Daniel T. S. Pak

A transcriptionally active DNA-binding site for human p53 protein complexes.

Recent studies have demonstrated transcriptional activation domains within the tumor suppressor protein p53, while others have described specific DNA-binding sites for p53, implying that the protein may act as a transcriptional regulatory factor. We have used a reiterative selection procedure (CASTing: cyclic amplification and selection of targets) to identify new specific binding sites for p53, using nuclear extracts from normal human fibroblasts as the source of p53 protein. The preferred consensus is the palindrome GGACATGCCCGGGCATGTCC. In vitro-translated p53 binds to this sequence only when mixed with nuclear extracts, suggesting that p53 may bind DNA after posttranslational modification or as a complex with other protein partners. When placed upstream of a reporter construct, this sequence promotes p53-dependent transcription in transient transfection assays.

Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes.

The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 were found in physical complex. The association was shown biochemically to require the chromodomain and shadow domains of HP1. The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

Regulation of Dendritic Spine Morphology by SPAR, a PSD-95-Associated RapGAP

The PSD-95/SAP90 family of scaffold proteins organizes the postsynaptic density (PSD) and regulates NMDA receptor signaling at excitatory synapses. We report that SPAR, a Rap-specific GTPase-activating protein (RapGAP), interacts with the guanylate kinase-like domain of PSD-95 and forms a complex with PSD-95 and NMDA receptors in brain. In heterologous cells, SPAR reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin. In hippocampal neurons, SPAR localizes to dendritic spines and causes enlargement of spine heads, many of which adopt an irregular appearance with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. The effects of SPAR on spine morphology depend on the RapGAP and actin-interacting domains, implicating Rap signaling in the regulation of postsynaptic structure.

LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses

Leukocyte common antigen–related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-α (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin–β-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, β-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-α and GRIP from rat brain extracts. We propose that the cadherin-β-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-α to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.

Rap2-JNK Removes Synaptic AMPA Receptors during Depotentiation

The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.

Interaction of Reelin with Amyloid Precursor Protein Promotes Neurite Outgrowth

The processing of amyloid precursor protein (APP) to Aβ is an important event in the pathogenesis of Alzheimers disease, but the physiological function of APP is not well understood. Our previous work has shown that APP processing and Aβ production are regulated by the extracellular matrix protein Reelin. In the present study, we examined whether Reelin interacts with APP, and the functional consequences of that interaction in vitro. Using coimmunoprecipitation, we found that Reelin interacted with APP through the central domain of Reelin (repeats 3–6) and the E1 extracellular domain of APP. Reelin increased cell surface levels of APP and decreased endocytosis of APP in hippocampal neurons in vitro. In vivo, Reelin levels were increased in brains of APP knock-out mice and decreased in APP-overexpressing mice. RNA interference knockdown of APP decreased neurite outgrowth in vitro and prevented Reelin from increasing neurite outgrowth. Knock-out of APP or Reelin decreased dendritic arborization in cortical neurons in vivo, and APP overexpression increased dendritic arborization. APP and Reelin have previously been shown to promote neurite outgrowth through interactions with integrins. We confirmed that APP interacted with α3β1 integrin, and α3β1 integrin altered APP trafficking and processing. Addition of an α3β1 integrin antibody prevented APP and Reelin-induced neurite outgrowth. These findings demonstrate that Reelin interacts with APP, potentially having important effects on neurite development.

A Drosophila Homolog of the Yeast Origin Recognition Complex

Genes from Drosophila melanogaster have been identified that encode proteins homologous to Orc2p and Orc5p of the Saccharomyces cerevisiae origin recognition complex (ORC). The abundance of the Drosophila Orc2p homolog DmORC2 is developmentally regulated and is greatest during the earliest stages of embryogenesis, concomitant with the highest rate of DNA replication. Fractionation of embryo nuclear extracts revealed that DmORC2 is found in a tightly associated complex with five additional polypeptides, much like the yeast ORC. These studies will enable direct testing of the initiator-based model of replication in a metazoan.

Glutamate receptor anchoring proteins and the molecular organization of excitatory synapses.

ABSTRACT: Ionotropic glutamate receptors are concentrated at postsynaptic sites in excitatory synapses. The cytoplasmic C‐terminal tail of certain glutamate receptor subunits interact with specific PDZ domain‐containing proteins. NMDA receptor NR2 subunits bind to the PSD‐95 family of proteins, whereas AMPA receptor subunits GluR2/3 bind to GRIP. These interactions may underlie the clustering, targeting, and immobilization of the glutamate receptors at postsynaptic sites. By virtue of their multiple protein‐binding domains (e.g., three PDZs in PSD‐95 and seven PDZs in GRIP), PSD‐95 and GRIP can function as multivalent proteins that organize a specific cytoskeletal and signaling complex associated with each class of glutamate receptor. The network of protein‐protein interactions mediated by these abundant PDZ proteins is likely to contribute significantly to the molecular scaffold of the postsynaptic density.

Reelin supplementation enhances cognitive ability, synaptic plasticity, and dendritic spine density

Apolipoprotein receptors belong to an evolutionarily conserved surface receptor family that has intimate roles in the modulation of synaptic plasticity and is necessary for proper hippocampal-dependent memory formation. The known lipoprotein receptor ligand Reelin is important for normal synaptic plasticity, dendritic morphology, and cognitive function; however, the in vivo effect of enhanced Reelin signaling on cognitive function and synaptic plasticity in wild-type mice is unknown. The present studies test the hypothesis that in vivo enhancement of Reelin signaling can alter synaptic plasticity and ultimately influence processes of learning and memory. Purified recombinant Reelin was injected bilaterally into the ventricles of wild-type mice. We demonstrate that a single in vivo injection of Reelin increased activation of adaptor protein Disabled-1 and cAMP-response element binding protein after 15 min. These changes correlated with increased dendritic spine density, increased hippocampal CA1 long-term potentiation (LTP), and enhanced performance in associative and spatial learning and memory. The present study suggests that an acute elevation of in vivo Reelin can have long-term effects on synaptic function and cognitive ability in wild-type mice.

The effects of amyloid precursor protein on postsynaptic composition and activity.

The amyloid precursor protein (APP) is cleaved to produce the Alzheimer disease-associated peptide Aβ, but the normal functions of uncleaved APP in the brain are unknown. We found that APP was present in the postsynaptic density of central excitatory synapses and coimmunoprecipitated with N-methyl-d-aspartate receptors (NMDARs). The presence of APP in the postsynaptic density was supported by the observation that NMDARs regulated trafficking and processing of APP; overexpression of the NR1 subunit increased surface levels of APP, whereas activation of NMDARs decreased surface APP and promoted production of Aβ. We transfected APP or APP RNA interference into primary neurons and used electrophysiological techniques to explore the effects of APP on postsynaptic function. Reduction of APP decreased (and overexpression of APP increased) NMDAR whole cell current density and peak amplitude of spontaneous miniature excitatory postsynaptic currents. The increase in NMDAR current by APP was due to specific recruitment of additional NR2B-containing receptors. Consistent with these findings, immunohistochemical experiments demonstrated that APP increased the surface levels and decreased internalization of NR2B subunits. These results demonstrate a novel physiological role of postsynaptic APP in enhancing NMDAR function.