Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.     

Amyloid β Induces Adhesion of Erythrocytes to Endothelial Cells and Affects Endothelial Viability and Functionality

It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer’s disease.     

COPS5 (Jab1) Protein Increases β Site Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation by Stabilizing RanBP9 Protein Levels

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer''s disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.     

In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.     

Selection of Aptamers for Amyloid β-Protein,the Causative Agent of Alzheimer's Disease

Alzheimer''s disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult¹. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies^2,3.Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD^4,5. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood⁶. Specific molecular recognition tools may unravel the relationships amongst Aβ assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies^7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)^9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).Despite emergence of aptamers as tools in modern biotechnology and medicine¹¹, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)^12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-β¹⁷, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils¹⁸. Aptamers generated using monomeric and several forms of fibrillar β₂-microglobulin (β₂m) were found to bind fibrils of certain other amyloidogenic proteins besides β₂m fibrils¹⁹. Ylera et al. described RNA aptamers selected against immobilized monomeric Aβ40²⁰. Unexpectedly, these aptamers bound fibrillar Aβ40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Aβ40²¹ generated using photo-induced cross-linking of unmodified proteins (PICUP)^22,23. Similar to previous findings^17,19,20, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope²¹. Here, we present the SELEX methodology used in production of these aptamers²¹.Download video file.^{(175M, mp4)}     

Packing Density of the Amyloid Precursor Protein in the Cell Membrane

Plasma membrane proteins organize into structures named compartments, microdomains, rafts, phases, crowds, or clusters. These structures are often smaller than 100 nm in diameter. Despite their importance in many cellular functions, little is known about their inner organization. For instance, how densely are molecules packed? Being aware of the protein compaction may contribute to our general understanding of why such structures exist and how they execute their functions. In this study, we have investigated plasma membrane crowds formed by the amyloid precursor protein (APP), a protein well known for its involvement in Alzheimer’s disease. By combining biochemical experiments with conventional and super-resolution stimulated emission depletion microscopy, we quantitatively determined the protein packing density within APP crowds. We found that crowds occurring with reasonable frequency contain between 20 and 30 molecules occupying a spherical area with a diameter between 65 and 85 nm. Additionally, we found the vast majority of plasmalemmal APP residing in these crowds. The model suggests a high molecular density of protein material within plasmalemmal APP crowds. This should affect the protein’s biochemical accessibility and processing by nonpathological α-secretases. As clustering of APP is a prerequisite for endocytic entry into the pathological processing pathway, elucidation of the packing density also provides a deeper understanding of this part of APP’s life cycle.     

Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.     

Degradation of Amyloid �� Protein by Purified Myelin Basic Protein

The progressive accumulation of β-amyloid (Aβ) in senile plaques and in the cerebral vasculature is the hallmark of Alzheimer disease and related disorders. Impaired clearance of Aβ from the brain likely contributes to the prevalent sporadic form of Alzheimer disease. Several major pathways for Aβ clearance include receptor-mediated cellular uptake, blood-brain barrier transport, and direct proteolytic degradation. Myelin basic protein (MBP) is the major structural protein component of myelin and plays a functional role in the formation and maintenance of the myelin sheath. MBP possesses endogenous serine proteinase activity and can undergo autocatalytic cleavage liberating distinct fragments. Recently, we showed that MBP binds Aβ and inhibits Aβ fibril formation (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952–9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720–4727). Here we show that Aβ40 and Aβ42 peptides are degraded by purified human brain MBP and recombinant human MBP, but not an MBP fragment that lacks autolytic activity. MBP-mediated Aβ degradation is inhibited by serine proteinase inhibitors. Similarly, Cos-1 cells expressing MBP degrade exogenous Aβ40 and Aβ42. In addition, we demonstrate that purified MBP also degrades assembled fibrillar Aβ in vitro. Mass spectrometry analysis identified distinct degradation products generated from Aβ digestion by MBP. Lastly, we demonstrate in situ that purified MBP can degrade parenchymal amyloid plaques as well as cerebral vascular amyloid that form in brain tissue of Aβ precursor protein transgenic mice. Together, these findings indicate that purified MBP possesses Aβ degrading activity in vitro.The progressive accumulation of β-amyloid (Aβ)³ in senile/neuritic plaques and the cerebral vasculature is the hallmark of Alzheimer disease (AD) and is widely used in the pathological diagnosis of the disease. Aβ is generated by proteolytic cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase (1, 2). The main species of Aβ are 40 and 42 amino acids in length. Aβ42 is much more amyloidogenic than Aβ40 because of its two additional hydrophobic amino acids at the carboxyl-terminal end of the peptide (3). The Aβ42 peptide is the predominant form in senile plaques, forming a β-sheet structure, which is insoluble and resistant to proteolysis.Although increased production of Aβ has been implicated in the onset of familial forms of AD, it has been hypothesized that the more common sporadic forms of AD may be caused by the impaired clearance of Aβ peptides from the CNS. Several major pathways for Aβ clearance have been proposed including receptor-mediated cellular uptake, blood-brain barrier transport into the circulation, and direct proteolytic degradation (4–6). In the latter case, several proteinases or peptidases have been identified that are capable of degrading Aβ, including neprilysin (7, 8), insulin-degrading enzyme (9), the urokinase/tissue plasminogen activator-plasmin system (10), endothelin-converting enzyme (11), angiotensin-converting enzyme (12), gelatinase A (matrix metalloproteinase-2) (13, 14), gelatinase B (matrix metalloproteinase-9) (15), and acylpeptide hydrolase (16). Each of these enzymes has been shown to cleave Aβ peptides at multiple sites (5). However, only neprilysin, insulin-degrading enzyme, endothelin-converting enzyme, and matrix metalloproteinase-9 have been shown to have a significant role in regulating Aβ levels in the brains of experimental animal models (8, 17, 18).The “classic” myelin basic proteins (MBP) are major structural components of myelin sheaths accounting for 30% of total myelin protein. There are four different major isoforms generated from alternative splicing with molecular masses of 17.3, 18.5, 20.2, and 21.5 kDa. The 18.5-kDa variant, composed of 180 amino acids including 19 Arg and 12 Lys basic residues, is most abundant in mature myelin (19). One of the major functions of MBP is to hold together the cytoplasmic leaflets of myelin membranes to maintain proper compaction of the myelin sheath through the electrostatic interaction between the positive Arg and Lys residues of MBP and the negatively charged phosphate groups of the membrane lipid (20). MBP plays an important role in the pathology of multiple sclerosis, which is an autoimmune disease characterized by demyelination within white matter (21). Recently, it was reported that purified MBP exhibits autocleavage activity, generating distinct peptide fragments (22). In this study, serine 151 was reported as the active site serine residue involved in autocatalysis.In the early stages of AD, appreciable and diffuse myelin breakdown in the white matter is observed (23). Also, in white matter regions there are much fewer fibrillar amyloid deposits than are commonly found in gray matter regions. Recently, our laboratory has shown that MBP strongly interacts with Aβ peptides and prevents their assembly into mature amyloid fibrils (24, 25). Through the course of these studies we observed that upon longer incubations the levels of Aβ peptides were reduced upon treatment with MBP. In light of this observation, coupled with the report that MBP possesses proteolytic activity, we hypothesized that MBP may degrade Aβ peptides. In the present study, we show that purified human brain MBP and recombinantly expressed human MBP can degrade soluble Aβ40 and Aβ42 peptides in vitro. Purified MBP also degraded fibrillar Aβ in vitro. Mass spectrometry analysis identified distinct degradation products generated from soluble and fibrillar Aβ digestion by MBP. Furthermore, purified MBP degraded parenchymal and vascular fibrillar amyloid deposits in situ in the brain tissue of APP transgenic mice. Together, these findings indicate that purified MBP possesses Aβ degrading activity in vitro.     

The Role of the Amyloid Protein Precursor (APP) in Alzheimer's Disease: Does the Normal Function of APP Explain the Topography of Neurodegeneration?

Alzheimer's disease (AD) is the most common form of dementia in the aged population. Early-onset familial AD (FAD) involves mutations in a gene on chromosome 21 encoding the amyloid protein precursor or on chromosomes 14 or 1 encoding genes known as presenilins. All mutations examined have been found to increase the production of amyloidogenic forms of the amyloid protein (A), a 4 kDa peptide derived from APP. Despite the remarkable progress in elucidating the biochemical mechanisms responsible for AD, little is known about the normal function of APP. A model of how APP and A are involved in pathogenesis is presented. This model may explain why certain neuronal populations are selectively vulnerable in AD. It is suggested that those neurons which more readily undergo neuritic sprouting and synaptic remodelling are more vulnerable to A neurotoxicity.     

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β₂-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.     

Mitochondrial Mislocalization Underlies Aβ42-Induced Neuronal Dysfunction in a Drosophila Model of Alzheimer's Disease

The amyloid-β 42 (Aβ42) is thought to play a central role in the pathogenesis of Alzheimer''s disease (AD). However, the molecular mechanisms by which Aβ42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Aβ42 induces mitochondrial mislocalization, which contributes to Aβ42-induced neuronal dysfunction in a transgenic Drosophila model. In the Aβ42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Aβ42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Aβ42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Aβ42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Aβ42 in vivo.     

Amyloid β Oligomeric Species Present in the Lag Phase of Amyloid Formation

Alzheimer’s disease (AD)-associated amyloid β peptide (Aβ) is one of the main actors in AD pathogenesis. Aβ is characterized by its high tendency to self-associate, leading to the generation of oligomers and amyloid fibrils. The elucidation of pathways and intermediates is crucial for the understanding of protein assembly mechanisms in general and in conjunction with neurodegenerative diseases, e.g., for the identification of new therapeutic targets. Our study focused on Aβ42 and its oligomeric assemblies in the lag phase of amyloid formation, as studied by sedimentation velocity (SV) centrifugation. The assembly state of Aβ during the lag phase, the time required by an Aβ solution to reach the exponential growth phase of aggregation, was characterized by a dominant monomer fraction below 1 S and a population of oligomeric species between 4 and 16 S. From the oligomer population, two major species close to a 12-mer and an 18-mer with a globular shape were identified. The recurrence of these two species at different initial concentrations and experimental conditions as the smallest assemblies present in solution supports the existence of distinct, energetically favored assemblies in solution. The sizes of the two species suggest an Aβ42 aggregation pathway that is based on a basic hexameric building block. The study demonstrates the potential of SV analysis for the evaluation of protein aggregation pathways.     

Novel 5′ Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer''s disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer''s disease.     

Mint Proteins Are Required for Synaptic Activity-dependent Amyloid Precursor Protein (APP) Trafficking and Amyloid β Generation

Aberrant amyloid β (Aβ) production plays a causal role in Alzheimer disease pathogenesis. A major cellular pathway for Aβ generation is the activity-dependent endocytosis and proteolytic cleavage of the amyloid precursor protein (APP). However, the molecules controlling activity-dependent APP trafficking in neurons are less defined. Mints are adaptor proteins that directly interact with the endocytic sorting motif of APP and are functionally important in regulating APP endocytosis and Aβ production. We analyzed neuronal cultures from control and Mint knockout neurons that were treated with either glutamate or tetrodotoxin to stimulate an increase or decrease in neuronal activity, respectively. We found that neuronal activation by glutamate increased APP endocytosis, followed by elevated APP insertion into the cell surface, stabilizing APP at the plasma membrane. Conversely, suppression of neuronal activity by tetrodotoxin decreased APP endocytosis and insertion. Interestingly, we found that activity-dependent APP trafficking and Aβ generation were blocked in Mint knockout neurons. We showed that wild-type Mint1 can rescue APP internalization and insertion in Mint knockout neurons. In addition, we found that Mint overexpression increased excitatory synaptic activity and that APP was internalized predominantly to endosomes associated with APP processing. We demonstrated that presenilin 1 (PS1) endocytosis requires interaction with the PDZ domains of Mint1 and that this interaction facilitates activity-dependent colocalization of APP and PS1. These findings demonstrate that Mints are necessary for activity-induced APP and PS1 trafficking and provide insight into the cellular fate of APP in endocytic pathways essential for Aβ production.     

Activation of LA-N-2 Cell Phospholipase D by Amyloid Beta Protein (25–35)

Amyloid protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25–35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and AP (25–35) was calcium-independent. The AP (25–35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AP (25–35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AP (25–35). Only NBQX was effective with an IC₅₀ of 75 M for AP (25–35) and quisqualate. Activation of phospholipase D by AP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AP (25–35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AP (25–35).