Hilmar Bading

Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways.

Here we report that synaptic and extrasynaptic NMDA (N-methyl-D-aspartate) receptors have opposite effects on CREB (cAMP response element binding protein) function, gene regulation and neuron survival. Calcium entry through synaptic NMDA receptors induced CREB activity and brain-derived neurotrophic factor (BDNF) gene expression as strongly as did stimulation of L-type calcium channels. In contrast, calcium entry through extrasynaptic NMDA receptors, triggered by bath glutamate exposure or hypoxic/ischemic conditions, activated a general and dominant CREB shut-off pathway that blocked induction of BDNF expression. Synaptic NMDA receptors have anti-apoptotic activity, whereas stimulation of extrasynaptic NMDA receptors caused loss of mitochondrial membrane potential (an early marker for glutamate-induced neuronal damage) and cell death. Specific blockade of extrasynaptic NMDA receptors may effectively prevent neuron loss following stroke and other neuropathological conditions associated with glutamate toxicity.

The Yin and Yang of NMDA receptor signalling.

Ca(2+) entry through the NMDA subtype of glutamate receptors has the power to determine whether neurons survive or die. Too much NMDA receptor activity is harmful to neurons - but so is too little. Is it a case of too much or too little Ca(2+) influx causing cell death or do other factors, such as receptor location or receptor-associated proteins, play a role? Understanding the mechanisms behind this dichotomous signalling is an important area of molecular neuroscience with direct clinical implications.

Stimulation of protein tyrosine phosphorylation by NMDA receptor activation

The N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate receptors, plays a key role in synaptic plasticity in the nervous system. After NMDA receptor activation, calcium entry into the postsynaptic neuron is a critical initial event. However, the subsequent mechanisms by which the NMDA receptor signal is processed are incompletely understood. Stimulation of cultured rat hippocampal cells with glutamate resulted in the rapid and transient tyrosine phosphorylation of a 39-kilodalton protein (p39). Tyrosine phosphorylation of p39 was triggered by the NMDA receptor and required an influx of Ca2+ from the extracellular medium. Because p39 was found to be highly related or identical to the microtubule-associated protein 2 kinase, the NMDA receptor signal may be processed by a sequential activation of protein kinases.

Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity

Information storage in the nervous system requires transcription triggered by synaptically evoked calcium signals. It has been suggested that translocation of calmodulin into the nucleus, initiated by submembranous calcium transients, relays synaptic signals to CREB. Here we show that in hippocampal neurons, signaling to CREB can be activated by nuclear calcium alone and does not require import of cytoplasmic proteins into the nucleus. The nucleus is particularly suited to integrate neuronal firing patterns, and specifies the transcriptional outputs through a burst frequency-to-nuclear calcium amplitude conversion. Calcium release from intracellular stores promotes calcium wave propagation into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors. Pharmacological or genetic modulation of nuclear calcium may directly affect transcription-dependent memory and cognitive functions.

Control of Recruitment and Transcription-Activating Function of CBP Determines Gene Regulation by NMDA Receptors and L-Type Calcium Channels

Recruitment of the coactivator CBP by signal-regulated transcription factors and stimulation of CBP activity are key regulatory events in the induction of gene transcription following Ca2+ flux through ligand- and/or voltage-gated ion channels in hippocampal neurons. The mode of Ca2+ entry (L-type Ca2+ channels versus NMDA receptors) differentially controls the CBP recruitment step to CREB, providing a molecular basis for the observed Ca2+ channel type-dependent differences in gene expression. In contrast, activation of CBP is triggered irrespective of the route of Ca2+ entry, as is activation of c-Jun, that recruits CBP independently of phosphorylation at major regulatory c-Jun phosphorylation sites, serines 63 and 73. This control of CBP recruitment and activation is likely relevant to other CBP-interacting transcription factors and represents a general mechanism through which Ca2+ signals associated with electrical activity may regulate the expression of many genes.

A calcium microdomain near NMDA receptors: on switch for ERK-dependent synapse-to-nucleus communication

A single second messenger, calcium, controls gene expression triggered by neuronal activity, and the spatial properties of calcium signals determine the type of transcriptional response. Nuclear calcium is a central regulator of transcription, though synaptic activity may elicit calcium transients that are confined to a space near the site of entry. Here we show that a calcium pool in the immediate vicinity of synaptic NMDA receptors is the on switch for extracellular signal-regulated kinase (ERK1/2)-mediated synapse-to-nucleus signaling; this signal propagates to the nucleus independently of global increases in calcium concentration, stimulates SRE-dependent gene expression and prolongs the transcriptionally active state of CREB following brief synaptic stimuli.

Nuclear Ca2+ and the cAMP Response Element-Binding Protein Family Mediate a Late Phase of Activity-Dependent Neuroprotection

The mechanism by which physiological synaptic NMDA receptor activity promotes neuronal survival is not well understood. Here, we show that that an episode of synaptic activity can promote neuroprotection for a long time after that activity has ceased. This long-lasting or “late phase” of neuroprotection is dependent on nuclear calcium signaling and cAMP response element (CRE)-mediated gene expression. In contrast, neuroprotection evoked acutely by ongoing synaptic activity relies solely on the activation of the phosphatidylinositol 3-kinase/Akt pathway. This “acute phase” does not require nuclear calcium signaling and is independent of activation of the CRE-binding protein (CREB) family of transcription factors. Thus, activity-dependent neuroprotection comprises two mechanistically distinct phases that differ in their spatial requirements for calcium and in their reliance on the CREB family.

Neuronal activity-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5.

The class II histone deacetylases, HDAC4 and HDAC5, directly bind to and repress myogenic transcription factors of the myocyte enhancer factor‐2 (MEF‐2) family thereby inhibiting skeletal myogenesis. During muscle differentiation, repression of gene transcription by MEF‐2/HDAC complexes is relieved due to calcium/calmodulin‐dependent (CaM) kinase‐induced translocation of HDAC4 and HDAC5 to the cytoplasm. MEF‐2 proteins and HDACs are also highly expressed in the nervous system and have been implicated in neuronal survival and differentiation. Here we investigated the possibility that the subcellular localization of HDACs, and thus their ability to repress target genes, is controlled by synaptic activity in neurones. We found that, in cultured hippocampal neurones, the localization of HDAC4 and HDAC5 is dynamic and signal‐regulated. Spontaneous electrical activity was sufficient for nuclear export of HDAC4 but not of HDAC5. HDAC5 translocation to the cytoplasm was induced following stimulation of calcium flux through synaptic NMDA receptors or L‐type calcium channels; glutamate bath application (stimulating synaptic and extrasynaptic NMDA receptors) antagonized nuclear export. Activity‐induced nucleocytoplasmic shuttling of both HDACs was partially blocked by the CaM kinase inhibitor KN‐62 with HDAC5 nuclear export being more sensitive to CaM kinase inhibition than that of HDAC4. Thus, the subcellular localization of HDACs in neurones is specified by neuronal activity; differences in the activation thresholds for HDAC4 and HDAC5 nuclear export provides a mechanism for input‐specific gene expression.

Decoding NMDA receptor signaling: identification of genomic programs specifying neuronal survival and death.

NMDA receptors promote neuronal survival but also cause cell degeneration and neuron loss. The mechanisms underlying these opposite effects on neuronal fate are unknown. Whole-genome expression profiling revealed that NMDA receptor signaling is decoded at the genomic level through activation of two distinct, largely nonoverlapping gene-expression programs. The location of the NMDA receptor activated specifies the transcriptional response: synaptic NMDA receptors induce a coordinate upregulation of newly identified pro-survival genes and downregulation of pro-death genes. Extrasynaptic NMDA receptors fail to activate this neuroprotective program, but instead induce expression of Clca1, a putative calcium-activated chloride channel that kills neurons. These results help explain the opposing roles of synaptic and extrasynaptic NMDA receptors on neuronal fate. They also demonstrate that the survival function is implemented in neurons through a multicomponent system of functionally related genes, whose coordinate expression is controlled by specific calcium signal initiation sites.

Preconditioning Doses of NMDA Promote Neuroprotection by Enhancing Neuronal Excitability

Neuroprotection can be induced by low doses of NMDA, which activate both synaptic and extrasynaptic NMDA receptors. This is in apparent contradiction with our recent findings that extrasynaptic NMDA receptor signaling exerts a dominant inhibitory effect on prosurvival signaling from synaptic NMDA receptors. Here we report that exposure to low preconditioning doses of NMDA results in preferential activation of synaptic NMDA receptors because of a dramatic increase in action potential firing. Both acute and long-lasting phases of neuroprotection in the face of apoptotic or excitotoxic insults are dependent on this firing enhancement. Key mediators of synaptic NMDA receptor-dependent neuroprotection, phosphatidylinositol 3 kinase-Akt (PI3 kinase-Akt) signaling to Forkhead box subgroup O (FOXO) export and glycogen synthase kinase 3β (GSK3β) inhibition and cAMP response element-binding protein-dependent (CREB-dependent) activation of brain-derived neurotrophic factor (BDNF), can be induced only by low doses of NMDA via this action potential-dependent route. In contrast, NMDA doses on the other side of the toxicity threshold do not favor synaptic NMDA receptor activation because they strongly suppress firing rates below baseline. The classic bell-shaped curve depicting neuronal fate in response to NMDA dose can be viewed as the net effect of two antagonizing (synaptic vs extrasynaptic) curves: via increased firing the synaptic signaling dominates at low doses, whereas firing becomes suppressed and extrasynaptic signaling dominates as the toxicity threshold is crossed.