EphB2 receptor cell-autonomous forward signaling mediates auditory memory recall and learning-driven spinogenesis.

While ephrin-B ligands and EphB receptors are expressed to high levels in the learning centers of the brain, it remains largely unknown how their trans-synaptic interactions contribute to memory. We find that EphB2 forward signaling is needed for contextual and sound-evoked memory recall and that constitutive over-activation of the receptor's intracellular tyrosine kinase domain results in enhanced memory. Loss of EphB2 expression does not affect the number of neurons activated following encoding, although a reduction of neurons activated after the sound-cued retrieval test was detected in the auditory cortex and hippocampal CA1. Further, spine density and maturation was reduced in the auditory cortex of mutants especially in the neurons that were dual-activated during both encoding and retrieval. Our data demonstrates that trans-synaptic ephrin-B-EphB2 interactions and forward signaling facilitate neural activation and structural plasticity in learning-associated neurons involved in the generation of memories.

TrkB Regulates N-Methyl-D-Aspartate Receptor Signaling by Uncoupling and Recruiting the Brain-Specific Guanine Nucleotide Exchange Factor, RasGrf1.

Brain-derived neurotrophic factor (BDNF) facilitates multiple aspects of neuronal differentiation and cellular physiology by activating the high-affinity receptor tyrosine kinase, TrkB. While it is known that both BDNF and TrkB modulate cellular processes involved in learning and memory, exactly how TrkB cross-talks and modulates signaling downstream of excitatory ionotropic receptors, such as the NMDA receptor (NMDAR), are not well understood. A model that we have investigated involves the signaling molecule RasGrf1, a guanine nucleotide exchange factor for both Ras and Rac. We previously identified RasGrf1 as a novel Trk binding partner that facilitates neurite outgrowth in response to both nerve growth factor (NGF) (Robinson et al. in J Biol Chem 280:225-235, 2005) and BDNF (Talebian et al. in J Mol Neurosci 49:38-51, 2013); however, RasGrf1 can also bind the NR2B subunit of the NMDAR (Krapivinsky et al. in Neuron 40:775-784, 2003) and stimulate long-term depression (LTD) (Li et al. in J Neurosci 26:1721-1729, 2006). We have addressed a model that TrkB facilitates learning and memory via two processes. First, TrkB uncouples RasGrf1 from NR2B and facilitates a decrease in NMDA signaling associated with LTD (p38-MAPK). Second, the recruitment of RasGrf1 to TrkB enhances neurite outgrowth and pERK activation and signaling associated with learning and memory. We demonstrate that NMDA recruits RasGrf1 to NR2B; however, co-stimulation with BDNF uncouples this association and recruits RasGrf1 to TrkB. In addition, activation of TrkB stimulates the tyrosine phosphorylation of RasGrf1 which increases neurite outgrowth (Talebian et al. in J Mol Neurosci 49:38-51, 2013), and the tyrosine phosphorylation of NR2B (Tyr1472) (Nakazawa et al. in J Biol Chem 276:693-699, 2001) which facilitates NMDAR cell surface retention (Zhang et al. in J Neurosci 28:415-24, 2008). Collectively, these data demonstrate that TrkB alters NMDA signaling by a dual mechanism that uncouples LTD and, in turn, stimulates neuronal growth and the signaling pathways associated with learning and memory.

Abnormalities in cortical interneuron subtypes in ephrin-B mutant mice.

To explore roles for ephrin-B/EphB signaling in cortical interneurons, we previously generated ephrin-B (Efnb1/b2/b3) conditional triple mutant (TMlz ) mice using a Dlx1/2.Cre inhibitory neuron driver and green fluorescent protein (GFP) reporters for the two main inhibitory interneuron groups distinguished by expression of either glutamic acid decarboxylase 1 (GAD1; GAD67-GFP) or 2 (GAD2; GAD65-GFP). This work showed a general involvement of ephrin-B in migration and population of interneurons into the embryonic neocortex. We now determined whether specific interneurons are selectively affected in the adult brains of TMlz .Cre mice by immunostaining with antibodies that identify the different subtypes. The results indicate that GAD67-GFP-expressing interneurons that also express parvalbumin (PV), calretinin (CR) and, to a lesser extent, somatostatin (SST) and Reelin (Rln) were significantly reduced in the cortex and hippocampal CA1 region in TMlz .Cre mutant mice. Neuropeptide Y (NPY) interneurons that also express GAD67-GFP were reduced in the hippocampal CA1 region, but much less so in the cortex, although these cells exhibited abnormal cortical layering. In GAD65-GFP-expressing interneurons, CR subtypes were reduced in both cortex and hippocampal CA1 region, whereas Rln interneurons were reduced exclusively in hippocampus, and the numbers of NPY and vasoactive intestinal polypeptide (VIP) subtypes appeared normal. PV and CR subtype interneurons in TMlz .Cre mice also exhibited reductions in their perisomatic area, suggesting abnormalities in dendritic/axonal complexity. Altogether, our data indicate that ephrin-B expression within forebrain interneurons is required in specific subtypes for their normal population, cortical layering and elaboration of cell processes.

Autonomous and non-autonomous roles for ephrin-B in interneuron migration.

While several studies indicate the importance of ephrin-B/EphB bidirectional signaling in excitatory neurons, potential roles for these molecules in inhibitory neurons are largely unknown. We identify here an autonomous receptor-like role for ephrin-B reverse signaling in the tangential migration of interneurons into the neocortex using ephrin-B (EfnB1/B2/B3) conditional triple mutant (TMlz) mice and a forebrain inhibitory neuron specific Cre driver. Inhibitory neuron deletion of the three EfnB genes leads to reduced interneuron migration, abnormal cortical excitability, and lethal audiogenic seizures. Truncated and intracellular point mutations confirm the importance of ephrin-B reverse signaling in interneuron migration and cortical excitability. A non-autonomous ligand-like role was also identified for ephrin-B2 that is expressed in neocortical radial glial cells and required for proper tangential migration of GAD65-positive interneurons. Our studies thus define both receptor-like and ligand-like roles for the ephrin-B molecules in controlling the migration of interneurons as they populate the neocortex and help establish excitatory/inhibitory (E/I) homeostasis.

Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells.

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.

The signaling adapter, FRS2, facilitates neuronal branching in primary cortical neurons via both Grb2- and Shp2-dependent mechanisms.

The neurotrophins are a family of closely related growth factors that regulate proliferation and differentiation in the developing and mature nervous systems. Neurotrophins stimulate a family of receptor tyrosine kinases (Trk receptors) and utilize an intracellular docking protein termed fibroblast growth factor (FGF) receptor substrate 2 (FRS2) as a major downstream adapter to activate Ras, phosphatidylinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling cascades. The goals of this study were twofold: first, to investigate the complexity of neurotrophin-induced FRS2 interactions in primary cortical neurons and to determine which pathway(s) are important in regulating neuronal growth and, second, to determine whether the related signaling adapter, FRS3, stimulates neuron growth comparable to FRS2. We find that neurotrophin treatment of primary cortical neurons stimulates the tyrosine phosphorylation of FRS2 and the subsequent recruitment of Shp2, Grb2, and Gab2. With FRS2 mutants deficient in Grb2 or Shp2 binding, we demonstrate that FRS2 binds Gab1 and Gab2 through Grb2, providing an alternative route to activate PI3 kinase and Shp2. Using recombinant adenoviruses expressing FRS2, we demonstrate that FRS2 overexpression promotes neurite outgrowth and branching in cortical neurons relative to controls. In contrast, overexpression of FRS3 does not stimulate neuronal growth. Moreover, we find that while loss of Shp2, but not Grb2, reduces brain-derived neurotrophic factor (BDNF)-induced MAPK activation, the loss of either pathway impairs neuronal growth. Collectively, these experiments demonstrate that FRS2 functions as an adapter of a multiprotein complex that is activated by the Trk receptors and that the activation of both Grb2- and Shp2-dependent pathways facilitates cortical neuronal growth.

Ras guanine nucleotide releasing factor 1 (RasGrf1) enhancement of Trk receptor-mediated neurite outgrowth requires activation of both H-Ras and Rac.

We previously demonstrated that the guanine nucleotide exchange factor, RasGrf1, binds nerve growth factor (NGF)-activated TrkA and facilitates neurotrophin-induced neurite outgrowth in PC12 cells. RasGrf1 can activate both Ras and Rac, via intrinsic Cdc25 and DH domains, respectively, suggesting that the activation of both could contribute to this process. Previous studies have assayed constitutive neurite outgrowth following RasGrf1 over-expression in PC12 cells, in either the absence or presence of ectopic H-Ras, and have suggested an essential role for either Ras or Rac depending on the presence of H-Ras over-expression. In contrast, in this study, we have addressed the mechanism of how RasGrf1 facilitates neurite outgrowth in response to the neurotrophins, NGF and BDNF. Using Ras/Rac activation assays and site-directed RasGrf1 mutants, we find that both Ras and Rac are essential to neurotrophin-induced neurite outgrowth. Moreover, we find that H-Ras over-expression rescues the loss of neurite outgrowth observed with a Rac minus mutant and that RasGrf1 differentially stimulates NGF-dependent activation of Rac or Ras, depending on cell type. Collectively, these studies clarify the mechanism of how RasGrf1 expression facilitates neurotrophin-induced neurite outgrowth. Moreover, they suggest that H-Ras over-expression should be used with caution to measure phenotypic responses.

Stanniocalcin-1 secretion and receptor regulation in kidney cells.

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.