PSD-95 alters microtubule dynamics via an association with EB3.

Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology.

Interplay of palmitoylation and phosphorylation in the trafficking and localization of phosphodiesterase 10A: implications for the treatment of schizophrenia.

Phosphodiesterase 10A (PDE10A) is a striatum-enriched, dual-specific cyclic nucleotide phosphodiesterase that has gained considerable attention as a potential therapeutic target for psychiatric disorders such as schizophrenia. As such, a PDE10A-selective inhibitor compound, MP-10, has recently entered clinical testing. Since little is known about the cellular regulation of PDE10A, we sought to elucidate the mechanisms that govern its subcellular localization in striatal medium spiny neurons. Previous reports suggest that PDE10A is primarily membrane bound and is transported throughout medium spiny neuron axons and dendrites. Moreover, it has been shown in PC12 cells that the localization of the major splice form, PDE10A2, may be regulated by protein kinase A phosphorylation at threonine 16 (Thr-16). Using an antibody that specifically recognizes phosphorylated Thr-16 (pThr-16) of PDE10A2, we provide evidence that phosphorylation at Thr-16 is critical for the regulation of PDE10A subcellular localization in vivo. Furthermore, we demonstrate in primary mouse striatal neuron cultures that PDE10A membrane association and transport throughout dendritic processes requires palmitoylation of cysteine 11 (Cys-11) of PDE10A2, likely by the palmitoyl acyltransferases DHHC-7 and -19. Finally, we show that Thr-16 phosphorylation regulates PDE10A trafficking and localization by preventing palmitoylation of Cys-11 rather than by interfering with palmitate-lipid interactions. These data support a model whereby PDE10A trafficking and localization can be regulated in response to local fluctuations in cAMP levels. Given this, we propose that excessive striatal dopamine release, as occurs in schizophrenia, might exert differential effects on the regulation of PDE10A localization in the two striatal output pathways.

Fragment-Based Discovery of an Apolipoprotein E4 (apoE4) Stabilizer.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses.

We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic post-synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post-synaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main post-synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4-7. An engineered deletion construct of GRIP1a/b (GRIP1a4-7), which both contains the aforementioned common region and binds to gephyrin, targets to the post-synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the post-synaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post-synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.

Activity-independent regulation of dendrite patterning by postsynaptic density protein PSD-95.

Dendritic morphology determines many aspects of neuronal function, including action potential propagation and information processing. However, the question remains as to how distinct neuronal dendrite branching patterns are established. Here, we report that postsynaptic density-95 (PSD-95), a protein involved in dendritic spine maturation and clustering of synaptic signaling proteins, plays a novel role in regulating dendrite outgrowth and branching, independent of its synaptic functions. In immature neurons, overexpression of PSD-95 decreases the proportion of primary dendrites that undergo additional branching, resulting in a marked reduction of secondary dendrite number. Conversely, knocking down PSD-95 protein in immature neurons increases secondary dendrite number. The effect of PSD-95 is activity-independent and is antagonized by cypin, a nonsynaptic protein that regulates PSD-95 localization. Binding of cypin to PSD-95 correlates with formation of stable dendrite branches. Finally, overexpression of PSD-95 in COS-7 cells disrupts microtubule organization, indicating that PSD-95 may modulate microtubules to regulate dendritic branching. Whereas many factors have been identified which regulate dendrite number, our findings provide direct evidence that proteins primarily involved in synaptic functions can also play developmental roles in shaping how a neuron patterns its dendrite branches.

Increased expression in dorsolateral prefrontal cortex of CAPON in schizophrenia and bipolar disorder.

BACKGROUND: We have previously reported linkage of markers on chromosome 1q22 to schizophrenia, a finding supported by several independent studies. Within this linkage region, we have identified significant linkage disequilibrium between schizophrenia and markers within the gene for carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON). Prior sequencing of the ten exons of CAPON failed to reveal a coding mutation associated with illness. METHODS AND FINDINGS: We screened a human fetal brain cDNA library and identified a new isoform of CAPON that consists of the terminal two exons of the gene, and verified the expression of the predicted corresponding protein in human dorsolateral prefrontal cortex (DLPFC). We examined the expression levels of both the ten-exon CAPON transcript and this new isoform in postmortem brain samples from the Stanley Array Collection. Quantitative real-time PCR analysis of RNA from the DLPFC in 105 individuals (35 with schizophrenia, 35 with bipolar disorder, and 35 psychiatrically normal controls) revealed significantly (p < 0.005) increased expression of the new isoform in both schizophrenia and bipolar disorder. Furthermore, this increased expression was significantly associated (p < 0.05) with genotype at three single-nucleotide polymorphisms previously identified as being in linkage disequilibrium with schizophrenia. CONCLUSION: Based on the known interactions between CAPON, neuronal nitric oxide synthase (nNOS), and proteins associated with the N-methyl-D-aspartate receptor (NMDAR) complex, overexpression of either CAPON isoform would be expected to disrupt the association between nNOS and the NMDAR, leading to changes consistent with the NMDAR hypofunctioning hypothesis of schizophrenia. This study adds support to a role of CAPON in schizophrenia, produces new evidence implicating this gene in the etiology of bipolar disorder, and suggests a possible mechanism of action of CAPON in psychiatric illness.

GABA(A) receptors and their associated proteins: implications in the etiology and treatment of schizophrenia and related disorders.

Gamma-aminobutyric acid type A (GABA(A)) receptors play an important role in mediating fast synaptic inhibition in the brain. They are ubiquitously expressed in the CNS and also represent a major site of action for clinically relevant drugs. Recent technological advances have greatly clarified the molecular and cellular roles played by distinct GABA(A) receptor subunit classes and isoforms in normal brain function. At the same time, postmortem and genetic studies have linked neuropsychiatric disorders including schizophrenia and bipolar disorder with GABAergic neurotransmission and various specific GABA(A) receptor subunits, while evidence implicating GABA(A)R-associated proteins is beginning to emerge. In this review we discuss the mounting genetic, molecular, and cellular evidence pointing toward a role for GABA(A) receptor heterogeneity in both schizophrenia etiology and therapeutic development. Finally, we speculate on the relationship between schizophrenia-related disorders and selected GABA(A) receptor associated proteins, key regulators of GABA(A) receptor trafficking, targeting, clustering, and anchoring that often carry out these functions in a subtype-specific manner.

Identification and characterization of two novel splice forms of GRIP1 in the rat brain.

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex.

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors.

We have found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the beta subunits of the gamma-aminobutyric acid type-A receptor (GABA(A)R). BIG2 is a Sec7 domain-containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C-terminal fragment of BIG2 and the large intracellular loop of the GABA(A)R beta subunits was revealed with a yeast two-hybrid assay. The native BIG2 and GABA(A)Rs interact in the brain since both coprecipitated from detergent extracts with either anti-GABA(A)R or anti-BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABA(A)Rs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans-Golgi network. BIG2 is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABA(A)Rs. BIG2 is present in both inhibitory GABAergic synapses that contain GABA(A)Rs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABA(A)R beta subunits plays a role in the exocytosis and trafficking of assembled GABA(A)R to the cell surface.

A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain.

We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is gamma-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with gamma-aminobutyric acid, type A, receptor (GABA(A)R) clusters than with alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA(A)R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.