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Keywords:

  • Alzheimer’s disease;
  • astrocytes;
  • membrane dynamics;
  • NADPH oxidase;
  • neurons;
  • phospholipases A2

Abstract

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

J. Neurochem. (2011) 116, 813–819.

Abstract

Phospholipases A2 (PLA2s) are essential enzymes in cells. They are not only responsible for maintaining the structural organization of cell membranes, but also play a pivotal role in the regulation of cell functions. Activation of PLA2s results in the release of fatty acids and lysophospholipids, products that are lipid mediators and compounds capable of altering membrane microdomains and physical properties. Although not fully understood, recent studies have linked aberrant PLA2 activity to oxidative signaling pathways involving NADPH oxidase that underlie the pathophysiology of a number of neurodegenerative diseases. In this paper, we review studies describing the involvement of cytosolic PLA2 in oxidative signaling pathways leading to neuronal impairment and activation of glial cell inflammatory responses. In addition, this review also includes information on the role of cytosolic PLA2 and exogenous secretory PLA2 on membrane physical properties, dynamics, and membrane proteins. Unraveling the mechanisms that regulate specific types of PLA2s and their effects on membrane dynamics are important prerequisites towards understanding their roles in the pathophysiology of Alzheimer’s disease, and in the development of novel therapeutics to retard progression of the disease.

Abbreviations used:

amyloid beta peptide

AA

arachidonic acid

APP

amyloid precursor protein

cPLA2

cytosolic PLA2

DHA

docosahexaenoic acid

iPLA2

calcium-independent PLA2

NF-κB

nuclear factor-kappa B

PLA2

phospholipase A2

ROS

reactive oxygen species

sPLA2

secretory PLA2

Phospholipids, together with proteins, cholesterol, and glycolipids, constitute integral components of what is considered to be the bilayer of cellular membranes. Different phospholipases, for example, PLA, PLC, and PLD are present in cells for hydrolysis of different moieties of the phospholipids, resulting in the generation of lipid mediators and second messengers that play important functions in regulating cellular activities. Among these different types of phospholipases, studies to understand the role of phospholipases A2 (PLA2s) in cells in the CNS have been the center of intense investigation. The presence of over 30 different types of PLA2s in different mammalian cells underscores their crucial role in regulating specific cell functions. The PLA2 family is classified into cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2), and secretory PLA2 (sPLA2) (Burke & Dennis 2009). This group of enzymes is responsible for the cleavage of the acyl groups in the sn-2 position of membrane phospholipids. While cPLA2 has been shown to preferentially release arachidonic acid (AA) (Ghosh et al. 2006), the iPLA2 seems to mediate the release of docosahexaenoic acid (DHA) (Strokin et al. 2003). While AA is a precursor for the biosynthesis of eicosanoids and has been linked to inflammatory responses, DHA is a precursor for the synthesis of neuroprotectin D (Lukiw et al. 2005; Niemoller & Bazan 2010). DHA also plays a role in regulating prostanoid production (Strokin et al. 2007).

Hydrolysis of membrane phospholipids by PLA2s also produces lysophospholipids, which are compounds possessing detergent-like properties. Lysophospholipids are involved in the modulation of membrane processes such as causing membrane buddings, formation of membrane raffles (Nakano et al. 2009), modulating protein complex assembly (Shin et al. 2010), and ion channel activity (Ben-Zeev et al. 2010).

Recent studies in our laboratories have focused on the role of cPLA2 in excitotoxic functions in neurons, and the role of sPLA2-IIA in response to inflammatory events within glial cells (Sun et al. 2010). In this review, we also presented evidence demonstrating how activated cPLA2 and sPLA2 may impact on cell membrane properties and dynamics, and how these changes may impact on cellular metabolism in Alzheimer’s disease (AD).

cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

The 87 kDa cPLA2 is comprised of a C2 domain for binding calcium and a catalytic domain with multiple phosphorylation sites (Ghosh et al. 2006). The requirement for calcium and protein kinases for cPLA2 suggests that this enzyme is under tight control by intracellular signaling pathways. Earlier studies from our laboratory, as well as those from other laboratories, have demonstrated activation of cPLA2 in astrocytes through the G-protein-coupled P2Y2 receptor, which is coupled to activation of protein kinases including the MAPKs and protein kinase C (Bruner & Murphy 1993; Chen & Chen 1998; Chen et al. 1999; Xu et al. 2002). Activation of cPLA2 is also observed downstream of the angiotensin II type 1 receptor pathway (Kobayashi et al. 2009). In fact, telmisartan, the angiotensis II receptor blocker, could inhibit cPLA2 expression and suppressed cerebral infarct in an experimental stroke model (Kobayashi et al. 2009). Both cPLA2 and iPLA2 are constitutively present in neurons, and inhibitions of their activities have led to the restriction of neurite outgrowth and loss of neuronal viability (Forlenza et al. 2007a; Mendes et al. 2005). Alteration of PLA2s in neuronal homeostasis can have an impact on memory function, and is thus an important implication in the pathogenesis of AD (Forlenza et al. 2007b). More recent studies have linked activation of cPLA2 and AA release in neurons to stimulation of responses by the NMDA receptor and its downstream signaling pathways involving NADPH oxidase (Shelat et al. 2008). Depending on the type of neurons, other protein kinases including calcium/calmodulin-dependent protein kinase (CAMK-II) and cGMP-dependent protein kinase (PKG), may also modulate cPLA2 activity (Chalimoniuk et al. 2009; Shimizu et al. 2008). The link between the NMDA receptor and production of reactive oxygen species (ROS) through activation of the superoxide producing enzyme NADPH oxidase is an important finding and the information supports the involvement of ROS in neuronal excitation (Brennan et al. 2009; Kishida & Klann 2007; Kishida et al. 2005). This source of ROS was shown to activate the signaling pathway leading to phosphorylation of extracellular signal-regulated kinases (Kishida et al. 2005; Serrano et al. 2009), an important MAPK for phosphorylation of cPLA2. As NADPH oxidase has been shown to occur in synaptic structures (Tejada-Simon et al. 2005), it is possible that this ROS signaling pathway may modulate synaptic membrane function by regulating cPLA2 and release of AA. Indeed, AA release was found in hippocampal neurons (Angelova & Muller 2006), and the application of AA as low as 1 pmol was shown to inhibit both pre-synaptic and post-synaptic types of potassium channel activity (Angelova & Muller 2009).

Despite an unknown mechanism, oligomeric amyloid beta peptide (Aβ) was shown to activate cPLA2 and increase AA in neurons (Sanchez-Mejia et al. 2008). Over stimulation of this event was shown to cause the impairment of mitochondrial function and neuronal apoptosis (Kriem et al. 2005). A study by Sun’s group further suggests that this toxic event of Aβ is initiated through a signaling pathway involving NMDA receptors and NADPH oxidase (Shelat et al. 2008). The increase in cPLA2 activity is in agreement with the observation that levels of AA are increased in AD transgenic mice and in the hippocampus of the AD brain (Sanchez-Mejia et al. 2008). A study with neuronal culture has also demonstrated the ability for Aβ to activate cPLA2, and inhibition of cPLA2 results in the reduction of Aβ-induced neurotoxicity (Sanchez-Mejia et al. 2008). Studies with neuronal cells providing evidence for oligomeric Aβ to produce ROS through the NADPH oxidase pathway are in good agreement with the oxidative hypothesis of AD (Block 2008; Simonyi et al. 2010; Sultana & Butterfield 2010). Indeed, an increase in 4-hydroxynonenal, a marker of lipid peroxidation, is found in amyloid plaques and elevated in the AD brain (Butterfield et al. 2010; Siegel et al. 2007). Taken together, these studies demonstrate the role for cPLA2 and AA as mediators for Aβ-induced neuronal impairment and possible use of pharmacological agents targeting this pathway for the treatment of AD (Sanchez-Mejia & Mucke 2010).

cPLA2 targets cellular membranes

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

Activation of cPLA2 has generally shown to cause the preferential release of AA from phosphatidylcholine (Ghosh et al. 2006). However, under specific conditions, this enzyme may also hydrolyze other phospholipids (Xue et al. 1999). For example, changes in the concentrations of various phospholipids were found in the brains of cPLA2 knockout mice, suggesting that cPLA2 is necessary to maintain a healthy ‘membrane environment’ (Rosenberger et al. 2003). In non-neural cells, cPLA2α has been shown to preferentially target phospholipids in the endoplasmic reticulum and Golgi membranes (Evans et al. 2004; Ghosh et al. 2007). Apparently, cPLA2 can also target other subcellular membranes, including mitochondrial, nuclear and plasma membranes. Studies with neutrophils indicated a transient recruitment of cPLA2 to the NADPH oxidase complex in plasma membrane (Shmelzer et al. 2003), possibly because of a relationship between the C2 domain of cPLA2 and the PX domain of p47phox (Shmelzer et al. 2008). Another study with neurons demonstrated the role of cPLA2 in the Aβ-induced alteration of mitochondrial dysfunction and apoptotic cell death (Kriem et al. 2005). In primary mouse neurons, cPLA2 was shown to mediate binding of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor to caveolin 1 through the caveolin 1 scaffolding protein domain, providing further evidence for the ability of cPLA2 to exert its action on different subcellular membranes (Gaudreault et al. 2004). Activation of cPLA2 and release of AA may also lead to activation of the sphingomyelinase-ceramide pathway, which in turn is involved in the neurotoxic effects of Aβ (Malaplate-Armand et al. 2006). As sphingomyelin is an important component of the neural cell plasma membrane, the ability for Aβ to activate the cPLA2/AA and sphingomyelinase pathways may offer an important explanation for the cytotoxic effects of Aβ on synaptic impairment and neuronal apoptosis (Florent-Bechard et al. 2009). Recent studies show an increase in sphingomyelinase and acid ceramidase in the AD brain, suggesting that deregulation of the sphingolipid metabolism is a part of AD pathology (He et al. 2010; Mielke and Lyketsos 2010). Although these studies suggest a relationship between PLA2 and other membrane lipids, more studies are needed to investigate how these changes lead to the alteration of membrane physical properties and membrane dynamics.

Role of PLA2 in modulating membrane physical properties and dynamics

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

Phospholipase A2 can target membrane phospholipids leading to changes in physical properties of cellular membranes (Schaeffer et al. 2005). There is evidence that cPLA2 and its hydrolytic products are activated in signaling pathways that modify actin dynamics (Moes et al. 2010). Studies with artificial giant liposomes have demonstrated the role for PLA2 to promote raft budding and fission (Staneva et al. 2004). PLA2 also causes a decrease in the liquid-disordered (Ld) and an increase in the liquid-ordered (Lo) phase (Simonsen 2008). Consistent with these biophysical studies, we used the fluorescence microscopy of LAURDAN to demonstrate membrane molecular changes because of oxidative stress induced by H2O2 (Zhu et al. 2005). In this study, oxidative stress was found to cause membranes to become more molecularly ordered through the activation of cPLA2 to target plasma membranes in primary rat astrocytes (Zhu et al. 2005). As Aβ activates cPLA2 through the NADPH oxidase signaling pathways in astrocytes, this activation was shown to produce more molecularly ordered membranes, leading to membrane domain reorganization (Hicks et al. 2008). In astrocytes, Aβ was shown to cause time-dependent activation of cPLA2 and iPLA2, and together, these PLA2s played a role in causing the loss of mitochondrial membrane potential, swelling of mitochondria, and the production of superoxide from mitochondria, which are characteristics of mitochondrial dysfunction (Zhu et al. 2006). These results further demonstrated the ability for intracellular PLA2 to target cellular membranes and alter cell functions under oxidative stress induced by Aβ.

Secretory PLA2 is involved in neuroinflammation

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

Secretory PLA2s comprises a superfamily of more than 10 different isoforms of low molecular mass (13–19 kDa) proteins that are widely present in mammalian cells, body fluids, and in secretagogues and venom (Murakami et al. 2010). Many isoforms are associated with dynamic physiological and pathological processes including inflammation and apoptosis (Olson et al. 2010). In the central nervous system, sPLA2-IIA has been shown to play a role in mediating several important physiological processes, including the regulation of neurotransmitter release, neuritogenesis, and neuronal apoptosis (Sun et al. 2010). Among the sPLA2s, sPLA2-IIA has been extensively studied because of its involvement in inflammatory diseases such as atherosclerosis, arthritis, and infection (Ibeas et al. 2009; Murakami et al. 1997). However, studies of sPLA2-IIA functions in rodents have been limited because a number of mouse strains, including the C57Bl/6 strain, do not express sPLA2-IIA because of a frame shift mutation (Kennedy et al. 1995). However, studies using the rat model have demonstrated up-regulation of this enzyme in the brain in response to injury and inflammation (Adibhatla & Hatcher 2008). Increased expression of sPLA2-IIA was also shown in rat spinal cord injury (Liu & Xu 2010; Titsworth et al. 2008; Titsworth et al. 2009). Interestingly, the increased expression in spinal cord injury was mainly attributed to an increased inflammatory response in the oligodendroglial cells (Titsworth et al. 2009). Our earlier study demonstrated an up-regulation of sPLA2-IIA mRNA in the rat brain after focal cerebral ischemia (Lin et al. 2004). In this model, increased immunoreactivity of sPLA2-IIA was found in reactive astrocytes within the penumbral region and not in microglial cells (Lin et al. 2004). A study with human AD brains also showed an increase in sPLA2-IIA mRNA expression in the hippocampal region in comparison with age-matched, non-demented controls (Moses et al. 2006). In this study, immunoreactivity of this enzyme was also confined to astrocytes and not microglial cells (Moses et al. 2006). In agreement with the up-regulation in the AD brain, an increase of sPLA2-IIA activity was observed in cerebrospinal fluid of AD patients, further suggesting the possibility of using sPLA2-IIA as a biomarker of neuroinflammation (Chalbot et al. 2009).

Studies with astrocytes, including both the immortalized rat cell line (DITNC) and primary rat astrocytes, indicated an increase in sPLA2-IIA mRNA expression in response to pro-inflammatory cytokines and lipopolysaccharides through activation of the nuclear factor-kappa B (NF-κB) pathway (Jensen et al. 2009; Xu et al. 2003). These cytokines and endotoxin can also induce other inflammatory responses including induction of cyclooxygenase-2 and inducible nitric oxide synthase (Jana et al. 2005). Besides cytokines, Aβ can stimulate induction of sPLA2-IIA expression in human astrocytes (Moses et al. 2006). Taken together, these studies well demonstrated the role of sPLA2-IIA among inflammatory responses in astrocytes. Although the mechanism needs to be further delineated, these astrocytic responses also involve oxidative pathways through activation of NADPH oxidase in these cells (Abramov et al. 2005; Jensen et al. 2009). The involvement of the NADPH oxidase pathway in glial cell inflammatory responses is important in explaining why some anti-oxidants are also anti-inflammatory agents.

Although microglial activation is considered an important pathology in AD, and that microglial cells are often found near the amyloid plaques (Mandrekar-Colucci & Landreth 2010), whether inflammatory responses in microglial cells include induction of sPLA2-IIA has not been clearly delineated. However, as there are many types of sPLA2 in the brain (Molloy et al. 1998), possible involvement of other sPLA2 subtypes in microglial inflammatory responses remains to be investigated.

In the normal rat brain, low levels of sPLA2-IIA protein are detected in the brainstem and spinal cord (Ma et al. 2010). sPLA2-IIA was also detected in the mitochondria of cerebellar granule cells (Mathisen et al. 2007) and in the mitochondria of cortical neurons (Chiricozzi et al. 2010). Although there is some evidence for activation of the mitochondrial sPLA2-IIA in response to neuronal excitation, the mechanism for its involvement in damaging mitochondrial membrane function remains to be further investigated.

Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

Perturbation of membrane phospholipids and fatty acid unsaturation can alter protein folding and their response (Ariyama et al. 2010). Study with erythrocytes indicated the susceptibility of membrane to the hydrolysis of phospholipids by sPLA2-IIA upon an increase in intracellular calcium and have demonstrated changes in lipid order by regulating the sPLA2 activity (Gonzalez et al. 2009). In monocytes, sPLA2-IIA enhances proliferative activity through binding to integrins (Saegusa et al. 2008). Amyloid precursor protein (APP), the precursor for production of Aβ fragments through hydrolysis by secretases, is a transmembrane protein that can be affected by membrane fluidity (Kojro et al. 2001). In fact, by applying both biochemical techniques and fluorescence microscopy of molecular rotor [Farnesyl-(2-carboxy-2-cyanovinyl)-julolidine, FCVJ] for the characterization of membrane fluidity, we demonstrated that sPLA2-III increases membrane fluidity, resulting in the enhanced secretion of α-secretase-cleaved soluble APP in SH-SY5Y cells (Yang et al. 2010). α-Secretase-cleaved soluble APP is then derived from the non-amyloidogenic pathway in which APP is cleaved by α-secretases.

Conclusion

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References

Biochemical and biophysical studies have demonstrated the involvement of cPLA2 and sPLA2-IIA in neurons and glial cells through receptor stimulation of oxidative signaling pathways involving NADPH oxidase, and through an increase in intracellular calcium and protein kinases (Fig. 1). Figure 1 further demonstrates action of Aβ on these pathways. As sPLA2-IIA is secretory, its release into the extracellular space can cause damage to neighboring cells. Transient activation of cPLA2 allows it to target many types of intracellular membranes, including mitochondrial membranes and plasma membranes, as demonstrated by changes in the membrane physical properties. Damage of mitochondrial membranes is linked to the production of more ROS and perturbation of respiratory chain activity leading to apoptotic pathways. In glial cells, Aβ may activate cytokine receptors involving stimulation of NF-κB and other transcriptional pathways, and through the subsequent activation of inflammatory responses, including synthesis of cyclooxygenases, inducible nitric oxide synthase, and sPLA2. Some transcriptional pathways induced by cytokines may be further enhanced by oxidative pathways, and subsequently promote inflammatory responses.

image

Figure 1.  A metabolic scheme depicting the involvement of different types of PLA2s in oxidative signaling pathways leading to neuronal impairment and glial cell inflammatory responses. This scheme is based on recent findings that in neurons and glial cells, specific cell surface receptors are coupled to the NADPH oxidase pathway in producing ROS and stimulation of protein kinases important in the activation of cPLA2 and release of AA. cPLA2 can target intracellular membranes and alter membrane physical properties and dynamics, including impairment of mitochondrial membrane, which triggers more ROS production and initiation of apoptotic processes. Although not well understood, these events may explain the underlying causes for the cytotoxic effects of Aβ. As shown in Box A, the NADPH oxidase pathway can also impact the NF-κB pathway induced by cytokines, and enhance inflammatory responses in glial cells. Box B: iPLA2s are abundant in neural cells and is considered a major source for the release of DHA. DHA is linked to production of neuroprotectin D1 (NPD1), a neuroprotective agent.

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Studies with astrocytes also demonstrated the involvement of cPLA2 in Aβ-induced biphasic changes in the membrane molecular order and mitochondrial dysfunction. Exogenous sPLA2 can alter neuronal cell membranes, leading to an alteration of APP processing. Taken together, these studies underscore the important role of PLA2s in altering cell functions, not only through their ability to produce lipid mediators, but also through biophysical pathways, such as lipid domain reorganization, membrane phase properties changes, and membrane fluidity. Although more future studies are needed, understanding the mechanisms underlying specific PLA2 action and membrane perturbation should prove to be important for developing novel therapeutic measures to suppress the neurotoxic and inflammatory responses associated with the progression of AD.

References

  1. Top of page
  2. Abstract
  3. cPLA2 is coupled to calcium mobilizing receptors and oxidative signaling pathways in neurons and glial cells
  4. cPLA2 targets cellular membranes
  5. Role of PLA2 in modulating membrane physical properties and dynamics
  6. Secretory PLA2 is involved in neuroinflammation
  7. Exogenous sPLA2s on membrane physical properties and amyloid precursor protein processing
  8. Conclusion
  9. Acknowledgement
  10. References