Giles E. Hardingham

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.

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.

Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses.

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPβ and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.

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.

Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening.

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.

Coupling of the NMDA receptor to neuroprotective and neurodestructive events

NMDA (N-methyl-D-aspartate) receptors are a subtype of ionotropic glutamate receptor with an important role in the physiology and pathophysiology of central neurons. Inappropriate levels of Ca(2+) influx through the NMDA receptor can contribute to neuronal loss in acute trauma such as ischaemia and traumatic brain injury, as well as certain neurodegenerative diseases such as Huntingtons disease. However, normal physiological patterns of NMDA receptor activity can promote neuroprotection against both apoptotic and excitotoxic insults. As a result, NMDA receptor blockade can promote neuronal death outright or render neurons vulnerable to secondary trauma. Thus responses to NMDA receptor activity follow a classical hormetic dose-response curve: both too much and too little can be harmful. There is a growing knowledge of the molecular mechanisms underlying both the neuroprotective and neurodestructive effects of NMDA receptor activity, as well as the factors that determine whether an episode of NMDA receptor activity is harmful or beneficial. It is becoming apparent that oxidative stress plays a role in promoting neuronal death in response to both hyper- and hypo-activity of the NMDA receptor. Increased understanding in this field is leading to the discovery of new therapeutic targets and strategies for excitotoxic disorders, as well as a growing appreciation of the harmful consequences of NMDA receptor blockade.