Rap2-JNK removes synaptic AMPA receptors during depotentiation

The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.     

Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1.

Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-regulated by another small G protein Rap. Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1. Transient transfection of cells with constitutively active forms of the known IL-1 signaling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK. Dominant negative forms of these were found to inhibit activation of p38 MAPK by IL-1. Dominant negative RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies downstream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway. Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominant negative mutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominant negative MKK3 or MKK6. In the same manner, the inhibitory effect of Rap on the activation of p38 by IL-1 occurred at a point downstream of MyD88, IRAK, and TRAF6, since the activation of p38 MAPK by these components was inhibited by overexpressing active Rap1AV12, while neither MKK3 nor MKK6 were affected. Active RasVHa associated with IRAK, IRAK2, and TRAF6, but not MyD88. In addition we found a role for TAK-1 in the activation of p38 MAPK by IL-1, with TAK-1 also associating with active Ras. Our study suggests that upon activation Ras becomes associated with IRAK, Traf-6, and TAK-1, possibly aiding the assembly of this multiprotein signaling complex required for p38 MAPK activation by IL-1.     

TRAF6 is a critical mediator of signal transduction by the viral oncogene latent membrane protein 1

The oncogenic latent membrane protein 1 (LMP1) of the Epstein-Barr virus recruits tumor necrosis factor-receptor (TNFR)-associated factors (TRAFs), the TNFR-associated death domain protein (TRADD) and JAK3 to induce intracellular signaling pathways. LMP1 serves as the prototype of a TRADD-binding receptor that transforms cells but does not induce apoptosis. Here we show that TRAF6 critically mediates LMP1 signaling to p38 mitogen-activated protein kinase (MAPK) via a MAPK kinase 6-dependent pathway. In addition, NF-kappaB but not c-Jun N-terminal kinase 1 (JNK1) induction by LMP1 involves TRAF6. The PxQxT motif of the LMP1 C-terminal activator region 1 (CTAR1) and tyrosine 384 of CTAR2 together are essential for full p38 MAPK activation and for TRAF6 recruitment to the LMP1 signaling complex. Dominant-negative TRADD blocks p38 MAPK activation by LMP1. The data suggest that entry of TRAF6 into the LMP1 complex is mediated by TRADD and TRAF2. In TRAF6-knockout fibroblasts, significant induction of p38 MAPK by LMP1 is dependent on the ectopic expression of TRAF6. We describe a novel role of TRAF6 as an essential signaling mediator of a transforming oncogene, downstream of TRADD and TRAF2.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

NMDA receptor-dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD

The activity-dependent removal of AMPA receptors from synapses underlies long-term depression in hippocampal excitatory synapses. In this study, we have investigated the role of the small GTPase Rab5 during this process. We propose that Rab5 is a critical link between the signaling cascades triggered by LTD induction and the machinery that executes the activity-dependent removal of AMPA receptors. We have found that Rab5 activation drives the specific internalization of synaptic AMPA receptors in a clathrin-dependent manner and that this activity is required for LTD. Interestingly, Rab5 does not participate in the constitutive cycling of AMPA receptors. Rab5 is able to remove both GluR1 and GluR2 AMPA receptor subunits, leading to GluR1 dephosphorylation. Importantly, NMDA receptor-dependent LTD induction produces a rapid and transient increase of active (GTP bound) Rab5. We propose a model in which synaptic activity leads to Rab5 activation, which in turn drives the removal of AMPA receptors from synapses.     

A novel form of synaptic plasticity in field CA3 of hippocampus requires GPER1 activation and BDNF release

Estrogen is an important modulator of hippocampal synaptic plasticity and memory consolidation through its rapid action on membrane-associated receptors. Here, we found that both estradiol and the G-protein–coupled estrogen receptor 1 (GPER1) specific agonist G1 rapidly induce brain-derived neurotrophic factor (BDNF) release, leading to transient stimulation of activity-regulated cytoskeleton-associated (Arc) protein translation and GluA1-containing AMPA receptor internalization in field CA3 of hippocampus. We also show that type-I metabotropic glutamate receptor (mGluR) activation does not induce Arc translation nor long-term depression (LTD) at the mossy fiber pathway, as opposed to its effects in CA1, and it only triggers LTD after GPER1 stimulation. Furthermore, this form of mGluR-dependent LTD is associated with ubiquitination and proteasome-mediated degradation of GluA1, and is prevented by proteasome inhibition. Overall, our study identifies a novel mechanism by which estrogen and BDNF regulate hippocampal synaptic plasticity in the adult brain.     

Sustained activation of metabotropic glutamate receptor 5 and protein tyrosine phosphatases mediate the expression of (S)-3,5-dihydroxyphenylglycine-induced long-term depression in the hippocampal CA1 region

Previous studies have shown that brief application of group I metabotropic glutamate receptor (mGluR) agonist (S)-3, 5-dihydroxyphenylglycine (DHPG) to hippocampal slices can induce a chemical form of long-term depression (DHPG-LTD) in the hippocampal CA1 region; however, the expression mechanisms of this LTD remain unclear. We show here that the expression of DHPG-LTD can be specifically reversed by application of the broad-spectrum mGluR antagonists, (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY341495, and mGluR5 antagonist, 2-methyl-6-(phenylethyl)pyridine, but not by NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid, mGluR1 antagonist, LY367385, group II mGluR antagonist, (2S)-alpha-ethylglutamic acid, or group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanic acid (MAP4). In addition, the ability of MCPG to reverse DHPG-LTD was mimicked by the protein tyrosine phosphatase inhibitors, phenylarsine oxide and orthovanadate, but not phospholipase C inhibitor, U73122, protein kinase C inhibitor, bisindolylmaleimide 1, p38 mitogen-activated protein kinase inhibitor, SB203580, or protein phosphatases 1/2 A inhibitor, okadaic acid. Moreover, MCPG reversed the DHPG-LTD without affecting the paired-pulse facilitation. The expression of DHPG-LTD was associated with the reduction of both tyrosine phosphorylation and surface expression of AMPA receptor GluR2 subunits. Together, these results suggest that sustained activation of mGluR5 and in turn triggering a protein tyrosine phosphatase-dependent regulation of postsynaptic expression of AMPA receptors may contribute to the expression of DHPG-LTD.     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.     

AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression

The signal transduction and molecular mechanisms underlying alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-mediated neuroprotection are unknown. In the present study, we determined a major AMPA receptor-mediated neuroprotective pathway. Exposure of cerebellar granule cells to AMPA (500 microM) + aniracetam (1 microM), a known blocker of AMPA receptor desensitization, evoked an accumulation of brain-derived neurotropic factor (BDNF) in the culture medium and enhanced TrkB-tyrosine phosphorylation following the release of BDNF. AMPA also activated the src-family tyrosine kinase, Lyn, and the downstream target of the phosphatidylinositol 3-kinase (PI3-K) pathway, Akt. Extracellular signal regulated kinase (ERK), a component of the mitogen-activated protein kinase (MAPK) pathway, was also activated. K252a, a selective inhibitor of neurotrophin signaling, blocked the AMPA-mediated neuroprotection. The involvement of BDNF release in protecting neurons by AMPA was confirmed using a BDNF-blocking antibody. AMPA-mediated neuroprotection is blocked by PP1, an inhibitor of src family kinases, LY294002, a PI3-K inhibitor, or U0126, a MAPK kinase (MEK) inhibitor. Neuroprotective concentrations of AMPA increased BDNF mRNA levels that was blocked by the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX). The increase in BDNF gene expression appeared to be the downstream target of the PI3-K-dependent activation of the MAPK cascade since MEK or the PI3-K inhibitor blocked the AMPA receptor-mediated increase in BDNF mRNA. Thus, AMPA receptors protect neurons through a mechanism involving BDNF release, TrkB receptor activation, and a signaling pathway involving a PI3-K dependent activation of MAPK that increases BDNF expression.     

Long-term depression of mGluR1 signaling

Glutamate produces both fast excitation through activation of ionotropic receptors and slower actions through metabotropic receptors (mGluRs). To date, ionotropic but not metabotropic neurotransmission has been shown to undergo long-term synaptic potentiation and depression. Burst stimulation of parallel fibers releases glutamate, which activates perisynaptic mGluR1 in the dendritic spines of cerebellar Purkinje cells. Here, we show that the mGluR1-dependent slow EPSC and its coincident Ca transient were selectively and persistently depressed by repeated climbing fiber-evoked depolarization of Purkinje cells in brain slices. LTD(mGluR1) was also observed when slow synaptic current was evoked by exogenous application of a group I mGluR agonist, implying a postsynaptic expression mechanism. Ca imaging further revealed that LTD(mGluR1) was expressed as coincident attenuation of both limbs of mGluR1 signaling: the slow EPSC and PLC/IP3-mediated dendritic Ca mobilization. Thus, different patterns of neural activity can evoke LTD of either fast ionotropic or slow mGluR1-mediated synaptic signaling.     

Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.     

Activation of receptors negatively coupled to adenylate cyclase is required for induction of long-term synaptic depression at Schaffer collateral-CA1 synapses

Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl) glycine (DCGIV; 5 microM), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 microM), resulted in a long-lasting depression of synaptic strength. When zaprinast (20 microM) was combined with a cell-permeant PKA inhibitor H-89 (10 microM), the need for mGluR IIs was bypassed. DCGIV, when combined with a "submaximal" low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 5 microM), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)-a-Cyclopropyl-[3- 3H]-4-phosphonophenylglycine (CPPG; 10 microM), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 microM), was sufficient to elicit CLTD. Inhibition of PKA with H-89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity.     

The cortico-basal ganglia-thalamocortical circuit with synaptic plasticity. I. Modification rules for excitatory and inhibitory synapses in the striatum

It is pointed out that Ca(2+)-dependent modification rules for NMDA-dependent (NMDA-independent) synaptic plasticity in the striatum are similar to those in the neocortex and hippocampus (cerebellum). A unitary postsynaptic mechanism of synaptic modification is proposed. It is based on the assumption that, in diverse central nervous system structures, long-term potentiation/depression (LTP/LTD) of excitatory transmission (depression/potentiation of inhibitory transmission, LTDi/LTPi) is the result of an increasing/decreasing the number of phosphorylated AMPA and NMDA (GABA(A)) receptors. According to the suggested mechanism, Ca(2+)/calmodulin-dependent protein kinase II and protein kinase C, whose activity is positively correlated with Ca(2+) enlargement, together with cAMP-dependent protein kinase A (cGMP-dependent protein kinase G, whose activity is negatively correlated with Ca(2+) rise) mainly phosphorylate ionotropic striatal receptors, if NMDA channels are opened (closed). Therefore, the positive/negative post-tetanic Ca(2+) shift in relation to a previous Ca(2+) rise must cause NMDA-dependent LTP+LTDi/LTD+LTPi or NMDA-independent LTD+LTPi/LTP+LTDi. Dopamine D(1)/D(2) or adenosine A(2A)/A(1) receptor activation must facilitate LTP+LTDi/LTD+LTPi due to an augmenting/lowering PKA activity. Activation of muscarinic M(1)/M(4) receptors must enhance LTP+LTDi/LTD+LTPi as a consequence of an increase/decrease in the activity of protein kinase C/A. The proposed mechanism is in agreement with known experimental data.     

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner

Insulin and its receptor are both present in the central nervous system and are implicated in neuronal survival and hippocampal synaptic plasticity. Here we show that insulin activates phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), and results in an induction of long-term depression (LTD) in hippocampal CA1 neurones. Evaluation of the frequency-response curve of synaptic plasticity revealed that insulin induced LTD at 0.033 Hz and LTP at 10 Hz, whereas in the absence of insulin, 1 Hz induced LTD and 100 Hz induced LTP. LTD induction in the presence of insulin required low frequency synaptic stimulation (0.033 Hz) and blockade of GABAergic transmission. The LTD or LTP induced in the presence of insulin was N-methyl-d-aspartate (NMDA) receptor specific as it could be inhibited by alpha-amino-5-phosphonopentanoic acid (APV), a specific NMDA receptor antagonist. LTD induction was also facilitated by lowering the extracellular Mg(2+) concentration, indicating an involvement of NMDA receptors. Inhibition of PI3K signalling or discontinuing synaptic stimulation also prevented this LTD. These results show that insulin modulates activity-dependent synaptic plasticity, which requires activation of NMDA receptors and the PI3K pathway. The results obtained provide a mechanistic link between insulin and synaptic plasticity, and explain how insulin functions as a neuromodulator.     

Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.