative stress has a crucial role in disease progression [86] but also can be used to activate such pro-electrophilic compounds to provide neuroprotection where it is needed. This approach represents a novel strategy against neurodegenerative disorders in which pro-electrophilic drugs are activated to electrophiles through pathological oxidative activity.

10. Conclusions

Sporadic forms of neurodegenerative diseases can be caused by excessive NMDA receptor activation and/or mitochondrial dysfunction that results in excessive nitrosative and oxidative stress. These pathological processes can result in malfunction of the UPS and/or molecular chaperones and contribute to abnormal protein accumulation and neuronal damage. Here we have described a mechanistic link between free radical production, abnormal protein accumulation, and neuronal cell injury in neurodegenerative disorders such as PD and AD. The elucidation of NO-mediated S-nitrosylation of parkin, PDI, and Drp1 in neurodegenerative disease may promote development of new drugs to prevent aberrant protein misfolding or excessive mitochondrial fission by targeted disruption of this process. Additionally, we describe the action of new memantine derivatives, NitroMemantines, that not only address the excitotoxicity damage caused by excessive Ca²⁺ influx via uncompetitive antagonism of the NMDA receptor, but also through their abilities to S-nitrosylate the NMDA receptor. We have also discovered a new class of electrophilic drugs that activate the endogenous protective mechanisms of the cell in response to oxidative stress. Both of these types of drugs, memantine and electrophiles, are preferentially activated by pathological conditions while being relatively inert during normal homeostasis of the cell. Thus, these next generation CNS drugs should be better tolerated clinically, making them both safe and effective.