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

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Characterization of novel GABAA receptor-associated proteins

The γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the mammalian brain. In order to begin to understand the mechanisms that direct the targeting and localization, clustering, and trafficking of brain GABAARs, I sought to identify cytosolic proteins that interact with the intracellular portion of the GABAAR subunits. Therefore, I used the yeast two-hybrid technique to screen an adult rat brain cDNA library using, as bait, the large intracellular loop (IL), of the GABAAR β3 subunit. I found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2), a protein known to be involved in vesicular and protein trafficking, interacts with the β subunits of the GABAA receptors. The native BIG2 and GABAARs both coprecipitated from detergent extracts with either anti-GABAAR or anti-BIG2 antibodies. Double label immunofluorescence of cultured hippocampal neurons showed that BIG2 concentrates in the trans-Golgi network and is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABAARs. The results are consistent with the hypotheses that the interaction of BIG2 with the GABAAR β subunits plays a role in the exocytosis and trafficking of assembled GABA AR to the cell surface. ^ I also isolated a clone corresponding to a novel splice form of the glutamate receptor interacting protein 1 (GRIP1). This splice form, called GRIP1c 4–7, contains 4 PDZ domains that are identical to PDZ domains 4–7 of GRIP1. GRIP1c 4–7 also contains 35 amino acids at the N-terminus and 12 amino acids at the C-terminus that differ from those of GRIP1a/b. Based on these peptide sequences, I have also isolated additional long and short GRIP1 splice variants called GRIP1d and GRIP1e 4–7. GRIP1c 4–7 interacted with GluR2/3 subunits of the AMPA receptor and with gephyrin but not with the GABA AR α1 subunit in detergent extracts of rat brain membranes. In low-density hippocampal cultures, GRIP1c 4–7 clusters colocalized with components of both GABAergic and glutamatergic synapses. GRIP1c 4–7-specific antibodies recognized a 75 kDa Mr protein that is enriched in a postsynaptic density (PSD) fraction isolated from brain. These results indicate that GRIP1c 4–7 plays functional role(s) in both GABAergic and glutamatergic synapses.

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

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 postsynaptic complex. Nevertheless the role that these GRIP1 protein isoforms play at the GABAergic postsynaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main postsynaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of HEK293 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 postsynaptic 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 postsynaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic postsynaptic complex and suggest that this interaction plays a role in GABAergic synaptic function

ACTB (Beta-Actin)–Normalized <i>CAPON</i> mRNA Short-Form Expression by Genotype

<p>Expression levels are least squares means. Individuals from all three diagnostic classifications are included, grouped only by genotype. Mean values per genotype for each SNP are plotted with 95% confidence intervals. The number of individuals per genotype is indicated within each bar. SNP alleles are given for forward strand sequence. All three SNPs exhibit significantly (<i>p</i> < 0.05) different levels of <i>CAPON</i> expression by genotype, with a dominant effect. Higher levels of <i>CAPON</i> are seen in individuals with one or two copies of alleles previously identified as associated with schizophrenia (T, rs1415263; C, rs4145621; and C, rs2661818). The mean (95% confidence interval lower bound, upper bound) for the three genotypes for each SNP are as follows. For rs1415263: 1.54 (1.29, 1.80) for CC; 2.07 (1.81, 2.34) for CT; and 1.83 (1.36, 2.29) for TT. For rs4145621: 1.51 (1.25, 1.78) for TT; 2.01 (1.74, 2.27) for TC; and 2.01 (1.61, 2.41) for CC. For rs2661818: 1.49 (1.21, 1.76) for GG; 1.96 (1.70, 2.23) for CG; and 2.04 (1.68, 2.41) for CC.</p

ACTB (Beta-Actin)–-Normalized <i>CAPON</i> mRNA Full-Length Expression by Diagnosis

<p>Expression levels are least squares means. Mean values per category are plotted with 95% confidence intervals. The number of individuals per sample is indicated within each bar. Level of expression does not differ significantly by diagnostic group. The mean (95% confidence interval lower bound, upper bound) for the control, schizophrenia, and bipolar groups are 1.28 (1.12, 1.45), 1.33 (1.17, 1.49), and 1.16 (0.99, 1.32), respectively.</p

ACTB (Beta-Actin)–Normalized <i>CAPON</i> mRNA Short-Form Expression by Diagnosis

<p>Expression levels are least squares means. Mean values per category are plotted with 95% confidence intervals. The number of individuals per sample is indicated within each bar. Expression is significantly higher in patients with schizophrenia (<i>p</i> = 0.0013) and bipolar (<i>p</i> = 0.0009) as compared to controls. The mean (95% confidence interval lower bound, upper bound) for the control, schizophrenia, and bipolar groups are 1.34 (1.05, 1.62), 2.02 (1.73, 2.30), and 2.05 (1.77, 2.34), respectively.</p

Western Blot of CAPON Protein Isoforms in DLPFC from Normal Control Individuals

<p>Tissue from Brodmann's area 46 (DLPFC) from five individuals was homogenized in TEE. Proteins were resolved by SDS-PAGE and transferred to PVDF membrane. Blots were probed with rabbit polyclonal antibodies to CAPON and actin, and proteins were detected using chemiluminescence. Band intensities for CAPON-L, CAPON-S, and CAPON-S + CAPON-S′ were calculated and normalized to the intensities of the corresponding actin bands. Untransfected COS-7 cells expressing CAPON-L and CAPON-S′ (COS-7/NT) and COS-7 cells expressing recombinant CAPON-S (COS-7/CAPON-S) and were used as controls for these proteins. CAPON-L appears to include multiple bands, possibly due to phosphorylation.</p

CAPON Gene Structure, Isoforms, and Protein Functional Domains

<div><p>CAPON full-length transcript and protein illustrations are based on NCBI reference sequences.</p> <p>(A) Genomic organization of <i>CAPON</i>. Exons are represented by numbered boxes; 5′ and 3′ UTR are represented by half-height boxes.</p> <p>(B) <i>CAPON</i> transcripts. 5′ and 3′ UTR are represented by yellow shaded boxes, exons by white boxes.</p> <p>(C) CAPON proteins. The phosphotyrosine-binding domain (PTB) is shaded green, and the PDZ-binding domain is shaded blue.</p></div