Amygdala Kindling Alters Protein Kinase C Activity in Dentate Gyrus

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.     

Pre-Synaptic Release Deficits in a DYT1 Dystonia Mouse Model

DYT1 early-onset generalized torsion dystonia (DYT1 dystonia) is an inherited movement disorder caused by mutations in one allele of DYT1 (TOR1A), coding for torsinA. The most common mutation is a trinucleotide deletion (ΔGAG), which causes a deletion of a glutamic acid residue (ΔE) in the C-terminal region of torsinA. Although recent studies using cultured cells suggest that torsinA contributes to protein processing in the secretory pathway, endocytosis, and the stability of synaptic proteins, the nature of how this mutation affects synaptic transmission remains unclear. We previously reported that theta-burst-induced long-term potentiation (LTP) in the CA1 region of the hippocampal slice is not altered in Dyt1 ΔGAG heterozygous knock-in (KI) mice. Here, we examined short-term synaptic plasticity and synaptic transmission in the hippocampal slices. Field recordings in the hippocampal Schaffer collaterals (SC) pathway revealed significantly enhanced paired pulse ratios (PPRs) in Dyt1 ΔGAG heterozygous KI mice, suggesting an impaired synaptic vesicle release. Whole-cell recordings from the CA1 neurons showed that Dyt1 ΔGAG heterozygous KI mice exhibited normal miniature excitatory post-synaptic currents (mEPSC), suggesting that action-potential independent spontaneous pre-synaptic release was normal. On the other hand, there was a significant decrease in the frequency, but not amplitude or kinetics, of spontaneous excitatory post-synaptic currents (sEPSC) in Dyt1 ΔGAG heterozygous KI mice, suggesting that the action-potential dependent pre-synaptic release was impaired. Moreover, hippocampal torsinA was significantly reduced in Dyt1 ΔGAG heterozygous KI mice. Although the hippocampal slice model may not represent the neurons directly associated with dystonic symptoms, impaired release of neurotransmitters caused by partial dysfunction of torsinA in other brain regions may contribute to the pathophysiology of DYT1 dystonia.     

Protease‐activated receptor‐1 modulates hippocampal memory formation and synaptic plasticity

Protease‐activated receptor‐1 (PAR1) is an unusual G‐protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1?/? mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1?/? mice have deficits in hippocampus‐dependent memory. We also show that while PAR1?/? mice have normal baseline synaptic transmission at Schaffer collateral‐CA1 synapses, they exhibit severe deficits in N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR‐mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR‐dependent processes subserving memory formation and synaptic plasticity.     

Protein kinase inhibition by omega-3 fatty acids

Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.     

Normal development and fertility of knockout mice lacking the tumor suppressor gene LRP1b suggest functional compensation by LRP1

LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family. At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling. Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth. In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors. LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis. Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species. Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b. In addition, we have found several potential ligands that bind to the extracellular domain. Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.     

Receptor clustering is involved in Reelin signaling

The Reelin signaling cascade plays a crucial role in the correct positioning of neurons during embryonic brain development. Reelin binding to apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) leads to phosphorylation of disabled 1 (Dab1), an adaptor protein which associates with the intracellular domains of both receptors. Coreceptors for Reelin have been postulated to be necessary for Dab1 phosphorylation. We show that bivalent agents specifically binding to ApoER2 or VLDLR are sufficient to mimic the Reelin signal. These agents induce Dab1 phosphorylation, activate members of the Src family of nonreceptor tyrosine kinases, modulate protein kinase B/Akt phosphorylation, and increase long-term potentiation in hippocampal slices. Induced dimerization of Dab1 in HEK293 cells leads to its phosphorylation even in the absence of Reelin receptors. The mechanism for and the sites of these phosphorylations are identical to those effected by Reelin in primary neurons. These results suggest that binding of Reelin, which exists as a homodimer in vivo, to ApoER2 and VLDLR induces clustering of ApoER2 and VLDLR. As a consequence, Dab1 becomes dimerized or oligomerized on the cytosolic side of the plasma membrane, constituting the active substrate for the kinase; this process seems to be sufficient to transmit the signal and does not appear to require any coreceptor.     

A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels

Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 alpha subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 alpha subunits. Co-expression of KChIPs1-3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice.     

Reelin and ApoE receptors cooperate to enhance hippocampal synaptic plasticity and learning

Two apolipoprotein E (apoE) receptors, the very low density lipoprotein (VLDL) receptor and apoE receptor 2 (apoER2), are also receptors for Reelin, a signaling protein that regulates neuronal migration during brain development. In the adult brain, Reelin is expressed by GABA-ergic interneurons, suggesting a potential function as a modulator of neurotransmission. ApoE receptors have been indirectly implicated in memory and neurodegenerative disorders because their ligand, apoE, is genetically associated with Alzheimer disease. We have used knockout mice to investigate the role of Reelin and its receptors in cognition and synaptic plasticity. Mice lacking either the VLDL receptor or the apoER2 show contextual fear conditioning deficits. VLDL receptor-deficient mice also have a moderate defect in long term potentiation (LTP), and apoER2 knockouts have a pronounced one. The perfusion of mouse hippocampal slices with Reelin has no effect on baseline synaptic transmission but significantly enhances LTP in area CA1. This Reelin-dependent augmentation of LTP is abolished in VLDL receptor and apoER2 knockout mice. Our results reveal a role for Reelin in controlling synaptic plasticity in the adult brain and suggest that both of its receptors are necessary for Reelin-dependent enhancement of synaptic transmission in the hippocampus. Thus, the impairment of apoE receptor-dependent neuromodulation may contribute to cognitive impairment and synaptic loss in Alzheimer disease.     

beta -Amyloid peptide activates alpha 7 nicotinic acetylcholine receptors expressed in Xenopus oocytes

The alpha7 nicotinic acetylcholine receptor is highly expressed in hippocampus and in cholinergic projection neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer's disease. Previous work suggests that beta-amyloid peptide can interact with alpha7 nicotinic acetylcholine receptors, although the nature of this interaction has not been well characterized. To test whether beta-amyloid peptide can activate alpha7 nicotinic acetylcholine receptors, we expressed these receptors in Xenopus oocytes and performed two-electrode voltage clamp recordings, characterizing the response to beta-amyloid peptide 1-42 applied at concentrations ranging from 1 pm to 100 nm. In alpha7-expressing oocytes, beta-amyloid peptide 1-42 elicits inward currents at low concentrations (1-100 pm), whereas at higher concentrations (nm), less effective receptor activation is observed, indicative of receptor desensitization. Preincubation with the alpha7-selective agents, the antagonist methyllycaconatine, and the agonist 4-OH-GTS-21 blocked beta-amyloid peptide-induced receptor activation. beta-amyloid peptide 1-42 at low concentrations was able to activate the L250T mutant alpha7 receptor. The endogenous Ca(2+)-activated chloride current in Xenopus oocytes is recruited upon receptor activation since replacing Ca(2+) with Ba(2+) in the recording solution reduced current amplitude. Thus, when beta-amyloid peptide activation of alpha7 receptors occurs, these currents are comprised, at least in part, of Ca(2+).     

Cutting edge: targeted ligation of CTLA-4 in vivo by membrane-bound anti-CTLA-4 antibody prevents rejection of allogeneic cells

Natural engagement of CTLA-4 on host B7 limits T cell activation. We hypothesized that therapeutic cross-linking of CTLA-4 in vivo may further inhibit T cell function and prevent allograft rejection. However, none of the currently available CTLA-4-binding reagents have ligating properties when injected in vivo. The observation that surface-immobilized anti-CTLA-4 mAb inhibits T cell activation in vitro prompted us to develop a membrane-bound single-chain anti-CTLA-4 Ab (7M). To model whether tissue expression of 7M could suppress allograft rejection, we examined the ability of H-2L(d)-specific TCR-transgenic T cells to reject 7M-expressing allogeneic tumor cells injected s.c. Expression of 7M significantly inhibited allogeneic rejection in mice that received CTLA-4(+/+) but not CTLA-4(-/-) T cells. Furthermore, CTLA-4(+/+) T cells that had encountered 7M-expressing tumors in vivo acquired defects in cytokine production and cytotoxicity. Thus, deliberate ligation of CTLA-4 in vivo potently inhibits allogeneic T cell responses.