Cannabinoid receptor activation reduces TNFalpha-induced surface localization of AMPAR-type glutamate receptors and excitotoxicity.

After injury or during neurodegenerative disease in the central nervous system (CNS), the concentration of tumor necrosis factor alpha (TNFalpha) rises above normal during the inflammatory response. In vitro and in vivo, addition of exogenous TNFalpha to neurons has been shown to induce rapid plasma membrane-delivery of AMPA-type glutamate receptors (AMPARs) potentiating glutamatergic excitotoxicity. Thus the discovery of drug targets reducing excess TNFalpha-induced AMPAR surface expression may help protect neurons after injury. In this study, we investigate the neuroprotective role of the CB1 cannabinoid receptor using quantitative immunofluorescent and real-time video microscopy to measure the steady-state plasma membrane AMPAR distribution and rate of AMPAR exocytosis after TNFalpha exposure in the presence or absence of CB1 agonists. The neuroprotective potential of CB1 activation with TNFalpha was measured in hippocampal neuron cultures challenged by an in vitro kainate (KA)-mediated model of Excitotoxic Neuroinflammatory Death (END). Here, we demonstrate that CB1 activation blocks the TNFalpha-induced increase in surface AMPARs and protects neurons from END. Thus, neuroprotective strategies which increase CB1 activity may help to reduce the END that occurs as a result of a majority of CNS insults.

Cell death after spinal cord injury is exacerbated by rapid TNF alpha-induced trafficking of GluR2-lacking AMPARs to the plasma membrane.

Glutamate, the major excitatory neurotransmitter in the CNS, is implicated in both normal neurotransmission and excitotoxicity. Numerous in vitro findings indicate that the ionotropic glutamate receptor, AMPAR, can rapidly traffic from intracellular stores to the plasma membrane, altering neuronal excitability. These receptor trafficking events are thought to be involved in CNS plasticity as well as learning and memory. AMPAR trafficking has recently been shown to be regulated by glial release of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) in vitro. This has potential relevance to several CNS disorders, because many pathological states have a neuroinflammatory component involving TNFalpha. However, TNFalpha-induced trafficking of AMPARs has only been explored in primary or slice cultures and has not been demonstrated in preclinical models of CNS damage. Here, we use confocal and image analysis techniques to demonstrate that spinal cord injury (SCI) induces trafficking of AMPARs to the neuronal membrane. We then show that this effect is mimicked by nanoinjections of TNFalpha, which produces specific trafficking of GluR2-lacking receptors which enhance excitotoxicity. To determine if TNFalpha-induced trafficking affects neuronal cell death, we sequestered TNFalpha after SCI using a soluble TNFalpha receptor, and significantly reduced both AMPAR trafficking and neuronal excitotoxicity in the injury penumbra. The data provide the first evidence linking rapid TNFalpha-induced AMPAR trafficking to early excitotoxic secondary injury after CNS trauma in vivo, and demonstrate a novel way in which pathological states hijack mechanisms involved in normal synaptic plasticity to produce cell death.

Rapid tumor necrosis factor alpha-induced exocytosis of glutamate receptor 2-lacking AMPA receptors to extrasynaptic plasma membrane potentiates excitotoxicity.

The postinjury inflammatory response in the CNS leads to neuronal excitotoxicity. Our previous studies show that a major component of this response, the inflammatory cytokine tumor necrosis factor alpha (TNFalpha), causes a rapid increase in AMPA glutamate receptors (AMPARs) on the plasma membrane of cultured hippocampal neurons. This may potentiate neuron death through an increased vulnerability to AMPAR-dependent excitotoxic stress. Here, we test this hypothesis with an in vitro lactose dehydrogenase death assay and examine in detail the AMPAR surface delivery time course, receptor subtype, and synaptic and extrasynaptic distribution after TNFalpha exposure. These data demonstrate that surface levels of glutamate receptor 2 (GluR2)-lacking Ca2+-permeable AMPARs peak at 15 min after TNFalpha treatment, and the majority are directed to extrasynaptic sites. TNFalpha also induces an increase in GluR2-containing surface AMPARs but with a slower time course. We propose that this activity contributes to excitotoxic neuron death because TNFalpha potentiation of kainate excitotoxicity is blocked by a Ca2+-permeable AMPAR antagonist [NASPM (1-naphthyl acetyl spermine)] and a specific phosphoinositide 3 kinase (PI3 kinase) inhibitor (LY294,002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]) previously shown to block the TNFalpha-induced increase in AMPAR surface delivery. This information forms the basis for future in vivo studies examining AMPAR-dependent potentiation of excitotoxic neuron death and dysfunction caused by TNFalpha after acute injury and during neurodegenerative or neuropsychiatric disorders.

Altered presymptomatic AMPA and cannabinoid receptor trafficking in motor neurons of ALS model mice: implications for excitotoxicity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving the selective loss of spinal cord motor neurons. Excitotoxicity mediated by glutamate has been implicated as a cause of this progressive degeneration. In this study we examined two types of receptors, the excitatory alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) and inhibitory cannabinoid receptor (CB1) with respect to their localization and total expression in spinal cord motor neurons. AMPAR and CB1 represent major excitatory and inhibitory transmission input, respectively, and their expression levels on the plasma membrane have direct relevance to the vulnerability of the motor neurons to glutamatergic excitotoxicity. We used quantitative immunofluorescence microscopy to comparatively measure the total cellular expression and the synaptic localization of specific subclasses of AMPARs [as determined by the presence of the subunits glutamate receptor 1 (GluR1) or glutamate receptor 2 (GluR2)] and CB1 in spinal cord motor neurons during disease progression in a G93ASOD1 mouse model of ALS. We found an increase in synaptic GluR1 and a decrease of synaptic and total GluR2 at early ages (6 weeks, prior to disease onset). Total CB1 receptor levels were decreased at 6 weeks old. We determined the gene expression of CB1, GluR1 and GluR2 using quantitative real-time reverse transcriptase-polymerase chain reaction. The decreased synaptic and total GluR2 and increased synaptic GluR1 levels may result in increased numbers of Ca2+-permeable AMPARs, thus contributing to neuronal death. Early alterations in CB1 expression may also predispose motor neurons to excitotoxicity. To our knowledge, this is the first demonstration of presymptomatic changes in trafficking of receptors that are in direct control of excitotoxicity and death in a mouse model of ALS.

Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors.

We directly resolved discrete exocytic fusion events mediating insertion of AMPA-type glutamate receptors (AMPARs) to the somatodendritic surface of rat hippocampal pyramidal neurons, in slice and dissociated cultures, using protein tagging with a pH-sensitive GFP (green fluorescent protein) variant and rapid (10 frames/s) fluorescence microscopy. AMPAR-containing exocytic events occurred under basal culture conditions in both the cell body and dendrites; potentiating chemical stimuli produced an NMDA receptor-dependent increase in the frequency of individual exocytic events. The number of AMPARs inserted per exocytic event, estimated using single-molecule analysis, was quite uniform but individual events differed significantly in kinetic properties affecting the subsequent surface distribution of receptors. "Transient" events, from which AMPARs dispersed laterally immediately after surface insertion, generated a pronounced but short-lived (dissipating within approximately 1 s) increase in surface AMPAR fluorescence extending locally (2-5 microm) from the site of exocytosis. "Persistent" events, from which inserted AMPARs dispersed slowly (typically over 5-10 s), affected local surface receptor concentration to a much smaller degree. Both modes of exocytic insertion occurred throughout the dendritic shaft, but remarkably, neither mode of insertion was observed directly into synaptic spines. AMPARs entered spines preferentially from transient events occurring in the adjoining dendritic shaft, driven apparently by mass action and short-range lateral diffusion, and locally delivered AMPARs remained mostly in the mobile fraction. These results suggest a highly dynamic mechanism for both constitutive and activity-dependent surface delivery of AMPARs, mediated by kinetically distinct exocytic modes that differ in propensity to drive lateral entry of receptors to nearby synapses.

Centrosome impairments and consequent cytokinesis defects are possible mechanisms of taxane drugs.

Taxol and taxotere are two of the most promising anticancer drugs. To determine the mechanisms responsible for cell death after exposure to low doses of taxane, PC3 cells were treated with taxol and taxotere, and observed with immunofluoroscence microscopy. Pericentriolar material dissociation and blockage of normal centrosome separation were found to result in two different abnormal spindle types; multipolar and monopolar spindles, respectively. The majority of abnormal spindles induced by taxol were monopolar spindles, whereas taxotere mostly induced abnormal multipolar spindles. Consequently, monopolar spindle mitosis resulted in cleavage failure, while multipolar spindle mitosis led to the formation of both cleavage failure and multipolar cell division. Multinucleation characterized interphase cells which had undergone cytokinesis defects. These cells subsequently became giant multinucleated cells after several rounds of cell cycle with sustained cleavage failure, and were gradually eliminated through cell death.

Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.

The proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) 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 TNFalpha 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, TNFalpha 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 TNFalpha can regulate neuronal circuit homeostasis in a manner that may exacerbate excitotoxic damage resulting from neuronal insults.

TNFalpha-induced AMPA-receptor trafficking in CNS neurons; relevance to excitotoxicity?

Injury and disease in the CNS increases the amount of tumor necrosis factor alpha (TNFalpha) that neurons are exposed to. This cytokine is central to the inflammatory response that occurs after injury and during prolonged CNS disease, and contributes to the process of neuronal cell death. Previous studies have addressed how long-term apoptotic-signaling pathways that are initiated by TNFalpha might influence these processes, but the effects of inflammation on neurons and synaptic function in the timescale of minutes after exposure are largely unexplored. Our published studies examining the effect of TNFalpha on trafficking of AMPA-type glutamate receptors (AMPARs) in hippocampal neurons demonstrate that glial-derived TNFalpha causes a rapid (<15 minute) increase in the number of neuronal, surface-localized, synaptic AMPARs leading to an increase in synaptic strength. This indicates that TNFalpha-signal transduction acts to facilitate increased surface localization of AMPARs from internal postsynaptic stores. Importantly, an excess of surface localized AMPARs might predispose the neuron to glutamate-mediated excitotoxicity and excessive intracellular calcium concentrations, leading to cell death. This suggests a new mechanism for excitotoxic TNFalpha-induced neuronal death that is initiated minutes after neurons are exposed to the products of the inflammatory response. Here we review the importance of AMPAR trafficking in normal neuronal function and how abnormalities that are mediated by glial-derived cytokines such as TNFalpha can be central in causing neuronal disorders. We have further investigated the effects of TNFalpha on different neuronal cell types and present new data from cortical and hippocampal neurons in culture. Finally, we have expanded our investigation of the temporal profile of the action of this cytokine relevant to neuronal damage. We conclude that TNFalpha-mediated effects on AMPAR trafficking are common in diverse neuronal cell types and very rapid in their onset. The abnormal AMPAR trafficking elicited by TNFalpha might present a novel target to aid the development of new neuroprotective drugs.

Control of synaptic strength by glial TNFalpha.

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.