Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3beta

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in the brains of individuals with Alzheimer’s disease (AD). We recently found that the glycosylation might precede hyperphosphorylation of tau in AD. In this study, we investigated the effect of glycosylation on phosphorylation of tau catalyzed by cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase-3β (GSK-3β). The phosphorylation of the longest isoform of recombinant human brain tau, tau₄₄₁, at various sites was detected by Western blots and by radioimmuno-dot-blot assay with phosphorylation-dependent and site-specific tau antibodies. We found that cdk5 phosphorylated tau₄₄₁ at Thr-181, Ser-199, Ser-202, Thr-205, Thr-212, Ser-214, Thr-217, Thr-231, Ser-235, Ser-396, and Ser-404, but not at Ser-262, Ser-400, Thr-403, Ser-409, Ser-413, or Ser-422. GSK-3β phosphorylated all the cdk5-catalyzed sites above except Ser-235. Deglycosylation by glycosidases depressed the subsequent phosphorylation of AD-tau (i) with cdk5 at Thr-181, Ser-199, Ser-202, Thr-205, and Ser-404, but not at Thr-212; and (ii) with GSK-3β at Thr-181, Ser-202, Thr-205, Ser-217, and Ser-404, but not at Ser-199, Thr-212, Thr-231, or Ser-396. These data suggest that aberrant glycosylation of tau in AD might be involved in neurofibrillary degeneration by promoting abnormal hyperphosphorylation by cdk5 and GSK-3β.     

Regulation of Phosphorylation of tau by Protein Kinases in Rat Brain

Microtubule associated protein tau is abnormally hyperphosphorylated in Alzheimer disease (AD) brain. To investigate the role of protein kinases involved in this lesion, metabolically active slices made from brains of adult rats were treated with or without various specific kinase activators in oxygenated artificial cerebrospinal fluid. The basal kinase activities of protein kinase-A (PKA), CaM Kinase II and GSK-3 were stimulated more than two-fold by isoproterenol, bradykinin and wortmannin, respectively. We found that cdk5 activity was co-stimulated with PKA by isoproterenol. Sequential activation of PKA (+cdk5), CaM Kinase II and GSK-3 produced hyperphosphorylation of tau at Ser-198/Ser-199/Ser-202, Ser-214, Thr-231/Ser-235, Ser-262, Ser-396/Ser-404 and Ser-422 sites. Like AD P-tau, the P-tau from brain slices bound to normal tau and its binding to tubulin was inhibited. These studies suggest that PKA, cdk5, CaM Kinase II and GSK-3 are involved in the regulation of phosphorylation of tau and that AD-type phosphorylation of tau is probably a product of the synergistic action of two or more of these kinases.     

Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3beta signaling

Zinc is an essential catalytic and structural element of many proteins and a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes severe neuronal cell death. We have previously observed that zinc-induced neuronal cell death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells. In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with 10 microM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was accompanied by increased Tyr-phosphorylated GSK-3beta as well as increased GSK-3beta kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably suppressed GSK-3beta activation and cell death. Furthermore, tau phosphorylation, but not the degradation of beta-catenin, was dependent upon zinc-induced GSK-3beta activation and contributed to cell death. The current data suggest that, following exposure to zinc, the sequential activation of Akt and GSK-3beta plays an important role directing hippocampal neural precursor cell death.     

Microtubule destruction induces tau liberation and its subsequent phosphorylation

Neurofibrillary tangle-bearing neurons, a pathological hallmark of Alzheimer’s disease, are mostly devoid of normal microtubule (MT) structure and instead have paired helical filaments that are composed of abnormal hyperphosphorylated tau. However, a causal relationship between tau phosphorylation and MT disruption has not been clarified. To examine whether MT disruption induces tau phosphorylation, stathmin, an MT-disrupting protein, was co-expressed with tau in COS-7 cells. Stathmin expression induced apparent MT catastrophe and tau hyperphosphorylation at Thr-181, Ser-202, Thr-205, and Thr-231 sites. In contrast, c-Jun N-terminal kinase activation, or phosphatase inhibition, led to significant tau phosphorylation without affecting MT structure. These findings suggest that MT disruption induces subsequent tau phosphorylation.     

Significance of the degree of synaptic Zn<Superscript>2+</Superscript> signaling in cognition

Zinc is a trace nutrient for the brain and a signal factor to serve for brain function. A portion of zinc is released from glutamatergic (zincergic) neuron terminals in the brain. Synaptic Zn²⁺ signaling is involved in synaptic plasticity such as long-term potentiaion (LTP), which is a cellular mechanism of memory. The block and/or loss of synaptic Zn²⁺ signaling in the hippocampus and amygdala with Zn²⁺ chelators affect cognition, while the role of synaptic Zn²⁺ signal is poorly understood, because zinc-binding proteins are great in number and multi-functional. Chronic zinc deficiency also affects cognition and cognitive decline induced by zinc deficiency might be associated with the increase in plasma glucocorticoid rather than the decrease in synaptic Zn²⁺ signaling. On the other hand, excess glutamatergic (zincergic) neuron activity induces excess influx of extracellular Zn²⁺ into hippocampal neurons, followed by cognitive decline. Intracellular Zn²⁺ dynamics, which is linked to presynaptic glutamate release, is critical for LTP and cognitive performance. This paper deals with insight into cognition from zinc as a nutrient and signal factor.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

Reduced protein phosphatase 2A activity induces hyperphosphorylation and altered compartmentalization of tau in transgenic mice

Hyperphosphorylated isoforms of the microtubule-associated protein tau are the major components of neurofibrillary lesions in Alzheimer's disease (AD). Protein phosphatase (PP) 2A is a major phosphatase implicated in tau dephosphorylation in vitro. Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor of PP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role of PP2A in tau phosphorylation and pathogenesis, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of Calpha L199P in cortical, hippocampal, and cerebellar neurons. PP2A activity in brain homogenates of transgenic mice was reduced to 66%. Endogenous tau protein was hyperphosphorylated at distinct sites including the AT8 epitope Ser-202/Thr-205, a major AD-associated tau phosphoepitope. AT8-positive tau aggregates accumulated in the soma and dendrites of cortical pyramidal cells and cerebellar Purkinje cells and co-localized with ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation. Our results also show that reduced PP2A activity is associated with altered compartmentalization and ubiquitination of tau, resembling a key pathological finding in AD.     

Asp664 cleavage of amyloid precursor protein induces tau phosphorylation by decreasing protein phosphatase 2A activity

Caspase cleavage of amyloid precursor protein (APP) has been reported to be important in amyloid beta protein (Aβ)‐mediated neurotoxicity. However, the underlying mechanisms are not clearly understood. In this study, we explored the effect of caspase cleavage of APP on tau phosphorylation in relation to Aβ. We found that Asp664 cleavage of APP increased tau phosphorylation at Thr212 and Ser262 in N2A cells and primary cultured hippocampal neurons. Compared with wild‐type APP, protein phosphatase 2A (PP2A) activity was significantly increased when Asp664 cleavage was blocked by the D664A point mutation. Furthermore, we found that over‐expression of C31 reduced PP2A activity. C31 binds directly to the PP2A catalytic subunit, through the asparagine, proline, threonine, tyrosine (NPTY) motif, which is essential for C31‐induced tau hyperphosphorylation. However, it appears that the other fragment produced by Asp664 cleavage, Jcasp, modulates neither PP2A activity nor tau hyperphosphorylation. Asp664 cleavage and accompanying tau hyperphosphorylation were remarkably diminished by blockage of Aβ production using a γ‐secretase inhibitor. Taken together, our results suggest that Asp664 cleavage of APP leads to tau hyperphosphorylation at specific epitopes by modulating PP2A activity as a downstream of Aβ. Direct binding of C31 to PP2A through the C31‐NPTY domain was identified as a mechanism underlying this effect.     

L-trans-PDC enhances hippocampal neuronal activity by stimulating glial glutamate release independently of blocking transporters

The glutamate transporter inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) reversibly enhanced hippocampal neuronal activity in the rat and mouse dentate gyrus. The PDC action was still found in mice lacking the glial glutamate transporter GLT-1. PDC did not influence the rate of spontaneous miniature excitatory postsynaptic currents and spontaneous inhibitory postsynaptic currents, ionotropic glutamate receptor currents, or GABA-evoked currents in cultured rat hippocampal neurons. PDC increased glutamate released from cultured hippocampal astrocytes from normal rats, normal mice, and GLT-1 knock-out mice, that is not inhibited by deleting extracellular Na(+), while the drug had no effect on the release from cultured rat hippocampal neurons. The results of the present study thus suggest that PDC stimulates glial glutamate release by a mechanism independent of inhibiting glutamate transporters, which perhaps causes an increase in synaptic glutamate concentrations, in part responsible for the enhancement in hippocampal neuronal activity.     

Protective Effects of Humanin on Okadaic Acid-Induced Neurotoxicities in Cultured Cortical Neurons

Neurofibrillary tangles are pathological hallmarks of Alzheimer’s disease (AD), which are mostly composed of hyperphosphorylated tau and directly correlate with dementia in AD patients. Okadaic acid (OA), a toxin extracted from marine life, can specifically inhibit protein phosphatases (PPs), including PP1 and Protein phosphatase 2A (PP2A), resulting in tau hyperphosphorylation. Humanin (HN), a peptide of 24 amino acids, was initially reported to protect neurons from AD-related cell toxicities. The present study was designed to test if HN could attenuate OA-induced neurotoxicities, including neural insults, apoptosis, autophagy, and tau hyperphosphorylation. We found that administration of OA for 24 h induced neuronal insults, including lactate dehydrogenase released, decreased of cell viability and numbers of living cells, neuronal apoptosis, cells autophagy and tau protein hyperphosphorylation. Pretreatment of cells with HN produced significant protective effects against OA-induced neural insults, apoptosis, autophagy and tau hyperphosphorylation. We also found that OA treatment inhibited PP2A activity and HN pretreatment significantly attenuated the inhibitory effects of OA. This study demonstrated for the first time that HN protected cortical neurons against OA-induced neurotoxicities, including neuronal insults, apoptosis, autophagy, and tau hyperphosphorylation. The mechanisms underlying the protections of HN may involve restoration of PP2A activity.     

Extrasynaptic membrane trafficking regulated by GluR1 serine 845 phosphorylation primes AMPA receptors for long-term potentiation

Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-D-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP.     

Regulation of s6 kinase 1 activation by phosphorylation at ser-411

Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G(1) phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1.     

Memantine inhibits and reverses the Alzheimer type abnormal hyperphosphorylation of tau and associated neurodegeneration

Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, reduces the clinical deterioration in moderate-to-severe Alzheimer disease (AD) for which other treatments are not available. The activity of protein phosphatase (PP)-2A is compromised in AD brain and is believed to be a cause of the abnormal hyperphosphorylation of tau and the consequent neurofibrillary degeneration. Here we show that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices. Such restorative effects of memantine were not detected either with 5,7-dichlorokynurenic acid or with D(-)-2-amino-5-phosphopentanoic acid, NMDA receptor antagonists active at the glycine binding site and at the glutamate binding site, respectively. These findings show (1) that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation of tau/neurofibrillary degeneration and (2) that this drug might be useful for the treatment of AD and related tauopathies.     

Inhibitory Interactions between Phosphorylation Sites in the C Terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor GluA1 Subunits

The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca²⁺ sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca²⁺ influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function.     

Promotion of hyperphosphorylation by frontotemporal dementia tau mutations

Mutations in the tau gene are known to cosegregate with the disease in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). However, the molecular mechanism by which these mutations might lead to the disease is not understood. Here, we show that four of the FTDP-17 tau mutations, R406W, V337M, G272V, and P301L, result in tau proteins that are more favorable substrates for phosphorylation by brain protein kinases than the wild-type, largest four-repeat protein tau4L and tau4L more than tau3L. In general, at all the sites studied, mutant tau proteins were phosphorylated faster and to a higher extent than tau4L and tau4L > tau3L. The most dramatic difference found was in the rate and level of phosphorylation of tau4L(R406W) at positions Ser-396, Ser-400, Thr-403, and Ser-404. Phosphorylation of this mutant tau was 12 times faster and 400% greater at Ser-396 and less than 30% at Ser-400, Thr-403, and Ser-404 than phosphorylation of tau4L. The mutated tau proteins polymerized into filaments when 4-6 mol of phosphate per mol of tau were incorporated, whereas wild-type tau required approximately 10 mol of phosphate per mol of protein to self-assemble. Mutated and wild-type tau proteins were able to sequester normal tau upon incorporation of approximately 4 mol of phosphate per mol of protein, which was achieved at as early as 30 min of phosphorylation in the case of mutant tau proteins. These findings taken together suggest that the mutations in tau might cause neurodegeneration by making the protein a more favorable substrate for hyperphosphorylation.     

The environmental toxin arsenite induces tau hyperphosphorylation

Abnormally hyperphosphorylated tau polymers known as paired helical filaments constitute one of the major characteristic lesions that lead to the demise of neurons in Alzheimer's disease. Here, we demonstrate that the environmental toxin arsenite causes a significant increase in the phosphorylation of several amino acid residues (Thr-181, Ser-202, Thr-205, Thr-231, Ser-262, Ser-356, Ser-396, and Ser-404) in tau, which are also hyperphosphorylated under pathological conditions. Complementary phosphopeptide mapping revealed a dramatic increase in the (32)P-labeling of many peptides in tau following arsenite treatment. Although arsenite activates extracellular-signal regulated kinases-1/-2 and stress-activated protein kinases, these enzymes did not contribute to the arsenite-increased phosphorylation, nor did they appear to normally modify tau in vivo. Tau phosphorylation induced by arsenite did not involve glycogen synthase kinase-3 or protein phosphatase-1 or -2, but the activity responsible for tau hyperphosphorylation could be inhibited with the protein kinase inhibitor roscovitine. The effects of arsenite on the phosphorylation of some tau mutations (DeltaKappa280, V337M, and R406W) associated with frontal-temporal dementia with parkinsonism linked to chromosome 17 was analyzed. The unchallenged and arsenite-induced phosphorylation of some mutant proteins, especially R406W, was altered at several phosphorylation sites, indicating that these mutations can significantly affect the structure of tau in vivo. Although the major kinase(s) involved in aberrant tau phosphorylation remains elusive, these results indicate that environmental factors, such as arsenite, may be involved in the cascade leading to deregulation of tau function associated with neurodegeneration.     

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.     

Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals

KCl and 4-aminopyridine (4-AP) evoke glutamate release from rat brain cortical nerve terminals by voltage clamping or by Na(+) channel-generated repetitive action potentials, respectively. Stimulation by 4-AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4-AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2-10. A strong correlation was observed for increasing concentrations of KCl and after application of phorbol 12-myristate 13-acetate (PMA) or staurosporine. The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC-independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4-AP, alone or with staurosporine, glutamate and FM2-10 release also correlated. However, higher concentrations of 4-AP or of 4-AP plus PMA greatly increased glutamate release but did not further increase FM2-10 release. This divergence suggests that 4-AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss-and-run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent; and (2) a kiss-and-run-like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.     

Glucose increases synaptic transmission from vagal afferent central nerve terminals via modulation of 5-HT3 receptors

Acute hyperglycemia has profound effects on vagally mediated gastrointestinal functions. We have reported recently that the release of glutamate from the central terminals of vagal afferent neurons is correlated directly with the extracellular glucose concentration. The present study was designed to test the hypothesis that 5-HT(3) receptors present on vagal afferent nerve terminals are involved in this glucose-dependent modulation of glutamatergic synaptic transmission. Whole-cell patch-clamp recordings were made from neurons of the nucleus tractus solitarius (NTS) in thin rat brainstem slices. Spontaneous and evoked glutamate release was decreased in a concentration-dependent manner by the 5-HT(3) receptor selective antagonist, ondansetron. Alterations in the extracellular glucose concentration induced parallel shifts in the ondansetron-mediated inhibition of glutamate release. The changes in excitatory synaptic transmission induced by extracellular glucose concentration were mimicked by the serotonin uptake inhibitor, fenfluramine. These data suggest that glucose alters excitatory synaptic transmission within the rat brainstem via actions on tonically active 5-HT(3) receptors, and the number of 5-HT(3) receptors on vagal afferent nerve terminals is positively correlated with the extracellular glucose concentration. These data indicate that the 5-HT(3) receptors present on synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose acts to modulate vagovagal reflexes.     

Indomethacin enhances learning and memory potential by interacting with CaMKII

The present study examined the effect of indomethacin (IM), a cyclooxygenase inhibitor, on learning and memory functions. IM activated Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) in cultured rat hippocampal neurons. IM (100 μM) significantly increased the rate of spontaneous AMPA receptor-mediated miniature excitatory postsynaptic currents elicited from CA1 pyramidal neurons of rat hippocampal slices, without affecting the amplitude, and enhanced extracellular high K(+) (20 mM)-induced glutamate release from rat hippocampal slices, indicating that IM stimulates presynaptic glutamate release. Those IM effects were clearly inhibited by the CaMKII inhibitor KN-93. IM persistently facilitated synaptic transmission monitored from the CA1 region of rat hippocampal slices in a concentration (1-100 μM)-dependent manner that was also abolished by KN-93. In the water maze test, IM (1 mg/kg, i.p.) enhanced spatial learning and memory ability for normal rats, and ameliorated scopolamine-induced spatial learning and memory impairment or age-related spatial learning and memory deterioration for senescence-accelerated mouse-prone 8 mice. In the test to learn 15 numbers consisting of three patterns of five digit number for healthy human subjects, oral intake with IM (25 mg/kg) significantly raised the scores of correct number arrangements that subjects memorized 5 min and 3 days after the test. The results of the present study indicate that IM could enhance learning and memory potential by facilitating hippocampal synaptic transmission as a result from stimulating presynaptic glutamate release under the control of CaMKII.