Activity-dependent modulation of synaptic efficacy in the brain contributes to neural circuit development and experience-dependent plasticity. Although glia are affected by activity and ensheathe synapses, their influence on synaptic strength has largely been ignored. Here, we show that a protein produced by glia, tumor necrosis factor alpha (TNFalpha), enhances synaptic efficacy by increasing surface expression of AMPA receptors. Preventing the actions of endogenous TNFalpha has the opposite effects. Thus, the continual presence of TNFalpha is required for preservation of synaptic strength at excitatory synapses. Through its effects on AMPA receptor trafficking, TNFalpha may play roles in synaptic plasticity and modulating responses to neural injury.

The proinflammatory cytokine tumor necrosis factor-␣ (TNF␣) causes a rapid exocytosis of AMPA receptors in hippocampal pyramidal cells and is constitutively required for the maintenance of normal surface expression of AMPA receptors. Here we demonstrate that TNF␣ acts on neuronal TNFR1 receptors to preferentially exocytose glutamate receptor 2-lacking AMPA receptors through a phosphatidylinositol 3 kinase-dependent process. This increases excitatory synaptic strength while changing the molecular stoichiometry of synaptic AMPA receptors. Conversely, TNF␣ causes an endocytosis of GABA A receptors, resulting in fewer surface GABA A receptors and a decrease in inhibitory synaptic strength. These results suggest that TNF␣ can regulate neuronal circuit homeostasis in a manner that may exacerbate excitotoxic damage resulting from neuronal insults.

The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

The endocytosis of AMPA receptors is thought to be important in the expression of long-term depression (LTD) triggered by NMDA receptor activation. Although signaling pathways necessary for LTD induction have been identified, those responsible for the regulated internalization of AMPA receptors are unknown. Here we show that activation of NMDA receptors alone can trigger AMPA receptor endocytosis through calcium influx and activation of the calcium-dependent protein phosphatase calcineurin. A distinct signaling mechanism mediates the AMPA receptor endocytosis stimulated by insulin. These results demonstrate that although multiple signaling pathways can induce AMPA receptor internalization, NMDA receptor activation enhances AMPA receptor endocytosis via a signaling mechanism required for the induction of LTD.

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

The survival, differentiation, and maintenance of responsive neurons are regulated by nerve growth factor (NGF), which is secreted by the target and interacts with receptors on the axon tip. It is uncertain how the NGF signal is communicated retrogradely from distal axons to neuron cell bodies. Retrograde transport of activated receptors in endocytic vesicles could convey the signal. However, little is known about endocytosis of NGF receptors, and there is no evidence that NGF receptors continue to signal after endocytosis. We have examined early events in the membrane traffic of NGF and its receptor, gp140(TrkA) (TrkA), in PC12 cells. NGF induced rapid and extensive endocytosis of TrkA in these cells, and the receptor subsequently moved into small organelles located near the plasma membrane. Some of these organelles contained clathrin and alpha-adaptin, which implies that TrkA is internalized by clathrin-mediated endocytosis. Using mechanical permeabilization and fractionation, intracellular organelles derived from endocytosis were separated from the plasma membrane. After NGF treatment, NGF was bound to TrkA in endocytic organelles, and TrkA was tyrosine-phosphorylated and bound to PLC-gamma1, suggesting that these receptors were competent to initiate signal transduction. These studies raise the possibility that NGF induces formation of signaling endosomes containing activated TrkA. They are an important first step in elucidating the molecular mechanism of NGF retrograde signaling.

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-Daspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.F ast excitatory synaptic transmission in the mammalian central nervous system is mediated primarily by ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type and Nmethyl-D-aspartate (NMDA)-type ionotropic glutamate receptors, which are coexpressed at many synapses and subserve distinct physiological functions in synaptic transmission (1-3). Although the vast majority of excitatory synapses in the hippocampus expresses functional NMDA receptors (NMDARs), electrophysiological and anatomical data suggest that the number of AMPA receptors (AMPARs) expressed at individual synapses on CA1 pyramidal cells is highly variable (4-8). Furthermore, recent evidence suggests that the surface expression of AMPARs at individual synapses is not fixed but is dynamically regulated by neuronal activity (2, 9-12). This activity-dependent regulation of the synaptic expression of AMPARs may contribute to the changes in synaptic strength that occur during NMDAR-dependent long-term potentiation and long-term depression (11, 12). Surprisingly, little is known about the detailed molecular mechanisms that regulate the number of AMPARs at excitatory synapses. Previously, we showed a pronounced redistribution of AMPARs away from synaptic sites within minutes after the triggering of long-term depression (12) or pharmacological activation of AMPARs (13). Herein, we show that this process is mediated by dynamin-dependent endocytosis and identify a role of NMDAR activation in promoting AMPAR endocytosis under physiological conditions. Materials and MethodsCell Culture and Immunocytochemistry. Hippocampal cultures were prepared as described (12, 13) and were used for experimentation at 2-3 weeks after plating. Surface AMPARs were stained with an antibody recognizing an extracellular epitope (amino acids 271-285) of the rat GluR1 subunit (Oncogene Research). Before treatment, antibody (5 g͞ml) was applied to live cells for 15 min at 37°C in conditioned cell medium. Cells were then briefly...

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.