Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. Here, we report that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms.

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). Here we show that NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission.

Recent work has shown substantial alterations in NMDA receptor subunit expression, assembly, and phosphorylation in the dopamine-depleted striatum of a rodent 6-hydroxydopamine model of Parkinson's disease. These modifications are hypothesized to result from the trafficking of NMDA receptors between subcellular compartments. Here we show that in rat striatal tissues the NR2A and NR2B subunits in the synaptosomal membrane, and not those in the light membrane and synaptic vesicle-enriched compartments, are tyrosine phosphorylated. The dopamine D1 receptor agonist SKF-82958 produces (1) an increase in NR1, NR2A, and NR2B proteins in the synaptosomal membrane fraction; (2) a decrease in NR1, NR2A, and NR2B proteins in the light membrane and synaptic vesicleenriched fractions; and (3) an increase in the tyrosine phosphorylation of NR2A and NR2B in the synaptosomal membrane compartment. The protein phosphatase inhibitor pervanadate reproduces the alterations in subcellular distribution and phosphorylation, whereas the effects of the dopamine D1 receptor agonist are blocked by genistein, a protein tyrosine kinase inhibitor. Dopamine D1 receptor agonist treatment does not change the subcellular distribution of the AMPA receptor subunits GluR1 or GluR2/3 in the striatum and has no effect on cortical or cerebellar NMDA receptor subunits. These data reveal a rapid dopamine D1 receptor-and tyrosine kinasedependent trafficking of striatal NMDA receptors between intracellular and postsynaptic sites. The subcellular trafficking of striatal NMDA receptors may play a significant role both in the pathogenesis of Parkinson's disease and in the development of adverse effects of chronic dopaminergic therapy in parkinsonian patients.

A monoclonal antibody (R1JHL) against the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor has been developed that recognizes an epitope in the region of the amino-terminal amino acids 341-561 (a region common to all splice variants of NR1). This monoclonal antibody identifies a broad band at 115 kDa in immunoblots using membranes from NR1-transfected cells and from rat brain tissue. No cross-reactivity with any NR2 subunit is seen. With the goal to determine quantitatively the subunit composition of cortical NMDA receptors, we used the monoclonal antibody to NR1 and polyclonal antibodies against the NR2A and NR2B subunits to perform immunoprecipitations of receptor subunits from solubilized adult rat cortical membranes. Solubilization of the receptor subunits was accomplished under both nondenaturing (native) conditions, under which the subunits seem to remain associated with one another, and denaturing conditions, under which the subunits are associated from each other. Although each of these antibodies selectively immunoprecipitates only its corresponding (cognate) subunit when the subunits have been solubilized under denaturing conditions, each of the antibodies immunoprecipitates a sizable fraction of the other two NMDA receptor subunits when membranes are solubilized under nondenaturing conditions, indicating an interaction in situ. Using quantitative immunoblot analysis of the three subunits in both the pellets and supernatants from the immunoprecipitations, we found 1) the dominant NMDA receptor complex in adult rat cortex contains at least three subunits, NR1/NR2A/NR2B; 2) a smaller fraction of NMDA receptors are composed of only two subunits, NR1/NR2B or NR1/NR2A; 3) there are no complexes that contain NR2A/NR2B that do not contain NR1; 4) only a small fraction of each subunit is not associated with any other NMDA receptor subunit; 5) no coimmunoprecipitation of noncognate subunits occurs unless the subunits are assembled with each other in situ; and 6) there is no physical interaction between these NMDA receptor subunits and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR2 or GluR3 subunits. These results suggest that functional studies with recombinant receptors composed of at least three subunits may be the most physiologically meaningful.

The a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors is subject to functionally distinct constitutive and regulated clathrin-dependent endocytosis, contributing to various forms of synaptic plasticity. In HEK293 cells transiently expressing GluR1 or GluR2 mutants containing domain deletions or point mutations in their intracellular carboxyl termini (CT), we found that deletion of the first 10 amino acids (834-843) selectively reduced the rate of constitutive AMPA receptor endocytosis, whereas truncation of the last 15 amino acids of the GluR2 CT, or point mutation of the tyrosine residues in this region, only eliminated the regulated (insulinstimulated) endocytosis. Moreover, in hippocampal slices, both insulin treatment and low-frequency stimulation (LFS) specifically stimulated tyrosine phosphorylation of the GluR2 subunits of native AMPA receptors, and the enhanced phosphorylation appears necessary for both insulin-and LFS-induced long-term depression of AMPA receptor-mediated excitatory postsynaptic currents. Thus, our results demonstrate that constitutive and regulated AMPA receptor endocytosis requires different sequences within GluR CTs and tyrosine phosphorylation of GluR2 CT is required for the regulated AMPA receptor endocytosis and hence the expression of certain forms of synaptic plasticity.

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (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-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-alpha/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation.

Synaptic adhesion molecules regulate multiple steps of synapse formation and maturation. The great diversity of neuronal synapses predicts the presence of a large number of adhesion molecules that control synapse formation through trans-synaptic and heterophilic adhesion. We identified a previously unknown trans-synaptic interaction between netrin-G ligand-3 (NGL-3), a postsynaptic density (PSD) 95-interacting postsynaptic adhesion molecule, and leukocyte common antigen-related (LAR), a receptor protein tyrosine phosphatase. NGL-3 and LAR expressed in heterologous cells induced pre- and postsynaptic differentiation in contacting axons and dendrites of cocultured rat hippocampal neurons, respectively. Neuronal overexpression of NGL-3 increased presynaptic contacts on dendrites of transfected neurons. Direct aggregation of NGL-3 on dendrites induced coclustering of excitatory postsynaptic proteins. Knockdown of NGL-3 reduced the number and function of excitatory synapses. Competitive inhibition by soluble LAR reduced NGL-3-induced presynaptic differentiation. These results suggest that the trans-synaptic adhesion between NGL-3 and LAR regulates excitatory synapse formation in a bidirectional manner.