NMDA receptor/amyloid precursor protein interactions: a comparison between wild-type and amyloid precursor protein mutations associated with familial Alzheimer's disease

Two recent reports showed that amyloid precursor protein (APP) may contribute to postsynaptic mechanisms via the regulation of the surface trafficking of excitatory N-methyl-D-aspartate (NMDA) receptors. Here we have investigated the interactions and surface trafficking of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptor subtypes with three APP mutations linked to familial Alzheimer's disease, APP695(Indiana), APP695(London) and APP695(Swedish). Flag-tagged mutated APP695s were generated and shown to be expressed at equivalent levels to wild-type APP695 in mammalian cells. Each APP mutant co-precipitated with NR1-1a/NR2A and NR1-1a/NR2B receptors following co-expression in mammalian cells. Further, as found for wild-type APP695, each enhanced NMDA receptor surface expression with no concomitant increase in total NR1-1a, NR2A or NR2B subunit expression. Thus these three familial APP mutations behave as wild-type APP695 with respect to their association with assembled NMDA receptors and their APP695-enhanced receptor cell surface trafficking.     

Immunohistochemical evidence for synaptic release of glutamate from orexin terminals in the locus coeruleus

Orexin (Orx or hypocretin) is critically important for maintaining wakefulness, since in its absence, narcolepsy with cataplexy occurs. In this role, Orx-containing neurons can exert their influence upon multiple targets through the brain by release of Orx but possibly also by release of other neurotransmitters. Indeed, evidence was previously presented to suggest that Orx terminals could utilize glutamate (Glu) in addition to Orx as a neurotransmitter. Using fluorescence and confocal laser scanning microscopy, we investigated whether Orx varicosities contain the presynaptic markers for synaptic release of Glu or GABA and come into contact with postsynaptic markers for excitatory synapses within the locus coeruleus of the rat brain. We found that a proportion of the Orx+ varicosities were immunostained for the vesicular transporter for Glu, VGluT2. None were immunostained for vesicular glutamate transporter 1 (VGluT1) or VGluT3 or for the vesicular transporter for GABA, vesicular GABA transporter (VGAT). Among the Orx+ varicosities, 4% of all and 28% of large varicosities contained VGluT2. A similar proportion of the large Orx+ varicosities contained synaptophysin (Syp), a presynaptic marker for synaptic vesicles. Orx+ varicosities also contacted elements immunostained for postsynaptic density protein-95 (PSD)-95, a postsynaptic marker for glutamatergic synapses. We thus conclude that synaptic release of Glu occurs from Orx terminals within the locus coeruleus and can thus be important for the engagement of noradrenergic neurons in stimulating and maintaining arousal.     

Neurotransmitter receptors and cognitive dysfunction in Alzheimer's disease and Parkinson's disease

Cognitive dysfunction is one of the most typical characteristics in various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (advanced stage). Although several mechanisms like neuronal apoptosis and inflammatory responses have been recognized to be involved in the pathogenesis of cognitive dysfunction in these diseases, recent studies on neurodegeneration and cognitive dysfunction have demonstrated a significant impact of receptor modulation on cognitive changes. The pathological alterations in various receptors appear to contribute to cognitive impairment and/or deterioration with correlation to diversified mechanisms. This article recapitulates the present understandings and concepts underlying the modulation of different receptors in human beings and various experimental models of Alzheimer's disease and Parkinson's disease as well as a conceptual update on the underlying mechanisms. Specific roles of serotonin, adrenaline, acetylcholine, dopamine receptors, and N-methyl-D-aspartate receptors in Alzheimer's disease and Parkinson's disease will be interactively discussed. Complex mechanisms involved in their signaling pathways in the cognitive dysfunction associated with the neurodegenerative diseases will also be addressed. Substantial evidence has suggested that those receptors are crucial neuroregulators contributing to cognitive pathology and complicated correlations exist between those receptors and the expression of cognitive capacities. The pathological alterations in the receptors would, therefore, contribute to cognitive impairments and/or deterioration in Alzheimer's disease and Parkinson's disease. Future research may shed light on new clues for the treatment of cognitive dysfunction in neurodegenerative diseases by targeting specific alterations in these receptors and their signal transduction pathways in the frontal-striatal, fronto-striato-thalamic, and mesolimbic circuitries.     

A framework to understand the variations of PSD-95 expression in brain aging and in Alzheimer's disease

The postsynaptic density protein PSD-95 is a major element of synapses. PSD-95 is involved in aging, Alzheimer's disease (AD) and numerous psychiatric disorders. However, contradictory data about PSD-95 expression in aging and AD have been reported. Indeed in AD versus control brains PSD-95 varies according to regions, increasing in the frontal cortex, at least in a primary stage, and decreasing in the temporal cortex. In contrast, in transgenic mouse models of aging and AD PSD-95 expression is decreased, in behaviorally aged impaired versus unimpaired rodents it can decrease or increase and finally, it is increased in rodents grown in enriched environments. Different factors explain these contradictory results in both animals and humans, among others concomitant psychiatric endophenotypes, such as depression. The possible involvement of PSD-95 in reactive and/or compensatory mechanisms during AD progression is underscored, at least before the occurrence of important synaptic elimination. Thus, in AD but not in AD transgenic mice, enhanced expression might precede the diminution commonly observed in advanced aging. A two-compartments cell model, separating events taking place in cell bodies and synapses, is presented. Overall these data suggest that AD research will progress by untangling pathological from protective events, a prerequisite for effective therapeutic strategies.     

Ammonia-induced alterations in glutamate and muscimol binding to cerebellar synaptic membranes

Binding of glutamate and muscimol (an agonist for GABA_A receptors) to their respective receptors has been studied in the cerebellum of normal and hyperammonemic rats. There was a decrease in both high- and low-affinity binding of glutamate in the cerebellum during hyperammonemia. Kinetic studies revealed that the decrease is due to a reduction in the number of binding sites, but not due to changes in the binding affinities. Further studies also revealed that the decrease was only in the (NMDA)-specific binding sites without any alterations in the binding to non-NMDA sites represented by kianic acid (KA)- and quisqualic acid (QQ)-sensitive receptor sites. These effects were also mimicked when the membrane preparations from the cerebellum of normal animals were incubated with ammonium acetate. Enhancement of muscimol binding was observed in animals injected with ammonium acetate. It is concluded that hyperammonemic states, even in the presence of a functional liver, are capable of altering amino acid neurotransmission and this might play an important role in cerebral dysfunction under these conditions.     

Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus

Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unopposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex.     

Neuroadaptations in the cellular and postsynaptic group 1 metabotropic glutamate receptor mGluR5 and Homer proteins following extinction of cocaine self-administration

This study examined the role of group1 metabotropic glutamate receptor mGluR5 and associated postsynaptic scaffolding protein Homer1b/c in behavioral plasticity after three withdrawal treatments from cocaine self-administration. Rats self-administered cocaine or saline for 14 days followed by a withdrawal period during which rats underwent extinction training, remained in their home cages, or were placed in the self-administration chambers in the absence of extinction. Subsequently, the tissue level and distribution of proteins in the synaptosomal fraction associated with the postsynaptic density were examined. Cocaine self-administration followed by home cage exposure reduced the mGluR5 protein in nucleus accumbens (NA) shell and dorsolateral striatum. While extinction training reduced mGluR5 protein in NAshell, NAcore and dorsolateral striatum did not display any change. The scaffolding protein PSD95 increased in NAcore of the extinguished animals. Extinction of drug seeking was associated with a significant decrease in the synaptosomal mGluR5 protein in NAshell and an increase in dorsolateral striatum, while that of NAcore was not modified. Interestingly, both Homer1b/c and PSD95 scaffolding proteins were decreased in the synaptosomal fraction after extinction training in NAshell but not NAcore. Extinguished drug-seeking behavior was also associated with an increase in the synaptosomal actin proteins in dorsolateral striatum. Therefore, extinction of cocaine seeking is associated with neuroadaptations in mGluR5 expression and distribution that are region-specific and consist of extinction-induced reversal of cocaine-induced adaptations as well as emergent extinction-induced alterations. Concurrent plasticity in the scaffolding proteins further suggests that mGluR5 receptor neuroadaptations may have implications for synaptic function.     

The intersection of amyloid beta and tau in glutamatergic synaptic dysfunction and collapse in Alzheimer's disease

The synaptic connections that form between neurons during development remain plastic and able to adapt throughout the lifespan, enabling learning and memory. However, during aging and in particular in neurodegenerative diseases, synapses become dysfunctional and degenerate, contributing to dementia. In the case of Alzheimer's disease (AD), synapse loss is the strongest pathological correlate of cognitive decline, indicating that synaptic degeneration plays a central role in dementia. Over the past decade, strong evidence has emerged that oligomeric forms of amyloid beta, the protein that accumulates in senile plaques in the AD brain, contribute to degeneration of synaptic structure and function. More recent data indicate that pathological forms of tau protein, which accumulate in neurofibrillary tangles in the AD brain, also cause synaptic dysfunction and loss. In this review, we will present the case that soluble forms of both amyloid beta and tau protein act at the synapse to cause neural network dysfunction, and further that these two pathological proteins may act in concert to cause synaptic pathology. These data may have wide-ranging implications for the targeting of soluble pathological proteins in neurodegenerative diseases to prevent or reverse cognitive decline.     

Growth hormone replacement in hypophysectomized rats affects spatial performance and hippocampal levels of NMDA receptor subunit and PSD-95 gene transcript levels

Clinical studies have demonstrated that growth hormone (GH) promotes learning and memory processes in GH-deficient (GHD) patients. In animal studies, GH also influences the N-methyl-D-aspartate (NMDA) receptor system in the hippocampus, an essential component of long-term potentiation (LTP), which is highly involved in memory acquisition. This study was designed to examine the beneficial effects of recombinant human GH (rhGH) on cognitive function in male rats with multiple hormone deficiencies resulting from hypophysectomy (Hx). The performance of an rhGH-treated group and an untreated control group was appraised in the Morris water maze (MWM). The rhGH-treated group performed significantly better in the spatial memory task than the control animals on the second and third trial days. Further training eliminated this difference between the groups. Hippocampal mRNA expression of the NMDA subunits NR1, NR2A and NR2B, insulin-like growth factor type 1 receptor (IGF-1R), and postsynaptic density protein-95 (PSD-95) was then measured in the animals by Northern blot analysis. The results suggest that there may be a relationship between the NMDA receptor subunit mRNA expression levels and learning ability, and that learning is improved by rhGH in Hx rats. Furthermore, a link between MWM performance and PSD-95 was also suggested by this study.     

Synaptic interactions involving acetylcholine,glutamate, and GABA in rat auditory cortex

Using electrophysiological techniques in the in vitro rat auditory cortex, we have examined how spontaneous acetylcholine (ACh) release modifies synaptic potentials mediated by glutamate and -aminobutyric acid (GABA). Single stimulus pulses to lower layer VI elicited in layer III a four-component (A-D) extracellular field response involving synaptic potentials mediated by glutamate and GABA. The cholinesterase inhibitor eserine (10–20 M) or the cholinergic agonist carbachol (25–50 M) depressed by 10–50% the glutamatergic components A and C, and the GABAergic components B and D. Atropine reversed the depressive effects of eserine and carbachol. A novel finding was that the degree of depression of component A varied inversely with stimulus intensity. However, during partial pharmacological antagonism of GABA_A receptors, depression of A varied directly, not inversely, with stimulus intensity. Normally, then, depression of A is offset by reduced GABAergic inhibition of A. We also tested for differential depression of responses mediated by N-methyl-d-aspartate (NMDA) versus non-NMDA glutamate receptors. Following physiological and pharmacological isolation of the responses, eserine depressed the non-NMDA, but not the NMDA, receptor-mediated potential. Since the isolated NMDA potential still could be depressed by carbachol, the data suggested that activation of NMDA receptors may reduce spontaneous ACh release. In support of this, preincubation of slices in NMDA (10–20 M) largely prevented eserine's, but not carbachol's, depression of components A and B.These results permit three conclusions of relevance to cortical information processing: (1) spontaneous ACh release tonically depresses synaptic potentials mediated by glutamate and GABA; (2) ACh depresses responses to weak inputs to a greater degree than responses to strong inputs; (3) activation of NMDA receptors may feed-back to reduce ACh release, a mechanism that could place regulation of local ACh release under glutamatergic afferent control.     

Role of NMDA and non-NMDA receptors in synaptic transmission in rat piriform cortex

Summary The pharmacology of synaptic transmission was studied in slices of rat piriform cortex using the selective non-NMDA glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) and the selective NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5). DNQX produced a dose-dependent blockade of synaptic transmission at both lateral olfactory tract and associational system synapses with half-maximal effects at about 2.5 M. D-AP5 had no significant effects on field potentials recorded in medium containing 2.5 mM Mg⁺⁺. However in low Mg⁺⁺ (100–200 M) medium, D-AP5 did reduce a slow component of postsynaptic responses in both synaptic systems. In Mg⁺⁺-free medium, 20 M DNQX did not completely block transmission; the remaining response components were blocked by D-AP5. These results suggest that normal synaptic transmission in the two main inputs to the superficial layers of piriform cortex is mediated by non-NMDA receptors but that NMDA receptors can also participate under conditions where the Mg⁺⁺ block of the NMDA channel is alleviated.     

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919–922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95–PMCA2 and syntaxin-1A–PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.     

Regulation of the NMDA receptor-mediated synaptic response by acetylcholinesterase inhibitors and its impairment in an animal model of Alzheimer's disease

The cholinergic system is crucial for cognitive processes and the deficient acetylcholine (ACh) function has been implicated in Alzheimer's disease (AD). Inhibitors of acetylcholinesterase (AChE), which act to enhance cholinergic function by prolonging the action of endogenously released ACh, have been used as the major therapy of AD. To understand the functional roles of cholinergic enhancement in prefrontal cortex (PFC), a key brain region for cognition, we examined the impact of AChE inhibitors in PFC neurons on synaptic responses mediated by the NMDA receptor (NMDAR), an important player in learning and memory. We found that AChE inhibitors produced a strong and persistent reduction of the amplitude of NMDA receptor-mediated excitatory postsynaptic current (NMDAR-EPSC). This effect was mainly mediated by nicotinic ACh receptors, and through a Ca²⁺-dependent mechanism. Inhibition of extracellular signal-regulated kinases (ERK) abolished the regulation of NMDAR function by AChE inhibitors, suggesting the involvement of ERK. In the transgenic mouse model of AD overexpressing mutant β-amyloid precursor protein (APP), the effect of AChE inhibitors on NMDAR-EPSC was significantly impaired, which was associated with their diminished effect on ERK activation. Taken together, these results suggest that one of the key targets of endogenous ACh involved in cognition is the NMDAR-mediated transmission. Loss of the regulation of synaptic NMDAR responses by endogenous ACh may contribute to the cognitive deficiency in AD.     

Siglec receptors and hiding plaques in Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease. One hallmark of this disease is the continuous increase in the numbers and size of aggregating amyloid plaques. The accumulation of extracellular plaques is an immunologically interesting phenomenon since microglial cells, brain-specific macrophages, should be able to cleanse the aggregating material from the human brain. Immunotherapy targeting β-amyloid peptides in plaques with antibodies represents a promising therapy in AD. Recent progress in pattern recognition receptors of monocytes and macrophages has revealed that the sialic acid-binding, immunoglobulin-like lectin (Siglec) family of receptors is an important recognition receptor for sialylated glycoproteins and glycolipids. Interestingly, recent studies have revealed that microglial cells contain only one type of Siglec receptors, Siglec-11, which mediates immunosuppressive signals and thus inhibits the function of other microglial pattern recognition receptors, such as TLRs, NLRs, and RAGE receptors. We will review here the recent literature which clearly indicates that aggregating amyloid plaques are masked in AD by sialylated glycoproteins and gangliosides. Sialylation and glycosylation of plaques, mimicking the cell surface glycocalyx, can activate the immunosuppressive Siglec-11 receptors, as well as hiding the neuritic plaques, allowing them to evade the immune surveillance of microglial cells. This kind of immune evasion can prevent the microglial cleansing process of aggregating amyloid plaques in AD.     

Muscarinic receptors in basal ganglia in dementia with Lewy bodies,Parkinson's disease and Alzheimer's disease

Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinson's disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.     

Staging of cytoskeletal and beta-amyloid changes in human isocortex reveals biphasic synaptic protein response during progression of Alzheimer's disease

We have examined the relationships between dementia, loss of synaptic proteins, changes in the cytoskeleton, and deposition of beta-amyloid plaques in the neocortex in a clinicopathologically staged epidemiological cohort using a combination of biochemical and morphometric techniques. We report that loss of synaptic proteins is a late-stage phenomenon, occurring only at Braak stages 5 and 6, or at moderate to severe clinical grades of dementia. Loss of synaptic proteins was seen only after the emergence of the full spectrum of tau and beta-amyloid pathology in the neocortex at stage 4, but not in the presence of beta-amyloid plaques alone. Contrary to previous studies, we report increases in the levels of synaptophysin, syntaxin, and SNAP-25 at stage 3 and of alpha-synuclein and MAP2 at stage 4. Minimal and mild clinical grades of dementia were associated with either unchanged or elevated levels of synaptic proteins in the neocortex. Progressive aggregation of paired helical filament (PHF)-tau protein could be detected biochemically from stage 2 onwards, and this was earliest change relative to the normal aging background defined by Braak stage 1 that we were able to detect in the neocortex. These results are consistent with the possibility that failure of axonal transport associated with early aggregation of tau protein elicits a transient adaptive synaptic response to partial de-afferentation that may be mediated by trophic factors. This early abnormality in cytoskeletal function may contribute directly to the earliest clinically detectable stages of dementia.     

Atrophy and dysfunction of parahippocampal white matter in mild Alzheimer's disease

In addition to atrophy of mesial temporal lobe structures critical for memory function, white matter projections to the hippocampus may be compromised in individuals with mild Alzheimer's disease (AD), thereby compounding the memory difficulty. In the present study, high-resolution structural imaging and diffusion tensor imaging techniques were used to examine microstructural alterations in the parahippocampal white matter (PWM) region that includes the perforant path. Results demonstrated white matter volume loss bilaterally in the PWM in patients with mild AD. In addition, the remaining white matter had significantly lower fractional anisotropy and higher mean diffusivity values. Both increased mean diffusivity and volume reduction in the PWM were associated with memory performance and ApoE ε4 allele status. These findings indicate that, in addition to partial disconnection of the hippocampus from incoming sensory information due to volume loss in PWM, microstructural alterations in remaining fibers may further degrade impulse transmission to the hippocampus and accentuate memory dysfunction. The results reported here also suggest that ApoE ε4 may exacerbate PWM changes.     

Preservation of 5-hydroxytryptamine1A receptor-G protein interactions in the cerebral cortex of patients with Alzheimer's disease.

The coupling of 5-hydroxytryptamine1A (5-HT1A) receptors to guanine nucleotide binding (G) proteins was investigated in membranes prepared from frontal and parietal cortices of control and Alzheimer's disease brains by characterising the effect of guanosine 5'-[beta gamma-imido]diphosphate (Gpp[NH]p) on [3H]8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]8-OH-DPAT) binding parameters. In the absence of guanine nucleotides, [3H]8-OH-DPAT bound to a single high affinity binding site in all membrane types. The number of [3H]8-OH-DPAT binding sites was significantly decreased in the parietal cortex of Alzheimer's disease samples compared with controls, whereas in the frontal cortex the number of binding sites remained unchanged. Gpp[NH]p reduced the [3H]8-OH-DPAT binding affinity and the number of binding sites to the same degree in both regions in control and Alzheimer's disease cases. [3H]8-OH-DPAT binding was inhibited in a concentration dependent manner with an IC50 value of approximately 1 microM in all cases. These results suggest that the 5-HT1A receptor-G protein complex is functionally intact in these regions in Alzheimer's disease brain.