A signaling cascade of nuclear calcium-CREB-ATF3 activated by synaptic NMDA receptors defines a gene repression module that protects against extrasynaptic NMDA receptor-induced neuronal cell death and ischemic brain damage.

Synapse-to-nucleus signaling triggered by synaptic NMDA receptors can lead to the buildup of a neuroprotective shield. Nuclear calcium activating the cAMP response element binding protein (CREB) plays a key role in neuroprotection acquired by synaptic activity. Here we show that in mouse hippocampal neurons, the transcription factor Atf3 (activating transcription factor 3) is a direct target of CREB. Induction of ATF3 expression by CREB in hippocampal neurons was initiated by calcium entry through synaptic NMDA receptors and required nuclear calcium transients and calcium/calmodulin-dependent protein kinase IV activity. Acting as a transcriptional repressor, ATF3 protects cultured hippocampal neurons from apoptosis and extrasynaptic NMDA receptor-induced cell death triggered by bath application of NMDA or oxygen-glucose deprivation. Expression of ATF3 in vivo using stereotaxic delivery of recombinant adeno-associated virus reduces brain damage following a cerebral ischemic insult in mice. Conversion of ATF3 to a transcriptional activator transforms ATF3 into a potent prodeath protein that kills neurons in cell culture and, when expressed in vivo in the hippocampus, ablates the neuronal cell layer. These results link nuclear calcium-CREB signaling to an ATF3-mediated neuroprotective gene repression program, indicating that activity-dependent shutoff of genes is an important process for survival. ATF3 supplementation may counteract age- and disease-related neuronal cell loss caused by a reduction in synaptic activity, malfunctioning of calcium signaling toward and within the nucleus ("nuclear calciopathy"), or increases in death signaling by extrasynaptic NMDA receptors.

Conjugation of peptides to the passivation shell of gold nanoparticles for targeting of cell-surface receptors.

We report the preparation of gold nanoparticles (AuNPs) functionalized with the peptide-toxin conantokin-G and their selective binding to N-methyl-d-aspartate (NMDA) receptors recombinantly expressed by transfected HEK 293 cells. The AuNPs are passivated with a mixed self-assembled monolayer of ω-carboxy- and ω-amino-polyethylene glycol (PEG) thiols. We compare two different passivation systems: the alkyl-PEG600 system is characterized by a C(11)-alkyl chain between the thiol group and the PEG segment, whereas the PEG3000 system lacks this alkyl-chain. We show that only the alkyl-PEG600 passivation system allows selective conjugation of cysteine-terminated peptides to the periphery of the passivation layer via a heterobifunctional linker strategy. In contrast, using the PEG3000 passivation system, peptides are immobilized both on the passivation layer and directly on the gold surface via concurrent place-exchange reaction. We therefore recommend the use of the alkyl-PEG600 system to precisely control the number of immobilized peptides on AuNPs. In fact, we show that the number of conjugated peptides per particle can be varied with good control simply by varying the composition of the self-assembled monolayer. Finally, we demonstrate that conjugation of the conantokin-G peptide to the solvent-exposed interface of the passivation layer results in maximal binding interaction between the peptide-functionalized AuNPs and the targeted NMDA receptors on the cell surface. Conantokin G-coupled AuNP may be used to spatially restrict NMDA-receptor-blockade on neuronal surfaces.

Synaptic activity and nuclear calcium signaling protect hippocampal neurons from death signal-associated nuclear translocation of FoxO3a induced by extrasynaptic N-methyl-D-aspartate receptors.

Synaptic activity and the generation of nuclear calcium signals promote neuronal survival through a transcription-dependent process that is not fully understood. Here we show that one mechanism of activity-induced acquired neuroprotection involves the Forkhead transcription factor, FoxO3a, which is known to induce genomic death responses upon translocation from the cytosol to the nucleus. Depletion of endogenous FoxO3a using RNA interference renders hippocampal neurons more resistant to excitotoxic cell death. Using a FoxO3a-green fluorescent protein (GFP) fusion protein to monitor in real time the localization of FoxO3a in hippocampal neurons, we found that several cell death inducing stimuli, including the stimulation of extrasynaptic N-methyl-D-aspartate receptors, growth factor withdrawal, and oxygen-glucose deprivation, caused a swift translocation of FoxO3a-GFP from the cytosol to the cell nucleus. This translocation was inhibited in hippocampal neurons that had undergone prolonged periods of synaptic activity before exposure to cell death-inducing conditions. The activity-dependent protection from death signal-induced FoxO3a-GFP nuclear translocation required synaptic N-methyl-D-aspartate receptor activation and was dependent on nuclear calcium signaling and calcium/calmodulin-dependent protein kinase IV. The modulation of nucleo-cytoplasmic shuttling of FoxO3a may represent one mechanism through which nuclear calcium-induced genomic responses affect cell death processes.

A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity.

BACKGROUND: Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic) may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death. RESULTS: Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours) periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6%) reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting. CONCLUSION: The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity-dependent neuroprotection. Other factors, in particular calcium signaling to the nucleus and the induction of survival promoting genes are more likely to mediate acquired neuroprotection.

The postsynaptic scaffold proteins ProSAP1/Shank2 and Homer1 are associated with glutamate receptor complexes at rat retinal synapses.

The postsynaptic density (PSD) at glutamatergic synapses is a macromolecular complex of various molecules that organize the different glutamate receptors spatially and link them to their appropriate downstream signaling pathways and to the cytoskeleton. Recently, a new family of multidomain proteins called Shanks or ProSAPs (proline-rich synapse-associated proteins) has been identified. They are suggested to be central adaptor proteins of the PSD of glutamatergic synapses, bridging different types of glutamate receptor complexes. With immunocytochemistry and light and electron microscopy, we examined the cellular, synaptic, and postnatal developmental expression of ProSAP1/Shank2 at the synapses of rat retina. With double-labeling experiments and confocal microscopy, we analyzed the association of ProSAP1/Shank2 with proteins specific for glutamatergic, glycinergic, and gamma-aminobutyric acid (GABA)ergic synapses and with proteins known to be involved in the structural and functional organization of PSDs containing N-methyl-D-aspartate receptors [95-kDa postsynaptic density protein (PSD-95)], group I metabotropic glutamate receptors (Homer1), and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors [glutamate receptor-interacting protein (GRIP)]. ProSAP1/Shank2 was present postsynaptically at the glutamatergic ribbon synapses of photoreceptor and bipolar cells, and it was absent from glycinergic and GABAergic amacrine cell synapses. The double-labeling experiments revealed a high rate of colocalization of ProSAP1/Shank2 with Homer1 and PSD-95, and little colocalization with GRIP. These data suggest that ProSAP1/Shank2 acts as an organizer at PSDs of different glutamatergic retinal synapses.

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina.

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.