Gang-Yi Wu

Maturation of a Central Glutamatergic Synapse

Whole-cell recordings from optic tectal neurons in Xenopus tadpoles were used to study the maturation of a glutamatergic synapse. The first glutamatergic transmission is mediated only by N-methyl-D-aspartate (NMDA) receptors and is silent at resting potentials. More mature synapses acquire transmission by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. This maturational program is mimicked by postsynaptic expression of constitutively active calcium-calmodulin-dependent protein kinase II (CaMKII). Newly formed synapses may be silent unless sufficient depolarization is provided by coincident activity that could activate postsynaptic CaMKII, resulting in the appearance of AMPA responses.

Regulation of Dendritic Morphogenesis by Ras–PI3K–Akt–mTOR and Ras–MAPK Signaling Pathways

Dendritic arborization and spine formation are critical for the functioning of neurons. Although many proteins have been identified recently as regulators of dendritic morphogenesis, the intracellular signaling pathways that control these processes are not well understood. Here we report that the Ras–phosphatidylinositol 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling pathway plays pivotal roles in the regulation of many aspects of dendrite formation. Whereas the PI3K–Akt–mTOR pathway alone controlled soma and dendrite size, a coordinated activation together with the Ras-mitogen-activated protein kinase signaling pathway was required for increasing dendritic complexity. Chronic inhibition of PI3K or mTOR reduced soma and dendrite size and dendritic complexity, as well as density of dendritic filopodia and spines, whereas a short-term inhibition promoted the formation of mushroom-shaped spines on cells expressing constitutively active mutants of Ras, PI3K, or Akt, or treated with the upstream activator BDNF. Together, our data underscore the central role of a spatiotemporally regulated key cell survival and growth pathway on trophic regulation of the coordinated development of dendrite size and shape.

Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Spaced stimuli stabilize MAPK pathway activation and its effects on dendritic morphology.

Memory storage in mammalian neurons probably depends on both biochemical events and morphological alterations in dendrites. Here we report an activity-dependent stabilization of the MAP kinase (MAPK) pathway, prominent in hippocampal dendrites. The longevity of the signal in these dendrites was increased to hours when multiple spaced stimuli were used. Likewise, spaced stimuli and MAPK activation were critical for protrusion of new dendritic filopodia that also remained stable for hours. Our experiments define a new role for stimulus-specific responses of MAPK signaling in activity-dependent neuronal plasticity. The local biochemical signaling in dendrites complements MAPK signaling in gene expression. Together, these processes may support long-lasting behavioral changes.

Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus.

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.

Calcium–Calmodulin-Dependent Kinase II Modulates Kv4.2 Channel Expression and Upregulates Neuronal A-Type Potassium Currents

Calcium–calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K+ channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K+ current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K+ channels.

The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein–protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein–protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.

Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins

Vaccinia virus can be used to infect cells in the CNS of frogs, Xenopus laevis, and Rana pipiens, both in vivo and in vitro. In vivo infections were accomplished by injection of viral solution into the tectal ventricle of stage 40-48 tadpoles or by local injections into distinct neural regions. Infections with high titer of virus injected into the ventricle resulted in the majority of cells in the brain expressing foreign protein, while cells in the retina and optic nerve showed no expression. Infection with lower viral titers resulted in fewer infected cells that were distributed throughout the otherwise normal tissue. Intense expression of foreign protein in the brain was observed 36 hr after injection and remained high for at least 4 days. Infected animals developed normally and had the same number of cells in the optic tectum as control animals. Infection with a recombinant virus carrying the gene for Green Fluorescent Protein labels neurons, so that infected cells can be observed in vivo. Vaccinia virus provides a versatile means to alter proteins in distinct populations of neurons in amphibia.