Treatment of Ischemic Brain Damage by Perturbing NMDA Receptor- PSD-95 Protein Interactions

Aarts, Michelle; Liu, Yitao; Liu, Lidong; Besshoh, Shintaro; Arundine, Mark; Gurd, James W.; Wang, Yu Tian; Salter, Michael W.; Tymianski, Michael

doi:10.1126/science.1072873

Cited by 901 publications

(900 citation statements)

References 20 publications

Supporting

Mentioning

854

Contrasting

Unclassified

Order By: Relevance

“…Four C-terminal residues of the ligand PDZ2-3 (STVV-COOH) identified in the current study with FRET hybrids are identical to those from the NR1 subunit of NMDA receptor, although Y2H assays have not identified this interaction (40). Finally, short peptides derived from the C terminus of NMDA receptor (NR2) have been shown to prevent neuronal death after stroke in a rat model (41). Using FRET hybrids, several ligands were identified that bound PDZ2 with higher affinity than the peptide derived from NR2, suggesting that this approach could enable discovery of therapeutically useful protein-interaction inhibitors (42).…”

Section: Discussionmentioning

confidence: 92%

Intracellular protein interaction mapping with FRET hybrids

You

Nguyen

Jabaiah

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

100

View full text Add to dashboard Cite

A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.cell sorting ͉ protein-ligand interactions T he ability to identify and quantitatively characterize proteinprotein interactions in living cells is essential for developing a detailed, system-level understanding of cellular function. The yeast two-hybrid (Y2H) system has served as the primary genetic tool to discover potential biological interaction partners for an enormous number of proteins (1, 2). Application of Y2H assays to each of the nearly 6,000 yeast ORFs enabled construction of the first large-scale protein interaction network in yeast (3, 4), and similar interaction studies were performed subsequently in Drosophila melanogaster and Caenorhabditis elegans (5, 6). Human-protein interaction networks are less well characterized and present difficult challenges to interaction mapping approaches. In fact, only a small fraction of an estimated 10 5 to 10 6 human-proteome interactions (7) have been unambiguously identified, primarily by using Y2H and mass spectrometry approaches (7,8). Unfortunately, high-throughput Y2H assays are typically prone to a high frequency of false positives that complicate the interpretation of interaction data. In some studies, it has been estimated that Ͼ50% of putative interactions identified are false positives (9).Several important challenges remain in the elucidation of protein-protein interaction networks and their influence on cell function (10). Large-scale interaction maps can portray basic network structure but lack quantitative features needed to understand network function. A predictive understanding of network function likely will require the elucidation of equilibrium and kinetic properties within a network, including individual equilibrium-binding constants. Furthermore, existing highthroughput approaches (e.g., Y2H) are not well suited to investigate real-time dynamics in protein networks essential for understanding cell function (11) because detection, in these cases, is ba...

show abstract

Section: Discussionmentioning

confidence: 92%

Intracellular protein interaction mapping with FRET hybrids

You

Nguyen

Jabaiah

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

100

View full text Add to dashboard Cite

show abstract

“…38 Likewise, in rats submitted to focal ischemia, the specific disruption of the interaction between the NR2B subunit of NMDA receptors and the PSD-95, which couples NMDA receptors to neuronal nitric oxid synthase (nNOS), reduced brain damage and improved the neurological function, without the negative consequences associated with blocking NMDA receptors. 39 Thus, dissection of the molecular pathway which couples Ca 2 þ -permeable GluR4-containing AMPA receptors to c-Jun/AP-1 will allow the development of selective therapeutic strategies aiming at the disruption of specific intracellular interactions coupled to glutamate receptors while maintaining the physiological actions of the receptor. Cell culture and transfection HEK293 cells, constitutively expressing the GluR4 flip subunit of AMPA receptors, were cultured in DMEM with 10% heat-inactivated fetal bovine serum (FBS), in the presence of geneticin (G418), and kept at 371C, in a humidified incubator with 5% CO 2 /95% air.…”

Section: Discussionmentioning

confidence: 99%

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor

et al. 2005

View full text Add to dashboard Cite

Cells preferentially expressing GluR4-containing a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca 2+ -permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4 flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca 2+ -permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

show abstract

“…6 B). The loss of NMDA receptor subunit interactions with the postsynaptic scaffold has been shown to block ischemia-induced toxicity (Aarts et al, 2002) but exerts no short term effect on basal synaptic transmission or long-term potentiation formation in hippocampal cultures (Lim et al, 2003). Previous studies have shown that actin depolymerization reduces NMDA channel activity (Rosenmund and Westbrook, 1993).…”

Section: Discussionmentioning

confidence: 99%

Ubiquitin–Proteasome-Mediated Synaptic Reorganization: A Novel Mechanism Underlying Rapid Ischemic Tolerance

Meller¹,

Thompson²,

Lusardi³

et al. 2008

J. Neurosci.

View full text Add to dashboard Cite

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (genemediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-␤-lactone].A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.

show abstract

Treatment of Ischemic Brain Damage by Perturbing NMDA Receptor- PSD-95 Protein Interactions

Cited by 901 publications

References 20 publications

Intracellular protein interaction mapping with FRET hybrids

Intracellular protein interaction mapping with FRET hybrids

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor

Ubiquitin–Proteasome-Mediated Synaptic Reorganization: A Novel Mechanism Underlying Rapid Ischemic Tolerance

Contact Info

Product

Resources

About