Sources of Calcium at Connexin 36 Gap Junctions in the Retina.

Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ, we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity.

Loss of Tpl2 activates compensatory signaling and resistance to EGFR/MET dual inhibition in v-RAS transduced keratinocytes.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer in the United States, affecting one million people per year. Patients with aggressive disease have limited treatment options and high mortality, highlighting the need to identify new biomarkers linked to poor clinical outcome. HRAS mutations are found in skin papillomas and cSCCs and increase in frequency when MAP3K family members are inhibited, suggesting a link between blockade of mitogen-activated protein kinase (MAPK) signaling and initiation of RAS-primed cells. Tpl2, a MAP3K gene, can serve as a tumor suppressor gene in cSCC. We have previously shown that upon Tpl2 ablation, mice have heightened sensitivity to aberrant RAS signaling. Tpl2-/- mice display significantly higher numbers of papillomas and cSCCs in two-stage chemical carcinogenesis studies and increased tumorigenicity of keratinocytes expressing oncogenic v-rasHa in nude mouse skin grafts. In part, this is mediated through increased mesenchymal-epithelial transition factor (MET) receptor activity. Epidermal Growth Factor Receptor (EGFR) is reported to be an essential factor for MET-driven carcinogenesis and MET activation may confer resistance to EGFR therapies, suggesting that the concurrent use of both an EGFR inhibitor and a MET inhibitor may show promise in advanced cSCCs. In this study we assessed whether normal or Ras-transformed Tpl2-/- keratinocytes have aberrant EGFR signaling and whether concomitant treatment with EGFR/MET tyrosine kinase inhibitors was more effective than single agents in reducing growth and angiogenic potential of Ras-transformed keratinocytes. Tpl2-/- keratinocytes exhibited increased HER-2 and STAT-3 under basal conditions and elevated p-MET and p-EGFR when transduced with oncogenic RAS. Inhibition of MET by Capmatinib increased p-EGFR in Tpl2-/- keratinocytes and papillomas, and inhibition of EGFR by Gefitinib increased HER2 and HER3 signaling in both genotypes. Treatment of keratinocytes with EGFR and MET inhibitors, in combination, significantly enhanced endothelial tube formation, MMP-9 activity and activation of other RTKs, with more pronounced effects when Tpl2 was ablated. These data indicate that Tpl2 cross-talks with both EGFR and MET signaling pathways. Upon inhibition of EGFR/MET signaling, a myriad of escape mechanisms exists in keratinocytes to overcome targeted drug effects.

Nonsynaptic NMDA receptors mediate activity-dependent plasticity of gap junctional coupling in the AII amacrine cell network.

Many neurons are coupled by electrical synapses into networks that have emergent properties. In the retina, coupling in these networks is dynamically regulated by changes in background illumination, optimizing signal integration for the visual environment. However, the mechanisms that control this plasticity are poorly understood. We have investigated these mechanisms in the rabbit AII amacrine cell, a multifunctional retinal neuron that forms an electrically coupled network via connexin 36 (Cx36) gap junctions. We find that presynaptic activity of glutamatergic ON bipolar cells drives increased phosphorylation of Cx36, indicative of increased coupling in the AII network. The phosphorylation is dependent on activation of nonsynaptic NMDA receptors that colocalize with Cx36 on AII amacrine cells, and is mediated by CaMKII. This activity-dependent increase in Cx36 phosphorylation works in opposition to dopamine-driven reduction of phosphorylation, establishing a local dynamic regulatory mechanism, and accounting for the nonlinear control of AII coupling by background illumination.

Illuminating synapses and circuitry in the retina.

In the central nervous system, space is at a premium. This is especially true in the retina, where synapses, cells, and circuitry have evolved to maximize signal-processing capacity within a thin, optically transparent tissue. For example, at some retinal synapses, single presynaptic active zones contact multiple postsynaptic targets; some individual neurons perform completely different tasks depending on visual conditions, while others execute hundreds of circuit computations in parallel; and the retinal network adapts, at various levels, to the ever-changing visual world. Each of these features reflects efficient use of limited cellular resources to optimally encode visual information.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Connexin 35/36 is phosphorylated at regulatory sites in the retina.

Connexin 35/36 is the most widespread neuronal gap junction protein in the retina and central nervous system. Electrical and/or tracer coupling in a number of neuronal circuits that express this connexin are regulated by light adaptation. In many cases, the regulation of coupling depends on signaling pathways that activate protein kinases such as PKA, and Cx35 has been shown to be regulated by PKA phosphorylation in cell culture systems. To examine whether phosphorylation might regulate Cx35/36 in the retina we developed phospho-specific polyclonal antibodies against the two regulatory phosphorylation sites of Cx35 and examined the phosphorylation state of this connexin in the retina. Western blot analysis with hybrid bass retinal membrane preparations showed Cx35 to be phosphorylated at both the Ser110 and Ser276 sites, and this labeling was eliminated by alkaline phosphatase digestion. The homologous sites of mouse and rabbit Cx36 were also phosphorylated in retinal membrane preparations. Quantitative confocal immunofluorescence analysis showed gap junctions identified with a monoclonal anti-Cx35 antibody to have variable levels of phosphorylation at both the Ser110 and Ser276 sites. Unusual gap junctions that could be identified by their large size (up to 32 microm2) and location in the IPL showed a prominent shift in phosphorylation state from heavily phosphorylated in nighttime, dark-adapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites showed significant changes in this manner. Under both lighting conditions, other gap junctions varied from non-phosphorylated to heavily phosphorylated. We predict that changes in the phosphorylation states of these sites correlate with changes in the degree of coupling through Cx35/36 gap junctions. This leads to the conclusion that connexin phosphorylation mediates changes in coupling in some retinal networks. However, these changes are not global and likely occur in a cell type-specific or possibly a gap junction-specific manner.