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Keywords:

  • aging brain;
  • molecular chaperone;
  • Parkinson's disease;
  • protein-disulfide isomerase;
  • S-nitrosylation;
  • ubiquitination

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

Glutamatergic hyperactivity, associated with Ca2+ influx and consequent production of nitric oxide (NO), is potentially involved in both normal brain aging and age-related neurodegenerative disorders. Many neurodegenerative diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Normal protein degradation by the ubiquitin-proteasome system can prevent accumulation of aberrantly folded proteins. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. In particular, molecular chaperones – such as protein disulfide isomerase, glucose regulated protein 78, and heat shock proteins – can provide neuroprotection from misfolded proteins by facilitating proper folding and thus preventing aggregation. Here, we present evidence for the hypothesis that NO contributes to normal brain aging and degenerative conditions by S-nitrosylating specific chaperones that would otherwise prevent accumulation of misfolded proteins.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

Inappropriate activation of signaling pathways that lead to neuronal cell injury and death is thought to be a major contributor to brain aging and neurodegenerative disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), polyglutamine (polyQ) diseases such as Huntington's disease, glaucoma, human immunodeficiency virus-associated dementia, multiple sclerosis, and ischemic brain injury, to name but a few. While many extracellular molecules and neurotransmitters may participate in neuronal damage, glutamate appears to be a principal factor contributing to what has been termed excitotoxicity (Olney, 1969; Olney et al., 1997). Toxicity evoked by glutamate is mediated at least in part by excessive activation of N-methyl-d-aspartate (NMDA)-type receptors with consequent Ca2+ influx (Lipton & Rosenberg, 1994). The intracellular Ca2+ triggers the generation of nitric oxide (NO) by activating neuronal NO synthase (nNOS) in a Ca2+/calmodulin (CaM)-dependent manner (Garthwaite et al., 1988; Bredt et al., 1991). Accumulation of nitrosative stress due to excessive generation of reactive nitrogen species (RNS) like NO is thought to play a causal role in neuronal cell damage and death, and therefore is recognized as a potential mediator of both aging and neurodegenerative disease in the brain. Moreover, additional NOS isoforms generate NO in a variety of other cell types. It has recently been proposed that mitochondrial cytochrome oxidase can also produce NO in a nitrite (inline image)- and pH-dependent but non-Ca2+-dependent manner (Castello et al., 2006). Importantly, normal mitochondrial respiration also generates free radicals, principally reactive oxygen species (ROS), and one such molecule, superoxide anion (O2) reacts rapidly with free radical NO• to form the very toxic product peroxynitrite (ONOO) (Beckman et al., 1990; Lipton et al., 1993). Notably, it has been postulated that the increasing burden of free radical production during the lifespan may mediate ‘normal’ brain aging as well as render the aged brain more susceptible to neurodegenerative disorders (reviewed in Finkel & Holbrook, 2000).

A common sign of many neurodegenerative diseases is the accumulation of misfolded proteins that adversely affect neuronal connectivity and plasticity and trigger cell death signaling pathways (Bence et al., 2001; Muchowski & Wacker, 2005). For example, degenerating brain contains aberrant accumulations of misfolded, aggregated proteins, such as α-synuclein and synphilin-1 in PD, and amyloid-β (Aβ) and tau in AD. Other diseases with inclusions include Huntington's, ALS, and prion diseases (Ciechanover & Brundin, 2003). Molecular chaperones are believed to provide a defense mechanism against the toxicity of misfolded proteins because chaperones can prevent inappropriate interactions within and between polypeptides and can promote refolding of proteins that have been misfolded because of cell stress. In addition to the quality control of proteins provided by molecular chaperones, the ubiquitin-proteasome system (UPS) is involved in the clearance of abnormal or aberrant proteins. When chaperones cannot repair misfolded proteins, they may be tagged via addition of polyubiquitin chains for degradation by the proteasome. In neurodegenerative conditions, intra- or extracellular protein aggregates are thought to accumulate in the brain as a result of a decrease in molecular chaperone or proteasome activities. Currently, soluble oligomers of these aberrant proteins are thought to be the most toxic forms via interference with normal cell activities, while frank aggregates may be an attempt by the cell to wall off potentially toxic material (Arrasate et al., 2004). Interestingly, neurotoxicity elicited by some aberrant proteins, such as Aβ1−42 peptide in AD, can augment NMDA-type glutamate receptor activity and intracellular Ca2+ levels (reviewed in Lipton, 2006). Furthermore, increased free Ca2+ levels and consequent nitrosative stress are associated with chaperone and proteasomal dysfunction, resulting in accumulation of misfolded aggregates (Isaacs et al., 2006; Zhang & Kaufman, 2006). However, until recently little was known regarding the molecular mechanism underlying the contribution of Ca2+ and NO to the formation of protein aggregates such as amyloid plaques in AD or Lewy bodies in PD.

A key theme of this article is the hypothesis that malfunction of Ca2+-mediated glutamatergic signaling pathways, which generate NO and ROS, contributes to both normal aging and neurodegeneration in the brain. In this review, we discuss specific examples showing that (i) oxidative/nitrosative stress is linked to brain aging, and (ii) S-nitrosylation of (a) ubiquitin E3 ligases such as parkin or (b) endoplasmic reticulum chaperones such as protein-disulfide isomerase (PDI) is critical for the accumulation of misfolded proteins in neurodegenerative diseases such as PD, AD and other conditions (Chung et al., 2004; Yao et al., 2004; Lipton et al., 2005; Uehara et al., 2006).

NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

It is well known that the amino acid glutamate is the major excitatory neurotransmitter in the brain. Glutamate is present in high concentrations in the adult central nervous system and is released for milliseconds from nerve terminals in a Ca2+-dependent manner. After glutamate enters the synaptic cleft, it diffuses across the cleft to interact with its corresponding receptors on the post-synaptic face of an adjacent neuron. Excitatory neurotransmission is necessary for the normal development and plasticity of synapses, and for some forms of learning or memory; however, excessive activation of glutamate receptors is implicated in neuronal damage in many neurological disorders ranging from acute hypoxic-ischemic brain injury to chronic neurodegenerative diseases. It is currently thought that overstimulation of extrasynaptic NMDA receptors mediate this neuronal damage, while, in contrast, synaptic activity may activate survival pathways (Hardingham et al., 2002; Papadia et al., 2005). Intense hyperstimulation of excitatory receptors leads to necrotic cell death, but more mild or chronic overstimulation can result in apoptotic or other forms of cell death (Ankarcrona et al., 1995; Bonfoco et al., 1995; Budd et al., 2000).

There are two large families of glutamate receptors in the nervous system, ionotropic receptors (representing ligand-gated ion channels), and metabotropic receptors (coupled to G-proteins). Ionotropic glutamate receptors are further divided into three broad classes, NMDA receptors, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, and kainate receptors, which are each named after synthetic ligands that can selectively activate these receptors. The NMDA receptor has attracted attention for a long period because it has several properties that set it apart from other ionotrophic glutamate receptors. One such characteristic, in contrast to most AMPA and kainate receptors, is that NMDA receptor-coupled channels are highly permeable to Ca2+, thus permitting Ca2+ entry after ligand binding if the cell is depolarized in order to relieve block of the receptor-associated ion channel by Mg2+ (Mayer et al., 1984; Nowak et al., 1984). Subsequent binding of Ca2+ to various intracellular molecules can lead to many significant consequences. In particular, excessive activation of NMDA receptors leads to the production of damaging free radicals (e.g., NO and ROS) and other enzymatic processes, contributing to cell death (Dawson et al., 1991; Lafon-Cazal et al., 1993; Lipton et al., 1993; Lipton & Rosenberg, 1994; Bonfoco et al., 1995; Budd et al., 2000).

Ca2+ influx and generation of NO

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

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 (Bredt et al., 1991; Abu-Soud & Stuehr, 1993) (Fig. 1). NO is a key molecule that plays a vital role in normal signal transduction but in excess can lead to neuronal cell damage and death. Three subtypes of NOS have been identified; two constitutive forms of NOS – nNOS and endothelial NOS (eNOS) – take their names from the cell type in which they were first found. The name of the third subtype – inducible NOS (iNOS) – indicates that expression of the enzyme is induced by acute inflammatory stimuli. All three isoforms are widely distributed in the brain. Each NOS isoform contains an oxidase domain at its amino-terminal end and a reductase domain at its carboxy-terminal end, separated by a Ca2+/CaM binding site (Bredt et al., 1991; Abu-Soud & Stuehr, 1993; Förstermann et al., 1998; Boucher & Moali, 1999; Groves & Wang, 2000). Constitutive and iNOS are also further distinguished by CaM binding: nNOS and eNOS bind CaM in a reversible Ca2+-dependent manner. In contrast, iNOS binds CaM so tightly at resting intracellular Ca2+ concentrations that its activity does not appear to be affected by transient variations in Ca2+ concentration.

image

Figure 1. Activation of the N-methyl-d-aspartate (NMDA) receptor by glutamate (Glu) and glycine (Gly) induces Ca2+ influx and consequent nitric oxide (NO) production via activation of neuronal NO synthase (nNOS). nNOS localizes to a complex attached to the NR1 subunit of the NMDA receptor via PDZ-domain binding to post-synaptic density protein (PSD-95). Subsequent effects of NO are mediated by chemical, enzymatic, and redox reactions within neurons. Specific interaction of NO with soluble guanylate cyclase (sGC) results in the production of cyclic guanosine monophosphate (cGMP), and cGMP could activate cGMP-dependent protein kinase to mediate the NO signaling. Excessive NMDA receptor activity, leading to the overproduction of NO can be neurotoxic. For example, S-nitrosylation of proteins such as protein disulfide isomerase (PDI) and parkin can contribute to neuronal cell damage and death. Neurotoxic effects of NO are also mediated by peroxynitrite (ONOO), a reaction product of NO and superoxide anion (inline image).

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Recently, a novel cellular mechanism for Ca2+-dependent release of NO was discovered in dorsal root ganglion neurons and pancreatic acinar cells. This Ca2+-dependent NO release occurs not as a result of de novo synthesis by NO but instead via liberation of NO from an S-nitrosothiol (SNO) pool, whereby NO is reversibly bound to specific cysteine residues (see below for additional chemical information regarding this reaction). Interestingly, NOS-independent release of NO was mediated by calpain (a Ca2+-dependent thiol protease) but not by CaM or protein kinase C (PKC) (Chvanov et al., 2006). This finding leads to the question of whether glutamatergic signaling in neuronal cells can also trigger NO release from SNO pools.

Regulation of brain function by NO

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

Early investigations indicated that NO plays a role in several aspects of brain function, including normal development, synaptic plasticity, and neuronal cell death (Dawson et al., 1991; O'Dell et al., 1991; Bredt & Snyder, 1994; Schuman & Madison, 1994). Physiological and some pathophysiological functions of NO are mediated in general via stimulation of guanylate cyclase to form cyclic guanosine-3′,5′-monophosphate (cGMP) or through S-nitrosylation of regulatory protein thiol groups (Garthwaite et al., 1988; Kandel & O'Dell, 1992; Lei et al., 1992; Stamler et al., 1992a; Lipton et al., 1993; Bredt & Snyder, 1994). S-nitros(yl)ation is defined as transfer of an NO group to a critical cysteine thiol/sulfhydryl (RSH or, more properly, thiolate anion, RS) to form an S-nitrosothiol derivative (R-SNO), which modulates protein function. Our group first identified the physiological relevance of S-nitrosylation by showing that NO and related RNS exert paradoxical effects via redox-based mechanisms – NO is neuroprotective via S-nitrosylation of NMDA receptors (as well as other subsequently discovered targets, including caspases), and yet can also be neurodestructive by formation of peroxynitrite (or, as later discovered, reaction with additional molecules such as glyceraldehyde 3-phosphate dehydrogenase) (Lipton et al., 1993; Dimmeler et al., 1997; Melino et al., 1997; Tenneti et al., 1997; Kim et al., 1999; Mannick et al., 1999; Choi et al., 2000; Hara et al., 2005). Over the past decade, accumulating evidence has suggested that S-nitrosylation can regulate the biological activity of a great variety of proteins, in some ways akin to phosphorylation (Lipton et al., 1993; Jaffrey et al., 2001; Gu et al., 2002; Haendeler et al., 2002; Chung et al., 2004; Yao et al., 2004; Hara et al., 2005; Sliskovic et al., 2005) (for reviews, see Stamler et al., 1992b; Stamler, 1994; Stamler et al., 2001; Lipton et al., 2002; Hess et al., 2005). Chemically, NO is often a good ‘leaving group’, facilitating further oxidation of critical thiol to disulfide bonds among neighboring (vicinal) cysteine residues or, via reaction with ROS, to sulfenic (–SO), sulfinic (–inline image) or sulfonic (–inline image) acid derivatization of the protein (Stamler & Hausladen, 1998; Gu et al., 2002; Yao et al., 2004; Uehara et al., 2006).

NO and the free radical theory of aging

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

In the mid-1950s, Denham Harman proposed a ‘free-radical theory’ of aging, speculating that excess amounts of free radicals (now known to include ROS and RNS) are generated in cells, and lead to a pattern of cumulative damage (Harman, 1956). More recent studies have suggested that deviation from the normal, controlled production of NO could be a factor in structural damage and functional impairment during brain aging (McCann, 1997). Indeed, some evidence for the increased activity of iNOS in aged brains has been reported (McCann et al., 1998; Vernet et al., 1998). However, controversial results have been obtained concerning nNOS; both increased and decreased activities have been reported in the aging brain (Mollace et al., 1995; Chalimoniuk & Strosznajder, 1998). A possible explanation for this is that nNOS activity varies in different regions of the brain during aging and associated neurodegenerative disorders such as AD, similar to the manner in which NMDA receptors have been reported to vary. One way that nitrosative stress could affect normal brain aging is through defects in mitochondrial function. NO, which in part mediates acute glutamate neurotoxicity, affects mitochondrial respiration by reversibly inhibiting complex IV (cytochrome c oxidase) (Cleeter et al., 1994; Clementi et al., 1998; Finkel & Holbrook 2000). Dysfunctional mitochondria may release ROS, probably via complexes I and III, and this in turn could possibly contribute to brain aging (Moncada & Erusalimsky, 2002). Additionally, such defects in mitochondrial function could be super-imposed on existing genetic predispositions, resulting in a relative excess of ROS/RNS in aging cells with potential pathological consequences. Therefore, glutamatergic signaling with consequent generation of NO may be implicated both in physiological aging and in pathological conditions associated with advancing age.

S-Nitrosylation and neurodegeneration

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

Analyses of mice deficient in either nNOS or iNOS confirmed that NO is an important mediator of cell injury and death after excitotoxic stimulation; NO generated from nNOS or iNOS is detrimental to neuronal survival (Huang et al., 1994; Iadecola et al., 1997). In addition, inhibition of NOS activity ameliorates the progression of disease pathology in animal models of PD, AD, and ALS, suggesting that excess generation of NO plays a pivotal role in the pathogenesis of several neurodegenerative diseases (Hantraye et al., 1996; Przedborski et al., 1996; Chabrier et al., 1999; Liberatore et al., 1999). Although the involvement of NO in neurodegeneration has been widely accepted, the chemical relationship between nitrosative stress and accumulation of misfolded proteins has remained obscure. However, recent findings have shed light on molecular events underlying this relationship. Specifically, we and others recently mounted physiological and chemical evidence that S-nitrosylation modulates the (i) ubiquitin E3 ligase activity of parkin (Chung et al., 2004; Yao et al., 2004; Lipton et al., 2005), and (ii) chaperone and isomerase activities of PDI (Uehara et al., 2006), contributing to protein misfolding and neurotoxicity in models of neurodegenerative disorders. Additionally, Cohen et al. (2006) recently demonstrated that insulin/insulin-like growth factor-1 (IGF-I) signaling, which influences longevity and lifespan in many species in part via downregulation of ROS/RNS generation, can affect aggregation of toxic proteins such as Aβ. This finding potentially provides an additional link between ROS/RNS production during the normal aging process and protein aggregation in neurodegenerative conditions.

Nitrosative stress impairs protein ubiquitination in PD

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

Ubiquitinated inclusion bodies are the hallmark of many neurodegenerative disorders. Age-associated defects in intracellular proteolysis of misfolded or aberrant proteins might lead to accumulation and ultimately deposition of aggregates within neurons or glial cells. Although such aberrant protein accumulation had been observed in patients with genetically encoded mutant proteins, recent evidence from our laboratory suggests that nitrosative and oxidative stress are potential causal factors for protein accumulation in the much more common sporadic form of PD. As illustrated below, nitrosative/oxidative stress, commonly found during normal aging, can mimic rare genetic causes of disorders, such as PD, by promoting protein misfolding in the absence of a genetic mutation (Chung et al., 2004; Yao et al., 2004; Lipton et al., 2005). For example, S-nitrosylation and further oxidation of parkin, a ubiquitin E3 ligase, or UCH-L1, a de-ubiquitinating enzyme that recycles ubiquitin, results in dysfunction of these enzymes and thus of the UPS (Nishikawa et al., 2003; Choi et al., 2004; Chung et al., 2004, 2005; Yao et al., 2004; Gu et al., 2005). We and others recently discovered that nitrosative stress triggers S-nitrosylation of parkin (forming SNO-parkin) not only in rodent models of PD but also in the brains of human patients with PD and Lewy bodies. SNO-parkin initially stimulates ubiquitin E3 ligase activity, resulting in enhanced ubiquitination as observed in Lewy bodies, followed by a decrease in enzyme activity, producing a futile cycle of dysfunctional UPS (Yao et al., 2004; Lim et al., 2005; Lipton et al., 2005). Additionally, S-nitrosylation appears to compromise the neuroprotective effect of parkin (Chung et al., 2004). It is likely that other ubiquitin E3 ligases with similar RING finger thiol motifs are S-nitrosylated in a similar manner to affect their enzymatic function; hence, S-nitrosylation of E3 ligases may be involved in a number of degenerative conditions.

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

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

The endoplasmic reticulum (ER) normally participates in protein processing and folding but undergoes a stress response when immature or misfolded proteins accumulate (Andrews & Johnson, 1996; Sidrauski et al., 1998; Ellgaard et al., 1999). ER stress stimulates two critical intracellular responses (Fig. 2). The first represents expression of chaperones that prevent protein aggregation via the unfolded protein response (UPR), and is implicated in protein re-folding, post-translational assembly of protein complexes, and protein degradation. This response is believed to contribute to adaptation during altered environmental conditions, promoting maintenance of cellular homeostasis. Another ER response involves attenuation of protein synthesis via eukaryotic initiation factor-2 (eIF2) kinase (Kaufman, 1999; Mori, 2000; Patil & Walter, 2001). Additionally, although severe ER stress can induce apoptosis, the ER withstands relatively mild insults via expression of stress proteins such as glucose-regulated protein (GRP) and protein-disulfide isomerase. These proteins behave as molecular chaperones that assist in the maturation, transport, and folding of secretory proteins. During protein folding in the ER, PDI catalyzes thiol/disulfide exchange, thus facilitating disulfide bond formation, re-arrangement reactions, and structural stability (Lyles & Gilbert, 1991). PDI has two domains that are homologous to the small, redox-active protein thioredoxin (TRX) (Edman et al., 1985). The two-thiol/disulfide centers of these thioredoxin-like domains function as independent active sites (Vuori et al., 1992). In neurodegenerative disorders and cerebral ischemia, the accumulation of immature and denatured proteins results in ER dysfunction (Hu et al., 2000; Conn et al., 2004; Rao & Bredesen, 2004; Atkin et al., 2006), but up-regulation of PDI represents an adaptive response promoting protein refolding and may offer neuronal cell protection (Tanaka et al., 2000; Ko et al., 2002; Conn et al., 2004; Hetz et al., 2005).

image

Figure 2. Possible mechanism of S-nitrosylated protein disulfide isomerase (SNO-PDI) contributing to the accumulation of aberrant proteins and neuronal cell damage or death. Endoplasmic reticulum (ER) stress is triggered when misfolded proteins accumulate within the ER lumen, inducing the unfolded protein response (UPR). The UPR is usually a transient homeostatic mechanism for cell survival, while prolonged UPR elicits neuronal cell death. Glucose regulated protein (GRP) 78 and PDI modulate the activity of UPR sensors, such as IRE1 and ATF6, by mediating proper protein folding. Activation of IRE1 induces splicing of XBP1 mRNA, resulting in increased stability of the subsequently translated Xbp-1 transcription factor (Yoshida et al., 2001). This spliced isoform of Xbp-1 protein up-regulates transcription of chaperones and cochaperones, as well as genes involved in protein degradation, to maintain ER function (Szegezdi et al., 2006). The basic leucine-zipper transcription factor ATF6 is involved, at least in part, in the induction of C/EBP-homologous protein (CHOP), which regulates the transition from pro-survival to pro-apoptotic signaling during ER stress. Proteins that fail to attain their native folded state are eventually retro-translocated across the ER membrane to be disposed of by cytosolic proteasomes. This process, known as ER-associated degradation (ERAD), is essential in preventing protein accumulation and aggregation in the ER. Under conditions of severe nitrosative stress, S-nitrosylation of neuronal PDI inhibits normal protein folding in the ER, activates ER stress, and induces a prolonged UPR, thus contributing to protein aggregation and cell damage or death. S-nitrosylation of one (of two) thioredoxin domains of PDI is shown for simplicity, resulting in SNO-PDI or possibly nitroxyl-PDI, as described in Uehara et al. (2006) and Forrester et al. (2006).

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Recently, we reported that excessive NO can lead to S-nitrosylation of the active site thiol groups of PDI, and this reaction inhibits both its isomerase and chaperone activities (Uehara et al., 2006). Moreover, we found that PDI is S-nitrosylated in the brains of AD and PD patients. Under pathological conditions, it is possible that both cysteine sulfhydryl groups in the TRX-like domains of PDI form S-nitrosothiols. Unlike formation of a single S-nitrosothiol, which is commonly seen after de-nitrosylation reactions catalyzed by PDI (Sliskovic et al., 2005), dual nitrosylation may be relatively more stable and prevent subsequent disulfide formation on PDI. Therefore, we speculate that these pathological S-nitrosylation reactions on PDI are more easily detected during neurodegenerative conditions. In order to determine the consequences of S-nitrosylated PDI (SNO-PDI) formation in neurons under pathological conditions, we exposed cultured cerebrocortical neurons to neurotoxic concentrations of NMDA, thus inducing excessive Ca2+ influx and NO production. Under these conditions, we found that PDI was S-nitrosylated in a NOS-dependent manner. SNO-PDI formation led to the accumulation of poly-ubiquitinated/misfolded proteins and activation of the UPR. Moreover, S-nitrosylation abrogated the inhibitory effect of PDI on aggregation of proteins observed in Lewy body inclusions (Chung et al., 2001; Uehara et al., 2006). S-nitrosylaton of PDI also prevented its attenuation of neuronal cell death triggered by ER stress, misfolded proteins, or proteasome inhibition (Fig. 2). Further evidence suggests that PDI may transport NO to the extracellular space, where it could conceivably exert additional adverse effects (Sliskovic et al., 2005). Additionally, NO can possibly mediate cell death or injury via S-nitrosylation or nitration reactions on other TRX-like proteins, such as TRX itself and glutaredoxin (Haendeler et al., 2002; Aracena-Parks et al., 2006; Tao et al., 2006).

In addition to PDI, S-nitrosylation is likely to affect critical thiol groups on other chaperones, such as HSP90 in the cytoplasm (Martínez-Ruiz et al., 2005) and possibly GRP in the ER. Normally, HSP90 stabilizes misfolded proteins and modulates the activity of cell signaling proteins including NOS and calreticulin (Muchowski & Wacker, 2005). In AD brains, levels of HSP90 are increased in both the cytosolic and membranous fractions, where HSP90 is thought to maintain tau and Aβ in a soluble conformation, thereby averting their aggregation (Kakimura et al., 2002; Dou et al., 2003). Martínez-Ruiz et al. (2005) recently demonstrated that S-nitrosylation of HSP90 can occur in endothelial cells, and this modification abolishes its ATPase activity, which is required for its function as a molecular chaperone. These studies imply that S-nitrosylation of HSP90 in neurons of AD brains may contribute to the accumulation of tau and Aβ aggregates.

The UPS is apparently impaired in the aging brain; additionally, inclusion bodies similar to those found in neurodegenerative disorders can appear in brains of normal aged individuals or those with subclinical manifestations of disease (Gray et al., 2003). These findings suggest that the activity of molecular chaperones and the UPS may decline in an age-dependent manner (Paz Gavilan et al., 2006). Given that SNO-parkin and SNO-PDI do not exist in detectable quantities in normal aged brain (Chung et al., 2004; Yao et al., 2004; Uehara et al., 2006), we speculate that S-nitrosylation of these and similar proteins may represent a key event that contributes to susceptibility of the aging brain to neurodegenerative conditions.

PDI activity in ALS

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

Recently, PDI has also been implicated in the pathophysiology of familial ALS (Atkin et al., 2006). Mutations in Cu/Zn superoxide dismutase (SOD1) are known to be involved in motor neuron death in some forms of familial ALS. SOD1 is an intracellular homodimeric metalloprotein that forms a stable intrasubunit disulfide bond. Biochemical evidence suggests that the disulfide-reduced monomer of mutant SOD1 (mt SOD1) forms inclusion bodies (Tiwari & Hayward, 2003; Arnesano et al., 2004; Doucette et al., 2004; Rakhit et al., 2004; Furukawa & O’Halloran, 2005), and aggregates of misfolded mt SOD1 are commonly associated with the disease as seen at post-mortem examination. In addition, although wt SOD1 is found predominantly in the cytoplasm, mt SOD1 forms monomers or insoluble high molecular weight multimers within the ER (Kikuchi et al., 2006). Atkin et al. (2006) recently showed that inhibition of PDI activity with bacitracin can increase aggregation of mt SOD1 in neuronal cells. Moreover, PDI colocalized and bound to intracellular aggregates of mt SOD1. Upregulation of the UPR was also observed in mt SOD1 mice. These findings suggest that ER stress may contribute to the pathophysiology of familial ALS, and PDI could potentially reduce mt SOD1 aggregation and affect neuronal survival. Interestingly, S-nitrosothiol levels have also been found to be abnormal in the spinal cords of mt SOD1 transgenic mice (Schonhoff et al., 2006). Whether SNO-PDI is involved in SOD1 aggregation and motor neuron injury in ALS remains to be studied.

Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

One mechanism that could potentially curtail excessive Ca2+ influx and resultant nNOS activity would be inhibition of NMDA receptor activity. Until recently, however, drugs in this class blocked virtually all NMDA receptor 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. In contrast, studies in our laboratory first showed that the adamantane derivative, memantine, preferentially blocks excessive (pathological) NMDA receptor activity while relatively sparing normal (physiological) activity. Memantine does this through its action as a low-affinity, uncompetitive antagonist with a relatively rapid off-rate. The inhibitory activity of memantine involves blockade of the ion channel when it is excessively open (termed open-channel block). The unique and subtle difference of the memantine blocking sites in the channel pore may explain the advantageous properties of memantine action.

An uncompetitive antagonist can be distinguished from a non-competitive antagonist, which acts allosterically at a non-competitive site, i.e. at a site other than the agonist-binding site. An uncompetitive antagonist is defined as an inhibitor whose action is contingent upon prior activation of the receptor by the agonist. Hence, the same amount of antagonist blocks higher concentrations of agonist relatively better than lower concentrations of agonist. Some open-channel blockers function as pure uncompetitive antagonists, depending on their exact properties of interaction with the ion channel. This uncompetitive mechanism of action coupled with a relatively fast off-rate from the channel yields a drug that preferentially blocks NMDA receptor-operated channels when they are excessively open while relatively sparing normal neurotransmission. In fact, the relatively fast off-rate is a major contributor to a drug like memantine's low affinity for the channel pore. While many factors determine a drug's clinical efficacy and tolerability, it appears that the relatively rapid off-rate is a predominant factor in memantine's tolerability in contrast to other NMDA-type receptor antagonists (reviewed Chen & Lipton, 2006; Lipton, 2006). Memantine has been used for many years in Europe to treat PD, and regulatory groups in both Europe and the USA recently voted its approval as the first treatment for moderate-to-severe AD. It is currently under study for a number of other neurodegenerative disorders.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References

It has been suggested that a major difference between the neurodegenerative brain and the ‘normal’ aging brain is a heavier burden of ROS/RNS. Excessive nitrosative stress triggered by excessive NMDA receptor activation and Ca2+ influx may result in dysfunction of ubiquitination and molecular chaperones, thus contributing to abnormal protein accumulation and neuronal damage in sporadic forms of neurodegenerative diseases. Our elucidation of an NO-mediated pathway to dysfunction of parkin and PDI by S-nitrosylation provides a mechanistic link between free radical production, abnormal protein accumulation, and neuronal cell injury in neurodegenerative disorders such as PD. Elucidation of this new pathway may lead to the development of additional new therapeutic approaches to prevent aberrant protein misfolding by targeted disruption or prevention of nitrosylation of specific proteins such as parkin and PDI.

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  1. Top of page
  2. Summary
  3. Introduction
  4. NMDA receptor-mediated glutamatergic signaling pathways induce Ca2+ influx
  5. Ca2+ influx and generation of NO
  6. Regulation of brain function by NO
  7. NO and the free radical theory of aging
  8. S-Nitrosylation and neurodegeneration
  9. Nitrosative stress impairs protein ubiquitination in PD
  10. S-Nitrosylation of PDI mediates protein misfolding and neurotoxicity in cell models of PD or AD
  11. PDI activity in ALS
  12. Potential treatment of excessive NMDA-induced Ca2+ influx and S-nitrosylation
  13. Conclusions
  14. Acknowledgments
  15. References
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