Faculty Bibliography

Background: Epigenetic dysregulation may be involved in the underlying molecular deficits in schizophrenia (SZ). Previous research by our group has show hypermethylation of GABAergic promoter gene in and increases in DNMT1 and DNMT3A in post mortem brain samples of patients with SZ. We have also shown that differences in DNMT1 and other epigenetically related enzymes are also found in the lymphocytes of living patients with chronic schizophrenia (CSZ). We now report preliminary results on epigenetic related mRNA's in lymphocytes on a larger sample of chronic schizophrenics and controls. Methods: CSZ (n =29) and non-psychotic controls (NPC) (n =31) subjects had a blood sample (50-60 cc) drawn, and lymphocyte pellet extracted by Ficoll gradient procedure. qPCR assays were used to measure epigenetically related mRNA's-DNMT1, DMNT3A, TET1, TET2, TET3, BDNF and NR3C (glucocorticoid receptor). We also assayed several immunological-related mRNA (which appeared as strong hits from RNA sequence analysis): T-Cell Surface Glycoprotein CD4, CCR1 (C-C motif chemokine receptor 1), FPRL3 (Formyl Peptide Receptor). Assays were performed using Taqman probes with B-Actin as housekeeping gene. Patients were also evaluated with psychopathology with PANSS, for cognitive function with MATRICS, and for odor identification and discrimination with Sniff and Sticks smell test. Results: In this sample CSZ showed significantly higher DMNT3A (P =.048) than NPC. Male CSZ showed significantly higher DNMT1 than NPC (P =.014), but in the total sample there was a trend, but no significant difference in DNMT1. Compared to NPC, CSZ subjects showed significantly higher levels of GABAergic enzymes measured by the GAD1 probe (which assayed GAD67 and GAD25 mRNA) (P =.030), higher levels of glucocorticoid receptor measured by the NRC3 probe (P =.006), and significantly lower levels of FRPRL3 (P =039). Higher levels of FPRL3 and CD4 were moderately correlated with PANSS positive symptoms in CSZ (FRPL3 r =+.43 P =.019, CD4 r =+.37 P =.054) and these correlations were slightly stronger in male CSZ. Higher DNMT1 and DNMT3A levels in lymphocytes of CSZ correlated negatively with scores on MATRICs battery (DNMT1-attention/vigilance-r =-.41, P =.031, working memory r =-.37, P =.048, composite score r =-.36, P =.063). Conclusions: CSZ demonstrate differences in epigenetically related mRNA's in their lymphocytes. In CSZ higher levels of DNMT were related to poorer cognitive performance and higher levels of immunological related mRNA to greater positive symptom scores. Some potential differences to our previously published results may be related to differences in mRNA's transcript variants detected by the Taqman probes and earlier research with specifically generated sequence probes

Deficits in N-methyl-D-aspartate receptor (NMDAR) function are increasingly linked to persistent negative symptoms and cognitive deficits in schizophrenia. Accordingly, clinical studies have been targeting the modulatory site of the NMDA receptor, based on the decreased function of NMDA receptor, to see whether increasing NMDA function can potentially help treat the negative and cognitive deficits seen in the disease. Glycine and D-serine are endogenous ligands to the NMDA modulatory site, but since high doses are needed to affect brain levels, related compounds are being developed, for example glycine transport (GlyT) inhibitors to potentially elevate brain glycine or targeting enzymes, such as D-amino acid oxidase (DAAO) to slow the breakdown and increase the brain level of D-serine. In the present study we further evaluated the effect of DAAO inhibitors 5-chloro-benzo[d]isoxazol-3-ol (CBIO) and sodium benzoate (NaB) in a phencyclidine (PCP) rodent mouse model to see if the inhibitors affect PCP-induced locomotor activity, alter brain D-serine level, and thereby potentially enhance D-serine responses. D-Serine dose-dependently reduced the PCP-induced locomotor activity at doses above 1000 mg/kg. Acute CBIO (30 mg/kg) did not affect PCP-induced locomotor activity, but appeared to reduce locomotor activity when given with D-serine (600 mg/kg); a dose that by itself did not have an effect. However, the effect was also present when the vehicle (Trappsol((R))) was tested with D-serine, suggesting that the reduction in locomotor activity was not related to DAAO inhibition, but possibly reflected enhanced bioavailability of D-serine across the blood brain barrier related to the vehicle. With this acute dose of CBIO, D-serine level in brain and plasma were not increased. Another weaker DAAO inhibitor NaB (400 mg/kg), and NaB plus D-serine also significantly reduced PCP-induced locomotor activity, but without affecting plasma or brain D-serine level, arguing against a DAAO-mediated effect. However, NaB reduced plasma L-serine and based on reports that NaB also elevates various plasma metabolites, for example aminoisobutyric acid (AIB), a potential effect via the System A amino acid carrier may be involved in the regulation of synaptic glycine level to modulate NMDAR function needs to be investigated. Acute ascorbic acid (300 mg/kg) also inhibited PCP-induced locomotor activity, which was further attenuated in the presence of D-serine (600 mg/kg). Ascorbic acid may have an action at the dopamine membrane carrier and/or altering redox mechanisms that modulate NMDARs, but this needs to be further investigated. The findings support an effect of D-serine on PCP-induced hyperactivity. They also offer suggestions on an interaction of NaB via an unknown mechanism, other than DAAO inhibition, perhaps through metabolomic changes, and find unexpected synergy between D-serine and ascorbic acid that supports combined NMDA glycine- and redox-site intervention. Although mechanisms of these specific agents need to be determined, overall it supports continued glutamatergic drug development.

Schizophrenic patients have a high rate of smoking and cognitive deficits which may be related to a decreased number or responsiveness of nicotinic receptors in their brains. Varenicline is a partial nicotinic agonist which is effective as an antismoking drug in cigarette smokers, although concerns have been raised about potential psychiatric side-effects. We conducted a double-blind placebo controlled study in 87 schizophrenic smokers to evaluate the effects of varenicline (2 mg/day) on measures of smoking, cognition, psychiatric symptoms, and side-effects in schizophrenic patients who were cigarette smokers. Varenicline significantly decreased cotinine levels (P<0.001), and other objective and subjective measures of smoking (P < .01), and responses on a smoking urges scale (P = .02), more than placebo. Varenicline did not improve scores on a cognitive battery designed to test the effect of drugs on cognitive performance in schizophrenia (the MATRICS battery), either in overall MATRICS battery Composite or individual Domain scores, more than placebo. There were no significant differences between varenicline vs. placebo effects on total symptom scores on psychiatric rating scales, PANSS, SANS, or Calgary Depression scales, and there were no significant drug effects in any of these scales sub-scores when we used Benjamin-Hochberg corrected significance levels (alpha = .05). Varenicline patients did not show greater side-effects than placebo treated patients at any time point when controlled for baseline side-effect scores. Our study supports the use of varenicline as a safe drug for smoking reduction in schizophrenia but not as a cognitive enhancer. TRIAL REGISTRATION: ClinicalTrials.gov 00802919.

Background: Deficits in Odor identification and discrimination have been found in previous studies of patients with schizophrenia (SZ), and structural and functional abnormalities in the olfactory system of schizophrenia are well documented. Some studies have reported relationship between odor deficits in SZ and negative symptoms. We investigated these questions in a new sample of chronic SZ and controls who are also participating in a study of epigenetic markers in the lymphocytes of SZ. Sequencing of activation and silencing of gene methylation by methylating enzymes (DNMT1 3a) and change in histone acetylation may be involved in epigenetic modifications of the development of the olfactory system. Methods: In this initial group of sample we studied 34 patients with chronic SZ and 23 controls. Odor identification discrimination was assessed with Sniff n Sticks smell test battery for Odor Identification and Odor Discrimination. Current psychiatric symptoms were assessed with PANSS interview. Cognition was assessed by MATRCIS battery. A blood sample was drawn for measurement of mRNA of enzyme related to methylation of genes (DNMT, TETT 1)and genes products heavily regular by promoter methylation( BDNF, glucocorticoid receptor),although assays on these samples have not been completed at this time. Results: SZ had significantly (P <0.01)lower scores than controls on the small identification and discrimination tests of the Sniff n Sticks battery. However, on the odor identification, but not on discrimination, there was a significant sex effect, with identification being deficient in male SZ but not females. There were no differences between male vs. female controls on the odor tests. There was a moderate statistically significant association between scores on odor discrimination and scores on the MATRICS battery (composite r=0.39 P=0.02, and working memory r=0.44 P=0.01), but this association was stronger in females than males. In controls there was only a significant association with higher odor identification with the scores on the MCCB domain of attention vigilance and no sex difference. In SZ patients poorer performance on odor discrimination was associated with higher scores on Negative symptoms (r=0.51, P <0.01), and higher scores on PANSS Total (r=0.39, P=o0.03) and no strong differences in effects in males vs. female SZ. Relationships to biological markers are being investigated. Conclusions: This current research confirms the previously reported deficits in olfaction in schizophrenia, and the relationship of olfactory deficits to negative symptoms. It suggests that sex differences may be important in odor identification test, but not the odor discrimination test in SZ. This is the first study to show olfactory deficits related to performance on the MATRCIS battery and suggest that there may be a sex difference in the strength of this relationship

Schizophrenia is characterized by cognitive deficits which persist after acute symptoms have been treated or resolved. Transcranial direct current stimulation (tDCS) has been reported to improve cognition and reduce smoking craving in healthy subjects but has not been as carefully evaluated in a randomized controlled study for these effects in schizophrenia. We conducted a randomized double-blind, sham-controlled study of the effects of 5 sessions of tDCS (2 milliamps for 20minutes) on cognition, psychiatric symptoms, and smoking and cigarette craving in 37 outpatients with schizophrenia or schizoaffective disorder who were current smokers. Thirty subjects provided evaluable data on the MATRICS Consensus Cognitive Battery (MCCB), with the primary outcome measure, the MCCB Composite score. Active compared to sham tDCS subjects showed significant improvements after the fifth tDCS session in MCCB Composite score (p=0.008) and on the MCCB Working Memory (p=0.002) and Attention-Vigilance (p=0.027) domain scores, with large effect sizes. MCCB Composite and Working Memory domain scores remained significant at Benjamini-Hochberg corrected significance levels (alpha=0.05). There were no statistically significant effects on secondary outcome measures of psychiatric symptoms (PANSS scores), hallucinations, cigarette craving, or cigarettes smoked. The positive effects of tDCS on cognitive performance suggest a potential efficacious treatment for cognitive deficits in partially recovered chronic schizophrenia outpatients that should be further investigated.

Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (alpha-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.

The epigenetic dysregulation of the brain genome associated with the clinical manifestations of schizophrenia (SZ) includes altered DNA promoter methylation of several candidate genes. We and others have reported that two enzymes that belong to the DNA-methylation/demethylation network pathways-DNMT1 (DNA-methyltransferase) and ten-eleven translocator-1(TET1) methylcytosine deoxygenase are abnormally increased in corticolimbic structures of SZ postmortem brain. The objective of this study was to investigate whether the expression of these components of the DNA-methylation-demethylation pathways known to be altered in the brain of SZ patients are also altered in peripheral blood lymphocytes (PBL). The data show that increases in DNMT1 and TET1 and in glucocorticoid receptor (GCortR) and brain derived neurotrophic factor (BDNF) mRNAs in PBL of SZ patients are comparable to those reported in the brain of SZ patients. The finding that the expressions of DNMT1 and TET1 are increased and SZ candidate genes such as BDNF and GCortR are altered in the same direction in both the brain and PBL together with recent studies showing highly correlated patterns of DNA methylation across the brain and blood, support the hypothesis that a common epigenetic dysregulation may be operative in the brain and peripheral tissues of SZ patients.