Michael A. Beazely

Activation of Pannexin-1 Hemichannels Augments Aberrant Bursting in the Hippocampus

Pannexin-1 (Px1) is expressed at postsynaptic sites in pyramidal neurons, suggesting that these hemichannels contribute to dendritic signals associated with synaptic function. We found that, in pyramidal neurons, N-methyl-d-aspartate receptor (NMDAR) activation induced a secondary prolonged current and dye flux that were blocked with a specific inhibitory peptide against Px1 hemichannels; knockdown of Px1 by RNA interference blocked the current in cultured neurons. Enhancing endogenous NMDAR activation in brain slices by removing external magnesium ions (Mg2+) triggered epileptiform activity, which had decreased spike amplitude and prolonged interburst interval during application of the Px1 hemichannel blocking peptide. We conclude that Px1 hemichannel opening is triggered by NMDAR stimulation and can contribute to epileptiform seizure activity.

Modulation of NMDA Receptors by Pituitary Adenylate Cyclase Activating Peptide in CA1 Neurons Requires Gαq, Protein Kinase C, and Activation of Src

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαq subunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nm) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.

D2-class dopamine receptor inhibition of NMDA currents in prefrontal cortical neurons is platelet-derived growth factor receptor-dependent

NMDA receptor function is modulated by both G‐protein‐coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet‐derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA‐evoked currents in a phospholipase C (PLC)‐dependent manner. We have investigated further the ability of D2‐class dopamine receptors to modulate NMDA‐evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF‐BB or quinpirole to isolated PFC neurons induces a slow‐onset and long‐lasting inhibition of NMDA‐evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA‐evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF‐BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCγ binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole‐induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.

Platelet-derived Growth Factor-mediated Induction of the Synaptic Plasticity Gene Arc/Arg3.1

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc.

Postsynaptic Clustering and Activation of Pyk2 by PSD-95

The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca2+ influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. We report that Pyk2 oligomerization by antibodies in vitro or overexpression of PSD-95 in PC6-3 cells induces trans-autophosphorylation of Tyr402, the first step in Pyk2 activation. In neurons, Ca2+ influx through NMDA-type glutamate receptors causes postsynaptic clustering and autophosphorylation of endogenous Pyk2 via Ca2+- and calmodulin-stimulated binding to PSD-95. Accordingly, Ca2+ influx promotes oligomerization and thereby autoactivation of Pyk2 by stimulating its interaction with PSD-95. We show that this mechanism of Pyk2 activation is critical for long-term potentiation in the hippocampus CA1 region, which is thought to underlie learning and memory.

Platelet-derived growth factor selectively inhibits NR2B-containing N-methyl-D-aspartate receptors in CA1 hippocampal neurons.

Platelet-derived growth factor (PDGF) β receptor activation inhibits N-methyl-d-aspartate (NMDA)-evoked currents in hippocampal and cortical neurons via the activation of phospholipase Cγ, PKC, the release of intracellular calcium, and a rearrangement of the actin cytoskeleton. In the hippocampus, the majority of NMDA receptors are heteromeric; most are composed of 2 NR1 subunits and 2 NR2A or 2 NR2B subunits. Using NR2B- and NR2A-specific antagonists, we demonstrate that PDGF-BB treatment preferentially inhibits NR2B-containing NMDA receptor currents in CA1 hippocampal neurons and enhances long-term depression in an NR2B subunit-dependent manner. Furthermore, treatment of hippocampal slices or cultures with PDGF-BB decreases the surface localization of NR2B but not of NR2A subunits. PDGFβ receptors colocalize to a higher degree with NR2B subunits than with NR2A subunits. After neuronal injury, PDGFβ receptors and PDGF-BB are up-regulated and PDGFβ receptor activation is neuroprotective against glutamate-induced neuronal damage in cultured neurons. We demonstrate that the neuroprotective effects of PDGF-BB are occluded by the NR2B antagonist, Ro25-6981, and that PDGF-BB promotes NMDA signaling to CREB and ERK1/2. We conclude that PDGFβR signaling, by preferentially targeting NR2B receptors, provides an important mechanism for neuroprotection by growth factors in the central nervous system.

Vasoactive intestinal peptide acts via multiple signal pathways to regulate hippocampal NMDA receptors and synaptic transmission.

Vasoactive intestinal peptide (VIP) is a 28‐amino acid peptide, which belongs to a superfamily of structurally related peptide hormones including pituitary adenylate cyclase‐activating polypeptide (PACAP). Although several studies have identified the involvement of PACAP in learning and memory, little work has been done to investigate such a role for VIP. At least three receptors for VIP have been identified including the PACAP receptor (PAC1‐R) and the two VIP receptors (VPAC receptors). VIP can activate the PAC1‐R only if it is used at relatively high concentrations (e.g., 100 nM); however, at lower concentrations (e.g., 1 nM) it is selective for the VPAC receptors. Our lab has showed that PAC1‐R activation signals through PKC/CAKβ/Src pathway to regulate NMDA receptors; however, there is little known about the potential regulation of NMDA receptors by VPAC receptors. Our studies demonstrated that application of 1 nM VIP enhanced NMDA currents by stimulating the VPAC receptors as the effect was blocked by VPAC receptor antagonist [Ac‐Tyr1, D‐Phe2]GRF (1–29). This enhancement of NMDA currents was blocked by both Rp‐cAMPS and PKI14–22 (they are highly specific PKA inhibitors), but not by the specific PKC inhibitor, bisindolylmaleimide I. In addition, the VIP‐induced enhancement of NMDA currents was accentuated by inhibition of phosphodiesterase 4, which inhibits the degradation of cAMP. This regulation of NMDA receptors also required the scaffolding protein AKAP. In contrast, the potentiation induced by high concentration of VIP (e.g., 100 nM) was mediated by PAC1‐R as well as by Src kinase. Overall, these results show that VIP can regulate NMDA receptors through different receptors and signaling pathways.

Development and evaluation of multifunctional agents for potential treatment of Alzheimer's disease: application to a pyrimidine-2,4-diamine template.

We investigated a group of 2-benzylpiperidin-N-benzylpyrimidin-4-amines with various electron-withdrawing or electron-donating groups (EWGs or EDGs, respectively) as multi-targeted Alzheimers disease (AD) therapeutics. The synthesized derivatives were screened for anti-cholinesterase (AChE and BuChE), anti-Aβ-aggregation (AChE- and self-induced) and anti-β-secretase (BACE-1) activities in an effort to identify lead, multifunctional candidates as part of our multi-targeted approach to treat AD. Biological assessment revealed that the nature of the substituent on the C-4 benzylamine group (e.g., halogen vs methoxy-based) greatly affected the biological profile. In vitro screening identified N(2)-(1-benzylpiperidin-4-yl)-N(4)-(3,4-dimethoxybenzyl)pyrimidine-2,4-diamine (7h) as the lead candidate with a dual ChE (AChE IC(50)=9.9 μM; BuChE IC(50)=11.4 μM), Aβ-aggregation (AChE-induced=59.3%; self-induced=17.4% at 100 μM) and BACE-1 (34% inhibition at 10 μM) inhibitory profile along with good cell viability (% neuroblastoma cell viability at 40 μM=81.0%). Molecular modeling studies indicate that a central pyrimidine-2,4-diamine ring serves as a suitable template to develop novel small molecule candidates to target multiple pathological routes in AD.

Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: Towards non-invasive glaucoma gene therapy

UNLABELLED Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

5-Hydroxytryptamine type 7 receptor neuroprotection against NMDA-induced excitotoxicity is PDGFβ receptor dependent

The serotonin (5‐HT) type 7 receptor is expressed throughout the CNS including the hippocampus. Long‐term (2–24 h) activation of 5‐HT7 receptors regulates growth factor receptor expression, including the expression of platelet‐derived growth factor (PDGF) β receptors. Direct activation of PDGFβ receptors in primary hippocampal and cortical neurons inhibits NMDA receptor activity and attenuates NMDA receptor‐induced neurotoxicity. Our objective was to investigate whether the 5‐HT7 receptor‐induced increase in PDGFβ receptor expression would be similarly neuroprotective. We demonstrate that 5‐HT7 receptor agonist treatment in primary hippocampal neurons also increases the expression of phospholipase C (PLC) γ, a downstream effector of PDGFβ receptors associated with the inhibition of NMDA receptor activity. To determine if the up‐regulation of PDGFβ receptors is neuroprotective, primary hippocampal neurons were incubated with the 5‐HT7 receptor agonist, LP 12, for 24 h. Indeed, LP 12 treatment prevented NMDA‐induced neurotoxicity and this effect was dependent on PDGFβ receptor kinase activity. Treatment of primary neurons with LP 12 also differentially altered NMDA receptor subunit expression, reducing the expression of NR1 and NR2B, but not NR2A. These findings demonstrate the potential for providing growth factor receptor‐dependent neuroprotective effects using small‐molecule ligands of G protein‐coupled receptors.