Differential participation of phospholipase A(2) isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800μM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxigenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.     

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.     

Phospholipase A(2) isoforms: a perspective

Several new PLA(2)s have been identified based on their nucleotide gene sequences. They were classified mainly into three groups: cytosolic PLA(2) (cPLA(2)), secretary PLA(2) (sPLA(2)), and intracellular PLA(2) (iPLA(2)). They differ from each other in terms of substrate specificity, Ca(2+) requirement and lipid modification. The questions that still remain to be addressed are the subcellular localization and differential regulation of the isoforms in various cell types and under different physiological conditions. It is required to identify the downstream events that occur upon PLA(2) activation, particularly target protein or metabolic pathway for liberated arachidonic acid or other fatty acids. Understanding the same will greatly help in the development of potent and specific pharmacological modulators that can be used for basic research and clinical applications.The information of the human and other genomes of PLA(2)s, combined with the use of proteomics and genetically manipulated mouse models of different diseases, will illuminate us about the specific and potentially overlapping roles of individual phospholipases as mediators of physiological and pathological processes. Hopefully, such understanding will enable the development of specific agents aimed at decreasing the potential contribution of individual secretary phospholipases to vascular diseases.The signaling cascades involved in the activation of cPLA(2) by mitogen activated protein kinases (MAPKs) is now evident. It has been demonstrated that p44 MAPK phosphorylates cPLA(2) and increases its activity in cells and tissues. The phosphorylation of cPLA(2) at ser505 occurs before the increase in intracellular Ca(2+) that facilitate the binding of the lipid binding domain of cPLA(2) to phospholipids, promoting its translocation to cellular membranes and AA release. Recently, a negative feed back loop for cPLA(2) activation by MAPK has been proposed. If PLA(2) activation in a given model depends on PKC, PKA, cAMP, or MAPK then inhibition of these phosphorylating enzymes may alter activities of PLA(2) isoforms during cellular injury. Understanding the signaling pathways involved in the activation/deactivation of PLA(2) during cellular injury will point to key events that can be used to prevent the cellular injury. Furthermore, to date, there is limited information available regarding the regulation of iPLA(2) or sPLA(2) by these pathways.     

Amyloid beta(1-42) and its beta(25-35) fragment induce activation and membrane translocation of cytosolic phospholipase A2 in bovine retina capillary pericytes

We investigated changes in cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) activities in bovine retina capillary pericytes after stimulation with 50 microM amyloid-beta (Abeta) (1-42) and its (25-35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Abeta peptides, we found that cPLA(2) activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Abeta(35-25) peptide did not stimulate or induce cPLA(2) translocation. Exposure to both Abeta peptides had no significant effect on cPLA(2) protein content as tested by Western immunoblot analysis. The addition of Abetas to quiescent pericytes was followed by phosphorylation of cPLA(2) and arachidonic acid release. Treatment with inhibitors (AACOCF(3), staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA(2) activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA(2), demonstrated that the stimulation of total PLA(2) activity by Abetas was mediated by both PLA(2) enzymes. Taken together with our previous observations that both Abeta peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA(2) activation/translocation and iPLA(2) activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation.     

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.     

Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Inhibition of phospholipase A2 reduces neurite outgrowth and neuronal viability

Phospholipase A2 (PLA(2)) has been implicated in neurodevelopmental processes and in the early development of the nervous system. We investigated the effects of the inhibition of calcium-dependent and calcium-independent subtypes of cytosolic PLA2 (cPLA2 and iPLA2) on the development and viability of primary cultures of cortical and hippocampal neurons. PLA2 in these cultures was continuously inhibited with methylarachidonyl-fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and iPLA2, or with bromoenol lactone (BEL), an irreversible selective iPLA2 inhibitor. The effect of PLA2 inhibitors on the development of neuronal cultures was ascertained by total cell count and morphological characterisation. Neuronal viability was quantified with MTT assays. Inhibition of PLA2 resulted in reduction of neuritogenesis and neuronal viability, disrupting neuronal homeostasis and leading to neuronal death. We conclude that the functional integrity of both calcium-dependent and calcium-independent cytosolic PLA2 is necessary for the in vitro development of cortical and hippocampal neurons.     

Peroxynitrite stimulates the activity of cytosolic phospholipase A2 in U937 cells: the extent of arachidonic acid formation regulates the balance between cell survival or death

Peroxynitrite stimulates in U937 cells release of arachidonic acid (AA) sensitive to various phospholipase A(2) (PLA(2)) inhibitors, including arachidonyl trifluoromethyl ketone (AACOCF(3)), which specifically inhibits cytosolic PLA(2) (cPLA(2)). This response linearly increases using non toxic concentrations of the oxidant, and reaches a plateau at levels at which toxicity becomes apparent. Three separate lines of evidence are consistent with the notion that AA generated by cPLA(2) promotes survival in cells exposed to peroxynitrite. Firstly, toxicity was suppressed by nanomolar levels of exogenous AA, or by AA generated by the direct PLA(2) activator melittin. Secondly AACOCF(3), or other PLA(2) inhibitors, promoted cell death after exposure to otherwise non toxic concentrations of peroxynitrite; exogenous AA abolished the enhancing effects mediated by the PLA(2) inhibitors. Finally, U937 cells transfected with cPLA(2) antisense oligonucleotides were killed by concentrations of peroxynitrite that were non-toxic for cells transfected with nonsense oligonucleotides. This lethal response was insensitive to AACOCF(3) and prevented by exogenous AA.     

Glutamate Signaling in Benign and Malignant Disorders: Current Status,Future Perspectives,and Therapeutic Implications

Glutamate, a nonessential amino acid, is the major excitatory neurotransmitter in the central nervous system. As such, glutamate has been shown to play a role in not only neural processes, such as learning and memory, but also in bioenergetics, biosynthetic and metabolic oncogenic pathways. Glutamate has been the target of intense investigation for its involvement not only in the pathogenesis of benign neurodegenerative diseases (NDDs) such as Parkinson''s disease, Alzheimer''s disease, schizophrenia, multiple sclerosis, and amyotropic lateral sclerosis (ALS), but also in carcinogenesis and progression of malignant diseases. In addition to its intracellular activities, glutamate in secreted form is a phylogenetically conserved cell signaling molecule. Glutamate binding activates multiple major receptor families including the metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in various signaling pathways in cancer. Inhibition of extracellular glutamate release or glutamate receptor activation via competitive or non-competitive antagonists decreases growth, migration and invasion and induces apoptosis in breast cancer, melanoma, glioma and prostate cancer cells. In this review, we discuss the current state of glutamate signaling research as it relates to benign and malignant diseases. In addition, we provide a synopsis of clinical trials using glutamate antagonists for the treatment of NDD and malignant diseases. We conclude that in addition to its potential role as a metabolic biomarker, glutamate receptors and glutamate-initiated signaling pathways may provide novel therapeutic opportunities for cancer.     

Calcium-independent phospholipase A(2) is required for human monocyte chemotaxis to monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A(2) (cPLA(2)) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA(2), calcium-independent PLA(2) (iPLA(2)) in comparison to cPLA(2). Pharmacological inhibitors of PLA(2) were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA(2) expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA(2) or cPLA(2) expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA(2)-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA(2)-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA(2)-deficient monocytes while having no effect on the iPLA(2)-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA(2) and cPLA(2) represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.     

Functional crosstalk between phospholipase D(2) and signaling phospholipase A(2)/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways

We performed reconstitution analyses of functional interaction between phospholipase A(2) (PLA(2)) and phospholipase D (PLD) enzymes. Cotransfection of HEK293 cells with cytosolic (cPLA(2)) or type IIA secretory (sPLA(2)-IIA) PLA(2) and PLD(2), but not PLD(1), led to marked augmentation of stimulus-induced arachidonate release. Interleukin-1-stimulated arachidonate release was accompanied by prostaglandin E(2) production via cyclooxygenase-2, the expression of which was augmented by PLD(2). Conversely, activation of PLD(2), not PLD(1), was facilitated by cPLA(2) or sPLA(2)-IIA. Thus, our results revealed functional crosstalk between signaling PLA(2)s and PLD(2) in the regulation of various cellular responses in which these enzymes have been implicated.     

Intracellular phospholipase A2 expression and location in human macrophages: influence of synthetic material surface chemistry

Phospholipase A(2) (PLA(2)) enzymes participate in a potent inflammatory pathway through the liberation of arachidonic acid upon hydrolysis of membrane glycerophospholipids. The presence of implanted polycarbonate-urethane (PCNU) materials, used in several medical applications, has the ability to influence inflammatory responses of human macrophages that are recruited to a tissue-material interface; however, the specific inflammatory pathways that are activated upon macrophage attachment to PCNU are largely unknown. Previous studies suggested the participation of PLA(2) pathways in material degradation with the use of chemical inhibitors, such as aristolochic acid (ARIST), however not accurately defining the specific PLA(2) enzymes involved. The current study aimed to establish specific groups of PLA(2) involved in the macrophage foreign body response to PCNU. ARIST was assessed for specific effects on secretory PLA(2) (sPLA(2)) protein expression and non-specific effects on key proteins, beta-actin and monocyte-specific esterase, implicated in the macrophage attack on PCNU materials. Macrophage attachment to PCNU materials induced increased intracellular expression of cytosolic PLA(2) (cPLA(2)), but not sPLA(2), relative to tissue culture polystyrene (TCPS) as detected by immunoblot analysis, demonstrating an early and delayed stimulation during the time course of increased cPLA(2) protein expression. Laser scanning confocal microscopy images indicated a change in location of cPLA(2) in macrophages adherent to PCNU surfaces compared to TCPS. This study has illustrated changes in macrophage cPLA(2) expression in response to cell-attachment to PCNU surfaces, demonstrating that the macrophage foreign body response to biomaterials induces a potent inflammatory pathway, which may lead to tissue damage near the site of material implantation.     

Differential effects of calcium-dependent and calcium-independent phospholipase A(2) inhibitors on kainate-induced neuronal injury in rat hippocampal slices

Brain tissue contains multiple forms of intracellular phospholipase A(2) (PLA(2)) activity that differ from each other in many ways including their response to specific inhibitors. The systemic administration of kainic acid to rats produces a marked increase in cPLA(2) activity in neurons and astrocytes. This is associated with increased lipid peroxidation as evidenced by accumulation of 4-hydroxynonenal (4-HNE) modified proteins. The present study describes the effect of specific inhibitors of Ca(2+)-dependent or Ca(2+)-independent PLA(2) on kainite-induced excitotoxic injury in rat hippocampal slices. Specific inhibitors of Ca(2+)-dependent PLA(2) prevented the decrease of a neuronal marker, GluR1, and increase in cPLA(2) and 4-HNE immunoreactivities in slices treated with kainate. This shows that cPLA(2) plays an important role in kainite-induced neurotoxicity and that cPLA(2) inhibitors can be used to protect hippocampal slices from damage induced by kainate.     

Delineating metabolic signatures of head and neck squamous cell carcinoma: Phospholipase A(2), a potential therapeutic target

A better understanding of molecular pathways involved in malignant transformation of head and neck squamous cell carcinoma (HNSCC) is essential for the development of novel and efficient anti-cancer drugs. To delineate the global metabolism of HNSCC, we report (1)H NMR-based metabolic profiling of HNSCC cells from five different patients that were derived from various sites of the upper aerodigestive tract, including the floor of mouth, tongue and larynx. Primary cultures of normal human oral keratinocytes (NHOK) from three different donors were used for comparison. (1)H NMR spectra of polar and non-polar extracts of cells were used to identify more than thirty-five metabolites. Principal component analysis performed on the NMR data revealed a clear classification of NHOK and HNSCC cells. HNSCC cells exhibited significantly altered levels of various metabolites that clearly revealed dysregulation in multiple metabolic events, including Warburg effect, oxidative phosphorylation, energy metabolism, TCA cycle anaplerotic flux, glutaminolysis, hexosamine pathway, osmo-regulatory and anti-oxidant mechanism. In addition, significant alterations in the ratios of phosphatidylcholine/lysophosphatidylcholine and phosphocholine/glycerophosphocholine, and elevated arachidonic acid observed in HNSCC cells reveal an altered membrane choline phospholipid metabolism (MCPM). Furthermore, significantly increased activity of phospholipase A(2) (PLA(2)), particularly cytosolic PLA(2) (cPLA(2)) observed in all the HNSCC cells confirm an altered MCPM. In summary, the metabolomic findings presented here can be useful to further elucidate the biological aspects that lead to HNSCC, and also provide a rational basis for monitoring molecular mechanisms in response to chemotherapy. Moreover, cPLA(2) may serve as a potential therapeutic target for anti-cancer therapy of HNSCC.     

Cellular components that functionally interact with signaling phospholipase A(2)s

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.     

Modulation of sensory input to the spinal cord by presynaptic ionotropic glutamate receptors

Sensory input from peripheral nerves to the dorsal horn of the spinal cord is mediated by a variety of agents released by the central terminals of dorsal root ganglion (DRG) neurons. These include, but are not limited to, amino acids, especially glutamate, peptides and purines. The unraveling of the mechanisms of synaptic transmission by central terminals of DRG neurons has to take into account various ways in which the message from the periphery can be modulated at the level of the first central synapse. These include postsynaptic and presynaptic mechanisms. Homomeric and heteromeric complexes of receptor subunits for the different transmitters released by DRG neurons and interneurons, clustered at the postsynaptic site of central synapses, can be expressed in different combinations and their rate of insertion into the postsynaptic membrane is activity-regulated. Inhibitory mechanisms are an important part of central modulation, especially via presynaptic inhibition, currently believed to involve GABA released by inhibitory intrinsic neurons. Recent work has established the occurrence of another way by which sensory input can be modulated, i.e. the expression of presynaptic ionotropic and metabotropic receptors in central terminals of DRG neurons. Microscopic evidence for the expression, in these terminals, of various subunits of ionotropic glutamate receptors documents the selective expression of glutamate receptors in functionally different DRG afferents. Electrophysiological and pharmacological data suggest that activation of presynaptic ionotropic glutamate receptors in central terminals of DRG neurons may result in inhibition of release of glutamate by the same terminals. Glutamate activating presynaptic receptors may spill over from the same or adjacent synapses, or may be released by processes of astroglial cells surrounding synaptic terminals. The wide expression of presynaptic ionotropic glutamate receptors, especially in superficial laminae of the dorsal horn, where Adelta- and C fibers terminate, provides an additional or alternative mechanism, besides GABA-mediated presynaptic inhibition, for the modulation of glutamate release by these fibers. Since, however, presynaptic ionotropic glutamate receptors are also expressed in terminals of GABAergic intrinsic interneurons, a decrease of GABA release resulting from activation of these receptors in the same laminae, may also play a role in central sensitization and hyperalgesia.     

The availability of surface GABA B receptors is independent of gamma-aminobutyric acid but controlled by glutamate in central neurons

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of gamma-aminobutyric acid type B (GABA(B)) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABA(B) receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABA(B) receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABA(B) receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABA(B) receptors in central neurons, consistent with their enrichment at glutamatergic synapses.     

Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.     

Excessive Glutamate Stimulation Impairs ACE2 Activity Through ADAM17-Mediated Shedding in Cultured Cortical Neurons

The excitotoxicity of glutamate plays an important role in the progression of various neurological disorders via participating in inflammation and neuronal damage. In this study, we identified the role of excessive glutamate stimulation in the modulation of angiotensin-converting enzyme type 2 (ACE2), a critical component in the compensatory axis of the renin–angiotensin system (RAS). In primary cultured cortical neurons, high concentration of glutamate (100 µM) significantly reduced the enzymatic activity of ACE2. The elevated activity of ADAM17, a member of the ‘A Disintegrin And Metalloprotease’ (ADAM) family, was found to contribute to this glutamate-induced ACE2 down-regulation. The decrease of ACE2 activity could be prevented by pre-treatment with antagonists targeting ionotropic glutamate receptors. In addition, the glutamate-induced decrease in ACE2 activity was significantly attenuated when the neurons were co-treated with MitoTEMPOL or blockers that target oxidative stress-mediated signaling pathway. In summary, our study reveals a strong relationship between excessive glutamate stimulation and ADAM17-mediated impairment in ACE2 activity, suggesting a possible cross-talk between glutamate-induced excitotoxicity and dysregulated RAS.     

Deregulation of excitatory neurotransmission underlying synapse failure in Alzheimer's disease

Alzheimer′s disease (AD) is the most common form of dementia in the elderly. Memory loss in AD is increasingly attributed to soluble oligomers of the amyloid‐β peptide (AβOs), toxins that accumulate in AD brains and target particular synapses. Glutamate receptors appear to be centrally involved in synaptic targeting by AβOs. Once bound to neurons, AβOs dysregulate the activity and reduce the surface expression of both N‐methyl‐d ‐aspartate (NMDA) and 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)propanoic acid (AMPA) types of glutamate receptors, impairing signaling pathways involved in synaptic plasticity. In the extracellular milieu, AβOs promote accumulation of the excitatory amino acids, glutamate and d ‐serine. This leads to overactivation of glutamate receptors, triggering abnormal calcium signals with noxious impacts on neurons. Here, we review key findings linking AβOs to deregulated glutamate neurotransmission and implicating this as a primary mechanism of synapse failure in AD. We also discuss strategies to counteract the impact of AβOs on excitatory neurotransmission. In particular, we review evidence showing that inducing neuronal hyperpolarization via activation of inhibitory GABA_A receptors prevents AβO‐induced excitotoxicity, suggesting that this could comprise a possible therapeutic approach in AD.