AKAP150-anchored PKA regulation of synaptic transmission and plasticity, neuronal excitability and CRF neuromodulation in the lateral habenula.

Numerous studies of hippocampal synaptic function in learning and memory have established the functional significance of the scaffolding A-kinase anchoring protein 150 (AKAP150) in kinase and phosphatase regulation of synaptic receptor and ion channel trafficking/function and hence synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a critical role in brain's processing of rewarding/aversive experiences. Here we focused on an unexplored role of AKAP150 in the lateral habenula (LHb), a diencephalic brain region that integrates and relays negative reward signals from forebrain striatal and limbic structures to midbrain monoaminergic centers. LHb aberrant activity (specifically hyperactivity) is also linked to depression. Using whole cell patch clamp recordings in LHb of male wildtype (WT) and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), we found that the genetic disruption of PKA anchoring to AKAP150 significantly reduced AMPA receptor (AMPAR)-mediated glutamatergic transmission and prevented the induction of presynaptic endocannabinoid (eCB)-mediated long-term depression (LTD) in LHb neurons. Moreover, ΔPKA mutation potentiated GABAA receptor (GABAAR)-mediated inhibitory transmission postsynaptically while increasing LHb intrinsic neuronal excitability through suppression of medium afterhyperpolarizations (mAHPs). Given that LHb is a highly stress-responsive brain region, we further tested the effects of corticotropin releasing factor (CRF) stress neuromodulator on synaptic transmission and intrinsic excitability of LHb neurons in WT and ΔPKA mice. As in our earlier study in rat LHb, CRF significantly suppressed GABAergic transmission onto LHb neurons and increased intrinsic excitability by diminishing small-conductance potassium (SK) channel-mediated mAHPs. ΔPKA mutation-induced suppression of mAHPs also blunted the synaptic and neuroexcitatory actions of CRF in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPAR and GABAAR synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPAR and potassium channel trafficking and eCB signaling within the LHb.

Factors affecting the outcome of delayed intravenous thrombolysis (>4.5hours).

INTRODUCTION: Evidence of the intravenous tissue plasminogen activator (tPA) efficacy beyond the 4.5hours window is emerging. We aim to study the factors affecting the outcome of delayed thrombolysis in patients of clear onset acute ischemic stroke (AIS). METHODS: Data of patients with AIS who received intravenous thrombolytic after 4.5hours were reviewed including: demographics, risk factors, clinical, laboratory, investigational and radiological data, evidence of mismatch, treatment type and onset, National Institutes of Health Stroke Scale (NIHSS) score at baseline, 24hours, 7days after thrombolysis and before discharge, and 3 months follow-up modified Rankin Scale (mRS). RESULTS: We report 136 patients treated by intravenous tPA between 4.53 and 19.75hours with average duration of 5.7h. The ASPECT score of our patients was≥7. Sixty-four cases showed intracranial arterial occlusion. Perfusion mismatch was detected in 117 (84.6%) patients, while clinical imaging mismatch was detected in 19 (15.4%). Early neurological improvement after 24hours occurred in 114 (83.8%) patients. At 90days, 91 patients (67%) achieved good outcome (mRS 0-2), while 45 (33%) had bad outcome (mRS 3-6). Age, endovascular treatment, NIHSS, AF, and HT were significantly higher in the bad outcome group. Age (P=0.001, OR: 1.099, 95% CI: 1.042-1.160) and baseline NIHSS were predictive of the poor outcome (P=0.002, OR: 1.151, 95% CI: 1.055-1.256). The best cutoff value of age was 72.5 with AUC of 0.76, sensitivity 73.3% and specificity 60.4%. While for NIHSS at admission, the cutoff value of 7 showed the best results with AUC of 0.73, sensitivity 71.1% and specificity 63.7%. Combination of age and admission NIHSS raised the sensitivity and specificity to 84.4% and 63.7%, respectively. CONCLUSION: Increased age and admission NIHSS may adversely affect the outcome of delayed thrombolysis and narrow the eligibility criteria. Age and baseline NIHSS based stratification of the patients may provide further evidence as regards the efficacy of the delayed thrombolysis.

Efficacy of mechanical thrombectomy in patients with ischemic stroke and cancer.

Cancer-related coagulopathy is a known cause of stroke and can lead to formation of thrombi with a unique composition. The effectiveness of mechanical thrombectomy in cancer patients is still unknown. The aim of the study was to evaluate the rate of successful reperfusion and the clinical outcome in cancer patients with stroke treated with endovascular therapies, compared to patients without cancer. We performed a retrospective analysis of consecutive patients with ischemic stroke treated with endovascular therapies at our hospital between January 2008 and January 2016. A sub-group analysis was performed including only patients with cryptogenic stroke. We included in the final analysis 14 patients with active cancer and 267 patients without cancer. Successful reperfusion was achieved in 79% of patients without cancer, and 71% of patients with active cancer (P = 0.68). Patients with cryptogenic stroke and active cancer had a lower reperfusion rate compared to patients with cryptogenic stroke without active cancer, although not significantly so (2/4 cancer patients, 50% vs 37/50, 74%, p: 0.31). Mortality rate was higher among cancer patients. Hemorrhagic transformation occurred in similar proportions in the two groups. Endovascular treatment in cancer patients seems, thus, effective.

Functional and molecular evidence of myelin- and neuroprotection by thyroid hormone administration in experimental allergic encephalomyelitis.

AIMS: Recent data in mouse and rat demyelination models indicate that administration of thyroid hormone (TH) has a positive effect on the demyelination/remyelination balance. As axonal pathology has been recognized as an early neuropathological event in multiple sclerosis, and remyelination is considered a pre-eminent neuroprotective strategy, in this study we investigated whether TH administration improves nerve impulse propagation and protects axons. METHODS: We followed up the somatosensory evoked potentials (SEPs) in triiodothyronine (T3)-treated and untreated experimental allergic encephalomyelitis (EAE) Dark-Agouti female rats during the electrical stimulation of the tail nerve. T3 treatment started on the 10th day post immunization (DPI) and a pulse administration was continued until the end of the study (33 DPI). SEPs were recorded at baseline (8 DPI) and the day after each hormone/ vehicle administration. RESULTS: T3 treatment was associated with better outcome of clinical and neurophysiological parameters. SEPs latencies of the two groups behaved differently, being briefer and closer to control values (=faster impulse propagation) in T3-treated animals. The effect was evident on 24 DPI. In the same groups of animals, we also investigated axonal proteins, showing that T3 administration normalizes neurofilament immunoreactivity in the fasciculus gracilis and tau hyperphosphorylation in the lumbar spinal cord of EAE animals. No sign of plasma hyperthyroidism was found; moreover, the dysregulation of TH nuclear receptor expression observed in the spinal cord of EAE animals was corrected by T3 treatment. CONCLUSIONS: T3 supplementation results in myelin sheath protection, nerve conduction preservation and axon protection in this animal model of multiple sclerosis.

Altered neocortical cell density and layer thickness in serotonin transporter knockout mice: a quantitation study.

The neurotransmitter serotonin (5-HT) plays morphogenetic roles during development, and their alteration could contribute to autism pathogenesis in humans. To further characterize 5-HT's contributions to neocortical development, we assessed the thickness and neuronal cell density of various cerebral cortical areas in serotonin transporter (5-HTT) knockout (ko) mice, characterized by elevated extracellular 5-HT levels. The thickness of layer IV is decreased in 5-HTT ko mice compared with wild-type (wt) mice. The overall effect on cortical thickness, however, depends on the genetic background of the mice. Overall cortical thickness is decreased in many cortical areas of 5-HTT ko mice with a mixed c129-CD1-C57BL/6J background. Instead, 5-HTT ko mice backcrossed into the C57BL/6J background display increases in supragranular and infragranular layers, which compensate entirely for decreased layer IV thickness, resulting in unchanged or even enhanced cortical thickness. Moreover, significant increases in neuronal cell density are found in 5-HTT ko mice with a C57BL/6J background (wt:hz:ko ratio = 1.00:1.04:1.17) but not in the mixed c129-CD1-C57BL/6J 5-HTT ko animals. These results provide evidence of 5-HTT gene effects on neocortical morphology in epistatic interaction with genetic variants at other loci and may model the effect of functional 5-HTT gene variants on neocortical development in autism.

Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate.

Protein kinases and phosphatases are targeted through association with anchoring proteins that tether the enzymes to subcellular structures and organelles. Through in situ fluorescent techniques using a Green Fluorescent Protein tag, we have mapped membrane-targeting domains on AKAP79, a multivalent anchoring protein that binds the cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and protein phosphatase 2B, calcineurin (CaN). Three linear sequences termed region A (residues 31-52), region B (residues 76-101) and region C (residues 116-145) mediate targeting of AKAP79 in HEK-293 cells and cortical neurons. Analysis of these targeting sequences suggests that they contain putative phosphorylation sites for PKA and PKC and are rich in basic and hydrophobic amino acids similar to a class of membrane-targeting domains which bind acidic phospholipids and calmodulin. Accordingly, the AKAP79 basic regions mediate binding to membrane vesicles containing acidic phospholipids including phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P2] and this binding is regulated by phosphorylation and calcium-calmodulin. Finally, AKAP79 was shown to be phosphorylated in HEK-293 cells following stimulation of PKA and PKC, and activation of PKC or calmodulin was shown to release AKAP79 from membrane particulate fractions. These findings suggest that AKAP79 might function in cells not only as an anchoring protein but also as a substrate and effector for the anchored kinases and phosphatases.

cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits.

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. Classification Of side chain determinants for anchoring and isoform selective association with AKAPs.

Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with A-Kinase Anchoring Proteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RIIalpha or RIIbeta, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RIIalpha decreased AKAP-binding up to 24 +/- 3 (n = 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RIIalpha reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n = 4) whereas introduction of an additional proline at position 6 in RIIbeta increased or conferred binding toward these anchoring proteins. Therefore, we conclude that beta-branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and prolines at positions 6 and 7 increase or stabilize RIIalpha interaction with selected anchoring proteins.

Transfected m2 muscarinic acetylcholine receptors couple to G alpha i2 and G alpha i3 in Chinese hamster ovary cells. Activation and desensitization of the phospholipase C signaling pathway.

Muscarinic acetylcholine receptor subtypes (m1-m5) differentially regulate phosphoinositide-specific phospholipase (PLC) through the activation of distinct guanine nucleotide-binding (G) proteins which can be distinguished on the basis of their sensitivity to inhibition by pertussis toxin (PTX). In transfected Chinese hamster ovary cells, the m2 receptor subtype regulates the stimulation of PLC and inhibition of adenylyl cyclase (AC) through PTX-sensitive G proteins. In this study, we utilized the ability of cholera toxin (CTX) to ADP-ribosylate PTX-sensitive alpha subunits as part of the ternary complex formed by heterotrimeric G proteins and agonist-bound receptors to detect and characterize the interactions between transfected m2 receptors and endogenous G proteins in Chinese hamster ovary cells. In membranes derived from cells expressing the m2, but not the m3 receptor, the cholinergic agonist carbachol stimulated CTX modification of a 40-kDa species (G alpha 40). Importantly, similar carbachol dose dependence values and PTX dose sensitivities were observed for m2 receptor-mediated PLC signaling and G alpha 40-CTX modification. High resolution urea-SDS-polyacrylamide gel electrophoresis analysis revealed that G alpha i2 (40 kDa) and G alpha i3 (41 kDa) were components of the G alpha 40 identified by m2 receptor-dependent CTX modification. Furthermore, the sensitivities of G alpha i2 and G alpha i3 to PTX modification were determined to be the same as those for PTX inhibition of G alpha 40 labeling by CTX and m2 receptor-mediated PLC signaling. Similarly, agonist-induced desensitization of m2 receptor-G protein signaling required doses of agonist associated with stimulation of PLC. Desensitization involved receptor sequestration and down-regulation of G alpha i3; however, only the reduction of G alpha i3 required prior activation PLC signaling. Finally, desensitization of m2-G protein coupling could be partially mimicked by treatment with thapsigargin, an inducer of intracellular Ca2+ release, without altering the number of cell surface receptors or G protein levels. These results demonstrate that m2 receptors couple to both G alpha i2 and G alpha i3 in vivo and that this interaction is integral to both positive and negative regulatory pathways leading to activation of PLC and desensitization of receptor signaling.