Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore

The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements.     

Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor

Phosphorylation of the GluA1 subunit of AMPA receptors has been proposed to regulate receptor trafficking and synaptic transmission and plasticity. However, it remains unclear whether GluA1 phosphorylation is permissive or sufficient for enacting these functional changes. Here we investigate the role of GluA1 phosphorylation at S831 and S845 residues in the hippocampus through the analyses of GluA1 S831D/S845D phosphomimetic knock-in mice. S831D/S845D mice showed normal total and surface expression and subcellular localization of GluA1 as well as intact basal synaptic transmission. In addition, theta-burst stimulation, a protocol that was sufficient to induce robust long-term potentiation (LTP) in WT mice, resulted in LTP of similar magnitude in S831D/S845D mice. However, S831D/S845D mice showed LTP induced with 10-Hz stimulation, a protocol that is weaker than theta-burst stimulation and was not sufficient to induce LTP in WT mice. Moreover, S831D/S845D mice exhibited LTP induced with spike-timing-dependent plasticity (STDP) protocol at a long pre-post interval that was subthreshold for WT mice, although a suprathreshold STDP protocol at a short pre-post interval resulted in similarly robust LTP for WT and S831D/S845D mice. These results indicate that phosphorylation of GluA1 at S831 and S845 is sufficient to lower the threshold for LTP induction, increasing the probability of synaptic plasticity.     

Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors

The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2⁺/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development.Activity-dependent plasticity at immature glutamatergic synapses is thought to underlie fine tuning of the synaptic circuitry and optimize the network for its adult functions. The synaptic mechanisms of plasticity at immature contacts differ from those in the adult because of developmental alterations in the expression of several molecules that are critical in mediating and modulating synaptic transmission. For example, in area CA1 of the hippocampus, the signaling cascades necessary for long-term potentiation (LTP) are altered during the first weeks of postnatal life, corresponding to the time of formation and maturation of glutamatergic synapses. In the neonate, LTP is dependent mainly on the activation of PKA, but later in development LTP requires the activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) together with other kinases (1, 2). In parallel, expression of the AMPA-receptor subunit GluA4 in the hippocampal pyramidal neurons is strongly down-regulated and replaced by other subunits, including GluA1 (3, 4).Both GluA4 and GluA1 and a splice variant of GluA2, GluA2_L, contain a long intracellular C-terminal domain (CTD) that is thought to be involved in activity-dependent synaptic incorporation of AMPA receptors (5–8, but also see ref. 9). Spontaneous synaptic activity and consequent activity-dependent PKA phosphorylation is sufficient to drive recombinant GluA4, but not GluA1, into synapses (4, 10), suggesting that the switch in the subunit composition of AMPA receptors may explain some of the developmental changes in the mechanisms of LTP. However, the exact role of the developmentally restricted expression of GluA4 in synaptic transmission and plasticity remains unknown.Here we show that GluA4 expression is sufficient to alter the signaling mechanisms underlying LTP and to confer PKA-dependent postsynaptic potentiation. Thus, the expression of GluA4 can explain fully the developmental switch in the LTP kinase dependency in CA1 pyramidal neurons.     

The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptors

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of ³⁵S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.     

Input-specific synaptic plasticity in the amygdala is regulated by neuroligin-1 via postsynaptic NMDA receptors

Despite considerable evidence for a critical role of neuroligin-1 in the specification of excitatory synapses, the cellular mechanisms and physiological roles of neuroligin-1 in mature neural circuits are poorly understood. In mutant mice deficient in neuroligin-1, or adult rats in which neuroligin-1 was depleted, we have found that neuroligin-1 stabilizes the NMDA receptors residing in the postsynaptic membrane of amygdala principal neurons, which allows for a normal range of NMDA receptor-mediated synaptic transmission. We observed marked decreases in NMDA receptor-mediated synaptic currents at afferent inputs to the amygdala of neuroligin-1 knockout mice. However, the knockout mice exhibited a significant impairment in spike-timing-dependent long-term potentiation (STD-LTP) at the thalamic but not the cortical inputs to the amygdala. Subsequent electrophysiological analyses indicated that STD-LTP in the cortical pathway is largely independent of activation of postsynaptic NMDA receptors. These findings suggest that neuroligin-1 can modulate, in a pathway-specific manner, synaptic plasticity in the amygdala circuits of adult animals, likely by regulating the abundance of postsynaptic NMDA receptors.     

GDE1/MIR16 is a glycerophosphoinositol phosphodiesterase regulated by stimulation of G protein-coupled receptors

Previously we identified MIR16 (membrane interacting protein of RGS16) as an integral membrane glycoprotein that interacts with regulator of G protein signaling proteins and shares significant sequence homology with bacterial glycerophosphodiester phosphodiesterases (GDEs), suggesting that it is a putative mammalian GDE. Here we show that MIR16 belongs to a large, evolutionarily conserved family of GDEs with a characteristic putative catalytic domain that shares a common motif (amino acids 92-116) with the catalytic domains of mammalian phosphoinositide phospholipases C. Expression of wild-type MIR16 (renamed GDE1), but not two catalytic domain mutants (E97A/D99A and H112A), leads to a dramatic increase in glycerophosphoinositol phosphodiesterase (GPI-PDE) activity in HEK 293T cells. Analysis of substrate specificity shows that GDE1/MIR16 selectively hydrolyzes GPI over glycerophosphocholine. The GPI-PDE activity of GDE1/MIR16 expressed in HEK 293T cells can be regulated by stimulation of G protein-coupled, alpha/beta-adrenergic, and lysophospholipid receptors. Membrane topology studies suggest a model in which the catalytic GDE domain faces the lumenextracellular space and the C terminus faces the cytoplasm. Our results suggest that by serving as a PDE for GPI with its activity regulated by G protein signaling, GDE1/MIR16 provides a link between phosphoinositide metabolism and G protein signal transduction.     

Wnt signaling in the thymus is regulated by differential expression of intracellular signaling molecules

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.     

Cleavage of the bacteriophage P1 packaging site (pac) is regulated by adenine methylation.

The packaging of bacteriophage PI DNA is initiated when the phage packaging site (pac) is recognized and cleaved and continues until the phage head is full. We have previously shown that pac is a 162-base-pair segment of P1 DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC). We show here that cleavage of pac is methylation sensitive. Both in vivo and in vitro experiments indicate that methylated pac is cleavable, whereas unmethylated pac is not. Moreover, DNA isolated from P1 phage and containing an uncut pac site was a poor substrate for in vitro cleavage until it was methylated by the Escherichia coli DNA adenine methyltransferase. Comparison of that uncut pac DNA with other viral DNA fragments by digestion with methylation-sensitive restriction enzymes indicated that the uncut pac DNA was preferentially undermethylated. In contrast, virion DNA containing a cut pac site was not undermethylated. We believe these results indicate that pac cleavage is regulated by adenine methylation during the phage lytic cycle.     

Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse

Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.     

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Ca(2+)-activated Cl(-) channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca(2+) has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca(2+) or by voltage in the absence of Ca(2+), but Ca(2+)- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca(2+) and voltage sensing. Deleting (448)EAVK in the first intracellular loop dramatically decreases apparent Ca(2+) affinity. In contrast, mutating the adjacent amino acids (444)EEEE abolishes intrinsic voltage dependence without altering the apparent Ca(2+)affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca(2+) was facilitated by anions with high permeability or by an increase in [Cl(-)](e). Our data show that the transition between closed and open states is governed by Ca(2+) in a voltage-dependent manner and suggest that anions allosterically modulate Ca(2+)-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.     

Dopamine responsiveness is regulated by targeted sorting of D2 receptors

Aberrant dopaminergic signaling is a critical determinant in multiple psychiatric disorders, and in many disease states, dopamine receptor number is altered. Here we identify a molecular mechanism that selectively targets D2 receptors for degradation after their activation by dopamine. The degradative fate of D2 receptors is determined by an interaction with G protein coupled receptor-associated sorting protein (GASP). As a consequence of this GASP interaction, D2 responses in rat brain fail to resensitize after agonist treatment. Disruption of the D2-GASP interaction facilitates recovery of D2 responses, suggesting that modulation of the D2-GASP interaction is important for the functional down-regulation of D2 receptors.     

Ethanol operant self-administration in rats is regulated by adenosine A2 receptors

BACKGROUND: Recent findings suggest that adenosine is involved in the neural and behavioral effects of ethanol (EtOH). Studies in neural cell culture show that EtOH, via activation of adenosine A2 receptors, triggers cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling and CRE (cAMP regulatory element)-mediated gene expression and that this effect is blocked by inhibiting G-protein betagamma subunits. Recently, we reported that expression of a betagamma inhibitor in the nucleus accumbens (NAc) reduces EtOH drinking in rats. The NAc expresses high levels of the adenosine A2A receptor in GABAergic medium spiny neurons. If the reinforcing effects of EtOH are mediated through an A2 activation of cAMP/PKA signaling via betagamma, then A2 receptor blockade should attenuate EtOH consumption. Here we tested this hypothesis. Because adenosine A2 and dopamine D2 receptors are coexpressed in neurons of the NAc, we compared the effects of A2 blockade with those of D2 receptor blockade. METHODS: Male Long-Evans rats were trained to self-administer 10% EtOH in daily 30-min sessions with an active and an inactive lever. Separate groups of rats were given the D2 antagonist eticlopride (0.005, 0.007, and 0.01 mg/kg), the A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX; 1, 3, 5, 7, 10, and 20 mg/kg), and the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.125, 0.25, and 0.5 mg/kg) by systemic injection. RESULTS: Eticlopride dose-dependently reduced EtOH drinking. DMPX showed a bimodal effect: 10 and 20 mg/kg decreased, but 1 mg/kg increased, EtOH consumption. DPCPX was without effect. CONCLUSIONS: In support of our hypothesis, the A2 antagonist DMPX attenuated EtOH self-administration. Low doses of the A2 antagonist enhanced EtOH drinking, consistent with the possibility that rats increase EtOH self-administration to overcome partial A2 blockade. The D2 antagonist eticlopride also decreased EtOH self-administration. These data provide the first evidence that pharmacological modulation of adenosine A2 receptors can regulate EtOH consumption in rats.     

A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis

Familial hemiplegic migraine (FHM)-causing mutations in the gene encoding the P/Q Ca²⁺ channel α_1A subunit (CACNA1A) locate to the pore and voltage sensor regions and normally involve gain-of-channel function. We now report on a mutation identified in the first intracellular loop of CACNA1A (α_1A(A454T)) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. α_1A(A454T) channels showed weakened regulation of voltage-dependent steady-state inactivation by Ca_Vβ subunits. More interestingy, A454T mutation suppressed P/Q channel modulation by syntaxin 1A or SNAP-25 and decreased exocytosis. Our findings reveal the importance of I-II loop structural integrity in the functional interaction between P/Q channel and proteins of the vesicle-docking/fusion machinery, and that genetic variation in CACNA1A may be not only a cause but also a modifier of migraine phenotype.     

Ethanol self-administration is regulated by CB1 receptors in the nucleus accumbens and ventral tegmental area in alcohol-preferring AA rats

Background: Endogenous cannabinoids and their receptors, CB1 receptors in particular, have been implicated in mediation of ethanol reinforcement. Previously, suppression of ethanol drinking by CB1 antagonists has been demonstrated in many experimental paradigms. However, the exact mechanism by which CB1 antagonists modulate ethanol drinking remains elusive. In the present study, we assessed the role of CB1 receptors within the key regions of the mesolimbic dopamine pathway, the nucleus accumbens (NAcc) and ventral tegmental area (VTA), in regulation of ethanol self‐administration. Methods: Adult male alcohol‐prefer AA rats were trained to self‐administer either 10% (w/v) ethanol or 0.1% (w/v) saccharin under an FR1 schedule during daily 30‐minute sessions. Following stable baseline responding, rats were tested after systemic administration of the CB1 antagonist SR141716A (0 to 10 mg/kg) and the agonist WIN55,212‐2 (0 to 2 mg/kg). Separate groups of rats were implanted with bilateral cannulas aimed at the NAcc or VTA, and tested after microinjections of SR141716A (0 to 3 μg) and WIN55,212‐2 (0 to 5 μg) into the NAcc or VTA. The highest intracerebral doses were tested also in rats responding for a 0.1% saccharin solution. Results: SR141617A dose‐dependently suppressed ethanol responding after systemic administration. Microinjections of SR141617A both into NAcc and VTA attenuated ethanol responding. In addition, intra‐NAcc injections of SR141617A suppressed saccharin intake. Although low doses of systemically given WIN55,212‐2 increased ethanol responding, no effects were seen after WIN55,212‐2 microinjections into NAcc or VTA. Conclusions: Bidirectional changes in ethanol self‐administration by the systematically administered CB1 agonist and antagonist show that ethanol reinforcement is controlled by CB1 receptors in alcohol‐preferring AA rats. Replication of the suppressive effects by CB1 antagonism in the NAcc and VTA suggests that endocannabinoids and their receptors mediate ethanol reinforcement through interaction with the mesolimbic dopamine pathway.     

Cyclin D1 expression is regulated by the retinoblastoma protein.

The product of the retinoblastoma susceptibility gene, pRb, acts as a tumor suppressor and loss of its function is involved in the development of various types of cancer. DNA tumor viruses are supposed to disturb the normal regulation of the cell cycle by inactivating pRb. However, a direct function of pRb in regulation of the cell cycle has hitherto not been shown. We demonstrate here that the cell cycle-dependent expression of one of the G1-phase cyclins, cyclin D1, is dependent on the presence of a functional Rb protein. Rb-deficient tumor cell lines as well as cells expressing viral oncoproteins (large tumor antigen of simian virus 40, early region 1A of adenovirus, early region 7 of papillomavirus) have low or barely detectable levels of cyclin D1. Expression of cyclin D1, but not of cyclins A and E, is induced by transfection of the Rb gene into Rb-deficient tumor cells. Cotransfection of a reporter gene under the control of the D1 promoter, together with the Rb gene, into Rb-deficient cell lines demonstrates stimulation of the D1 promoter by Rb, which parallels the stimulation of endogenous cyclin D1 gene expression. Our finding that pRb stimulates expression of a key component of cell cycle control, cyclin D1, suggests the existence of a regulatory loop between pRb and cyclin D1 and extends existing models of tumor suppressor function.