No abstract

SynGAP is a Ras GTPase activating protein present at the postsynaptic density (PSD) in quantities matching those of the core scaffold protein PSD-95. SynGAP is reported to inhibit synaptic accumulation of AMPA receptors. Here, we characterize by immunogold electron microscopy the distribution of SynGAP at the PSD under basal and depolarizing conditions in rat hippocampal neuronal cultures. The PSD core, extending up to 40 nm from the postsynaptic membrane, typically shows label for SynGAP, while half of the synapses exhibit additional labeling in a zone 40–120 nm from the postsynaptic membrane. Upon depolarization with high K+, labeling for SynGAP significantly decreases at the core of the PSD and concomitantly increases at the 40–120 nm zone. Under the same depolarization conditions, label for PSD-95, the presumed binding partner of SynGAP, does not change its localization at the PSD. Depolarization-induced redistribution of SynGAP is reversible and also occurs upon application of NMDA. Activity-induced movement of SynGAP could vacate sites in the PSD core allowing other elements to bind to these sites, such as transmembrane AMPA receptor regulatory proteins, and simultaneously facilitate access of SynGAP to CaMKII and Ras, elements of a regulatory cascade.

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D 1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.T ranscriptional regulation in eukaryotes is governed by the coordinated action of regulatory factors that bind to specific DNA elements. One class of these factors comprises zinc finger proteins of which Sp1 is a prototypical example, having three Cys-2-His-2 zinc finger motifs (1). Other family members, Sp2, Sp3, and Sp4, with similar structural and functional features also have been identified (2, 3). Sp1, Sp3, and Sp4 bind to the same recognition sequence (GC boxes) with similar affinities (3, 4). While Sp1 and Sp4 generally act as transcription activators, Sp3 can act as repressor or activator (5). Sp2, on the other hand, has a DNA-binding specificity different (2) from that of Sp1, Sp3, or Sp4. Several additional factors with the same zinc finger motif as Sp1 have been cloned and found to bind to the GC box sequence (6-8).Central dopaminergic neurotransmission is crucial for normal brain function, and its aberrations are intricately involved in several neuropsychiatric disorders. The specific biological effects of dopamine are determined at least in part by the complex spatial and temporal regulation of genes encoding its receptors. and D 2 genes have revealed a delicate balance among several nuclear factors that tightly regulate expression of these genes (10-12). For example, the D 2 gene promoter is under strong negative control (13). One of its silencing elements (nucleotides Ϫ116 to Ϫ76), which consists of an Sp1 consensus sequence (GC box) and three TGGG repeats (GT box), interacts with Sp1, Sp3 (10), and an unidentified factor (13). In the present investigation, we characterized the nature and function of this nuclear protein, which regulates the expression of dopamine receptor genes.

SynGAP, a protein abundant at the postsynaptic density (PSD) of glutamatergic neurons, is known to modulate synaptic strength by regulating the incorporation of AMPA receptors at the synapse. Two isoforms of SynGAP, α1 and α2, which differ in their C-termini, have opposing effects on synaptic strength. In the present study, antibodies specific for SynGAP-α1 and SynGAP-α2 are used to compare the distribution patterns of the two isoforms at the postsynaptic density (PSD) under basal and excitatory conditions. Western immunoblotting shows enrichment of both isoforms in PSD fractions isolated from adult rat brain. Immunogold electron microscopy of rat hippocampal neuronal cultures shows similar distribution of both isoforms at the PSD, with a high density of immunolabel within the PSD core under basal conditions. Application of NMDA promotes movement of SynGAP-α1 as well as SynGAP-α2 out of the PSD core. In isolated PSDs both isoforms of SynGAP can be phosphorylated upon activation of the endogenous CaMKII. Application of tatCN21, a cell-penetrating inhibitor of CaMKII, to hippocampal neuronal cultures blocks NMDA-induced redistribution of SynGAP-α1 and SynGAP-α2. Thus CaMKII activation promotes the removal of two distinct C-terminal SynGAP variants from the PSD.

Homer is a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity, calcium signaling and neurological disorders. Here, we use pre-embedding immunogold electron microscopy to illustrate the differential localization of three Homer gene products (Homer 1, 2, and 3) in different regions of the mouse brain. In cross-sectioned PSDs, Homer occupies a layer ~30–100 nm from the postsynaptic membrane lying just beyond the dense material that defines the PSD core (~30 nm thick). Homer is evenly distributed within the PSD area along the lateral axis, but not at the peri-PSD locations within 60 nm from the edge of the PSD, where mGluR1 and 5 are concentrated. This distribution of Homer matches that of Shank, another major PSD scaffold protein, but differs from those of other two major binding partners of Homer, type I mGluR and IP3 receptors. Many PSD proteins rapidly redistribute upon acute (2 min) stimulation. To determine whether Homer distribution is affected by acute stimulation, we examined its distribution in dissociated hippocampal cultures under different conditions. Both the pattern and density of label for Homer 1, the isoform that is ubiquitous in hippocampus, remained unchanged under high K+ depolarization (90 mM for 2–5 min), NMDA treatment (50 μM for 2 min), and calcium-free conditions (EGTA at 1 mM for 2 min). In contrast, Shank and CaMKII accumulate at the PSD upon NMDA treatment, and CaMKII is excluded from the PSD complex under low calcium conditions.

The human D 1A dopamine receptor is transcribed from a tissue-specific regulated gene under the control of two promoters. An activator region (AR1) located between nucleotides ؊1154 and ؊1136 (relative to the first ATG) enhances transcription from the upstream promoter that is active in the brain. In this investigation, we sought to identify the nuclear factors that regulate the D 1A gene through their binding to AR1 using yeast one-hybrid screening. Sp3 and Zic2 were among the positive clones isolated. Although Sp1 was not isolated from this screening and purified Sp1 alone does not bind to AR1 in gel shift experiments, this general transcription factor binds to AR1 in the presence of D 1A expressing NS20Y nuclear extract and activates the D 1A promoter. Thus, Sp1 appears to require an unknown factor(s) or post-translational modification to interact with AR1. On the other hand, Zic2 and Sp3 inhibit Sp1-induced activation of the D 1A gene in an AR1-dependent manner. Zic2 and D 1A genes have reciprocal brain regional distributions; Zic2 is expressed primarily in the cerebellum, and D 1A is highly expressed in corpus striatum. These observations collectively suggest that one of the physiologic functions of Zic2 is repression of D 1A gene transcription and that the intracellular balance among Sp1, Sp3 and Zic2 is important for regulating the tissuespecific expression of this dopamine receptor.Sp1 and Sp3 are ubiquitous transcription factors that play major roles in the expression of many cellular genes including constitutive housekeeping and inducible genes (1). Sp3 shares extensive structural and sequence homology with Sp1 and can function as a synergist or antagonist of Sp1-mediated activation of target promoters (2-5). In addition, internally translated isoforms of Sp3 function as potent inhibitors of Sp1-mediated transcription in vivo since such truncated isoforms lack substantial portions of the Sp3 transactivation domain (6). Thus, the balance between Sp1 and Sp3 is an important regulator of target genes (7).The murine zinc finger protein of the cerebellum (Zic) was cloned through a search for proteins involved in cerebellar development (8). Subsequently, Zic2 and Zic3 were cloned as members of the Zic gene family (9). Zic expression is highly restricted to the cerebellar granule cell lineage and in medulloblastoma cells (10). Furthermore, analysis of Zic knock-out mice confirms that this transcription factor is involved in cerebellar development (11). On the other hand, mutations in Zic2 have been associated with holoprosencephaly (12). Dopamine plays important roles in several physiologic functions including locomotion (13, 14), learning and memory (15, 16), neuroendocrine modulation (17) We had previously found that the human D 1A receptor gene is transcribed in the brain from two promoters (25). A cis-acting element located between nucleotides Ϫ1154 and Ϫ1136 relative to the translation start site (termed activator region 1, AR1) 1 mediates transactivation of the upstream promoter in neuronal cells (25). ...

Shank and GKAP are scaffold proteins and binding partners at the postsynaptic density (PSD). The distribution and dynamics of Shank and GKAP were studied in dissociated hippocampal cultures by pre-embedding immunogold electron microscopy. Antibodies against epitopes containing their respective mutual binding sites were used to verify the expected juxtapositioning of Shank and GKAP. If all Shank and GKAP molecules at the PSD were bound to each other, the distribution of label for the two proteins should coincide. However, labels for the mutual binding sites showed significant differences in distribution, with a narrow distribution for GKAP located close to the postsynaptic membrane, and a wider distribution for Shank extending deeper into the cytoplasm. Upon depolarization with high K+, neither the intensity nor distribution of label for GKAP changed, but labeling intensity for Shank at the PSD increased to ~150% of controls while the median distance of label from postsynaptic membrane increased by 7.5 nm. These results indicate a preferential recruitment of Shank to more distal parts of the PSD complex. Conversely, upon incubation in Ca2+-free medium containing EGTA, the labeling intensity of Shank at the PSD decreased to ~70% of controls and the median distance of label from postsynaptic membrane decreased by 9 nm, indicating a preferential loss of Shank molecules in more distal parts of the PSD complex. These observations identify two pools of Shank at the PSD complex, one relatively stable pool, closer to the postsynaptic membrane that can bind to GKAP, and another more dynamic pool at a location too far away to bind to GKAP.

Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteosomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity, would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.