The Alzheimer's Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid ß and tau.

The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aß42 and Aß40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high diagnostic performance for AD, including the early phases of the disease. However, between-center differences in pre-analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry. The aim of the group was to develop a simplified and standardized pre-analytical protocol for CSF collection and handling before analysis for routine clinical use, and ultimately to ensure high diagnostic performance and minimize patient misclassification rates. Widespread application of the protocol would help minimize variability in measurements, which would facilitate the implementation of unified cut-off levels across laboratories, and foster the use of CSF biomarkers in AD diagnostics for the benefit of the patients.

Gene Expression and Pharmacodynamic Changes in 1,760 Systemic Lupus Erythematosus Patients From Two Phase III Trials of BAFF Blockade With Tabalumab.

OBJECTIVE: To characterize baseline gene expression and pharmacodynamically induced changes in whole blood gene expression in 1,760 systemic lupus erythematosus (SLE) patients from 2 phase III, 52-week, randomized, placebo-controlled, double-blind studies in which patients were treated with the BAFF-blocking IgG4 monoclonal antibody tabalumab. METHODS: Patient samples were obtained from SLE patients from the ILLUMINATE-1 and ILLUMINATE-2 studies, and control samples were obtained from healthy donors. Blood was collected in Tempus tubes at baseline, week 16, and week 52. RNA was analyzed using Affymetrix Human Transcriptome Array 2.0 and NanoString. RESULTS: At baseline, expression of the interferon (IFN) response gene was elevated in patients compared with controls, with 75% of patients being positive for this IFN response gene signature. There was, however, substantial heterogeneity of IFN response gene expression and complex relationships among gene networks. The IFN response gene signature was a predictor of time to disease flare, independent of anti-double-stranded DNA (anti-dsDNA) antibody and C3 and C4 levels, and overall disease activity. Pharmacodynamically induced changes in gene expression following tabalumab treatment were extensive, occurring predominantly in B cell-related and immunoglobulin genes, and were consistent with other pharmacodynamic changes including anti-dsDNA antibody, C3, and immunoglobulin levels. CONCLUSION: SLE patients demonstrated increased expression of an IFN response gene signature (75% of patients had an elevated IFN response gene signature) at baseline in ILLUMINATE-1 and ILLUMINATE-2. Substantial heterogeneity of gene expression was detected among individual patients and in gene networks. The IFN response gene signature was an independent risk factor for future disease flares. Pharmacodynamic changes in gene expression were consistent with the mechanism of BAFF blockade by tabalumab.

Serotonin 2A Receptor SNP rs7330461 Association with Treatment Response to Pomaglumetad Methionil in Patients with Schizophrenia.

This study aims to confirm the initial pharmacogenetic finding observed within the clinical proof-of-concept trial of an enhanced response to treatment with pomaglumetad methionil (LY2140023 monohydrate) in Caucasian schizophrenia patients homozygous for T/T at single nucleotide polymorphism rs7330461 in the serotonin (5-hydroxytryptamine) 2A receptor gene compared to A/A homozygous patients. The effect of the rs7330461 genotype on the response to pomaglumetad methionil treatment was assessed in three additional clinical trials and in an integrated analysis. Overall, this study includes data from 1115 Caucasian patients for whom genotyping information for rs7330461 was available, consisting of 513 A/A homozygous, 466 A/T heterozygous and 136 T/T homozygous patients. Caucasian T/T homozygous patients showed significantly (p ≤ 0.05) greater improvement in Positive and Negative Syndrome Scale (PANSS) total scores during treatment with pomaglumetad methionil 40 mg twice daily compared to A/A homozygous patients. Additionally, T/T homozygous patients receiving pomaglumetad methionil had significantly (p ≤ 0.05) greater improvements in PANSS total scores compared to placebo and similar improvements as T/T homozygous patients receiving standard-of-care (SOC) treatment. The findings reported here in conjunction with prior reports show that in Caucasian patients with schizophrenia, the T/T genotype at rs7330461 is consistently associated with an increased treatment response to pomaglumetad methionil compared to the A/A genotype.

Genomic structure and expression of the human serotonin 2A receptor gene (HTR2A) locus: identification of novel HTR2A and antisense (HTR2A-AS1) exons.

BACKGROUND: The serotonin 2A receptor is widely implicated in genetic association studies and remains an important drug target for psychiatric, neurological, and cardiovascular conditions. RNA sequencing redefined the architecture of the serotonin 2A receptor gene (HTR2A), revealing novel mRNA transcript isoforms utilizing unannotated untranslated regions of the gene. Expression of these untranslated regions is modulated by common single nucleotide polymorphisms (SNPs), namely rs6311. Previous studies did not fully capture the complexity of the sense- and antisense-encoded transcripts with respect to novel exons in the HTR2A gene locus. Here, we comprehensively catalogued exons and RNA isoforms for both HTR2A and HTR2A-AS1 using RNA-Seq from human prefrontal cortex and multiple mouse tissues. We subsequently tested associations between expression of newfound gene features and common SNPs in humans. RESULTS: We find that the human HTR2A gene spans ~66 kilobases and consists of 7, rather than 4 exons. Furthermore, the revised human HTR2A-AS1 gene spans ~474 kilobases and consists of 18, rather than 3 exons. Three HTR2A exons directly overlap with HTR2A-AS1 exons, suggesting potential for complementary nucleotide interactions. The repertoire of possible mouse Htr2a splice isoforms is remarkably similar to humans and we also find evidence for overlapping sense-antisense transcripts in the same relative positions as the human transcripts. rs6311 and SNPs in high linkage disequilibrium are associated with HTR2A-AS1 expression, in addition to previously described associations with expression of the extended 5' untranslated region of HTR2A. CONCLUSIONS: Our proposed HTR2A and HTR2A-AS1 gene structures dramatically differ from current annotations, now including overlapping exons on the sense and anti-sense strands. We also find orthologous transcript isoforms expressed in mice, providing opportunities to elucidate the biological roles of the human isoforms using a model system. Associations between rs6311 and expression of HTR2A and HTR2A-AS1 suggest this polymorphism is capable of modulating the expression of the sense or antisense transcripts. Still unclear is whether these SNPs act directly on the expression of the sense or antisense transcripts and whether overlapping exons are capable of interacting through complimentary base-pairing. Additional studies are necessary to determine the extent and nature of interactions between the SNPs and the transcripts prior to interpreting these findings in the context of phenotypes associated with HTR2A.

Olanzapine Reversed Brain Gene Expression Changes Induced by Phencyclidine Treatment in Non-Human Primates.

The NMDA receptor antagonist phencyclidine (PCP) creates schizophrenia-like symptoms in normal controls. The effect of PCP on non-human primate brain gene expression was examined and compared to changes induced by olanzapine treatment. Experimental studies of PCP and antipsychotic drugs have direct relevance to understanding the patho-physiology and treatment of schizophrenia. Genome-wide changes in prefrontal cortex gene expression revealed alterations of 146 transcripts in the PCP treatment group compared to vehicle controls. Dysregulated genes were enriched in identified classes implicated in neurological and genetic disorders, including schizophrenia genes from the Psychiatric Genomics Consortium 108 loci as well as cell death in PCP-treated primates. Canonical pathway analysis revealed a significant overrepresentation of several groups including synaptic long-term potentiation and calcium signaling. Olanzapine coadministered with PCP normalized 34% of the 146 PCP-induced probe set expression changes, and a network of 17 olanzapine-normalized genes was identified enriched in schizophrenia candidate genes containing RGS4, SYN1 and AKT as nodes. The results of this study support the use of PCP administration in non-human primates as a glutamatergic model of schizophrenia and suggest that a large number of PCP-induced expression differences can be reversed by olanzapine. The results of this study may be informative for identification of potential candidates for pharmacogenetics and biomarker research related to the treatment of schizophrenia.

CYP2D6 predicted metabolizer status and safety in adult patients with attention-deficit hyperactivity disorder participating in a large placebo-controlled atomoxetine maintenance of response clinical trial.

Atomoxetine, which is indicated for treatment of attention-deficit hyperactivity disorder (ADHD), is predominantly metabolized by genetically polymorphic cytochrome P450 2D6 (CYP2D6). Based on identified CYP2D6 genotypes, individuals can be categorized into 4 phenotypic metabolizer groups as ultrarapid, extensive, intermediate, and poor. Previous studies have focused on observed differences between poor and extensive metabolizers, but it is not well understood whether the safety profile of intermediate metabolizers differs from that of ultrarapid and extensive metabolizers. This study compared safety and tolerability among the different CYP2D6 metabolizer groups in the 12-week open-label phase of an atomoxetine study in adult patients with ADHD. Genotyping identified 1039 patients as extensive/ultrarapid metabolizers, 780 patients as intermediate metabolizers, and 117 patients as poor metabolizers. Common (≥5% frequency) treatment-emergent adverse events did not significantly differ between extensive/ultrarapid and intermediate metabolizers (odds ratios were <2.0 or >0.5). Poor metabolizers had higher frequencies of dry mouth, erectile dysfunction, hyperhidrosis, insomnia, and urinary retention compared with the other metabolizer groups. There were no significant differences between extensive/ultrarapid and intermediate metabolizers in changes from baseline in vital signs. These results suggest that data from CYP2D6 intermediate and extensive/ultrarapid metabolizers can be combined when considering safety analyses related to atomoxetine.

Multiple regulatory variants modulate expression of 5-hydroxytryptamine 2A receptors in human cortex.

BACKGROUND: The 5-hydroxytryptamine 2A receptor, encoded by HTR2A, is a major postsynaptic target for serotonin in the human brain and a therapeutic drug target. Despite hundreds of genetic associations investigating HTR2A polymorphisms in neuropsychiatric disorders and therapies, the role of genetic HTR2A variability in health and disease remains uncertain. METHODS: To discover and characterize regulatory HTR2A variants, we sequenced whole transcriptomes from 10 human brain regions with massively parallel RNA sequencing and measured allelic expression of multiple HTR2A messenger (m)RNA transcript variants. Following discovery of functional variants, we further characterized their impact on genetic expression in vitro. RESULTS: Three polymorphisms modulate the use of novel alternative exons and untranslated regions (UTRs), changing expression of RNA and protein. The frequent promoter variant rs6311, widely implicated in human neuropsychiatric disorders, decreases usage of an upstream transcription start site encoding a longer 5'UTR with greater translation efficiency. rs76665058, located in an extended 3'UTR and unique to individuals of African descent, modulates allelic HTR2A mRNA expression. The third single nucleotide polymorphism, unannotated and present in only a single subject, directs alternative splicing of exon 2. Targeted analysis of HTR2A in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study reveals associations between functional variants and depression severity or citalopram response. CONCLUSIONS: Regulatory polymorphisms modulate HTR2A mRNA expression in an isoform-specific manner, directing the usage of novel untranslated regions and alternative exons. These results provide a foundation for delineating the role of HTR2A and serotonin signaling in central nervous system disorders.

Antipsychotic-induced gene regulation in multiple brain regions.

The molecular mechanism of action of antipsychotic drugs is not well understood. Their complex receptor affinity profiles indicate that their action could extend beyond dopamine receptor blockade. Single gene expression studies and high-throughput gene profiling have shown the induction of genes from several molecular classes and functional categories. Using a focused microarray approach, we investigated gene regulation in rat striatum, frontal cortex, and hippocampus after chronic administration of haloperidol or olanzapine. Regulated genes were validated by in situ hybridization, real-time PCR, and immunohistochemistry. Only limited overlap was observed in genes regulated by haloperidol and olanzapine. Both drugs elicited maximal gene regulation in the striatum and least in the hippocampus. Striatal gene induction by haloperidol was predominantly in neurotransmitter signaling, G-protein coupled receptors, and transcription factors. Olanzapine prominently induced retinoic acid and trophic factor signaling genes in the frontal cortex. The data also revealed the induction of several genes that could be targeted in future drug development efforts. The study uncovered the induction of several novel genes, including somatostatin receptors and metabotropic glutamate receptors. The results demonstrating the regulation of multiple receptors and transcription factors suggests that both typical and atypical antipsychotics could possess a complex molecular mechanism of action.

Preclinical pharmacology of FMPD [6-fluoro-10-[3-(2-methoxyethyl)-4-methyl-piperazin-1-yl]-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene]: a potential novel antipsychotic with lower histamine H1 receptor affinity than olanzapine.

FMPD [6-fluoro-10-[3-(2-methoxyethyl)-4-methyl-piperazin-1-yl]-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene] is a potential novel antipsychotic with high affinity for dopamine D2 (Ki= 6.3 nM), 5-HT(2A) (Ki= 7.3 nM), and 5-HT6 (Ki= 8.0 nM) human recombinant receptors and lower affinity for histamine H1 (Ki= 30 nM) and 5-HT2C (Ki= 102 nM) human recombinant receptors than olanzapine. Oral administration of FMPD increased rat nucleus accumbens 3,4-dihyroxyphenylacetic acid concentrations (ED200 = 6 mg/kg), blocked 5-HT2A agonist-induced increases in rat serum corticosterone levels (ED50= 1.8 mg/kg), and inhibited the ex vivo binding of [125I]SB-258585 [4-iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide] to striatal 5-HT6 receptors (ED50= 10 mg/kg) but failed to inhibit ex vivo binding of [3H]pyrilamine to hypothalamic histamine H1 receptors at doses of up to 30 mg/kg. In electrophysiology studies, acute administration of FMPD selectively elevated the number of spontaneously active A10 (versus A9) dopamine neurons and chronic administration selectively decreased the number of spontaneously active A10 (versus A9) dopamine neurons. FMPD did not produce catalepsy at doses lower than 25 mg/kg p.o. In Fos-induction studies, FMPD had an atypical antipsychotic profile in the striatum and nucleus accumbens and increased Fos expression in orexin-containing neurons of the hypothalamus. FMPD produced only a transient elevation of prolactin levels. These data indicate that FMPD is an orally available potent antagonist of dopamine D2, 5-HT2A, and 5-HT6 receptors and a weak antagonist of H1 and 5-HT2C receptors. FMPD has the potential to have efficacy in treating schizophrenia and bipolar mania with a low risk of treatment-emergent extrapyramidal symptoms, prolactin elevation, and weight gain. Clinical trials are needed to test these hypotheses.

Fast Fos: rapid protocols for single- and double-labeling c-Fos immunohistochemistry in fresh frozen brain sections.

Immunohistochemical localization of c-Fos immunoreactivity has been used successfully for over a decade to visualize patterns of neuronal activity in the brain and spinal cord. These experiments are extremely useful in identifying physiological or pharmacological activation of specific populations of neurons. Unfortunately, conventional c-Fos immunohistochemical protocols are very time and resource intensive. We have adapted and optimized established c-Fos immunohistochemistry (IHC) methodologies for use with fresh frozen brain tissue mounted directly onto slides. The resultant rapid protocols, which we refer to as "Fast Fos", include applications for single- and double-labeling, utilizing either enzyme-substrate or fluorescent detection systems. These protocols provide increased assay throughput and reproducibility, which can be further enhanced by use of an automated slide stainer. Taken as a whole, the c-Fos IHC protocols described in this report provide a flexible system for the identification of neuronal activation that substantially reduces time and resource expenditure while increasing quality and reproducibility of data.

Phencyclidine-induced changes in rat cortical gene expression identified by microarray analysis: implications for schizophrenia.

Acute phencyclidine induces schizophrenia-like symptoms in healthy humans and psychotic episodes in schizophrenics. Although phencyclidine is known as a N-methyl d-aspartate receptor antagonist (NMDA-R), the molecular events underlying the behavioral symptoms remain largely unknown. Statistical analysis of oligonucleotide microarray data was used to identify phencyclidine-induced alterations in rat cortical gene expression. Acute phencyclidine produced a statistically significant change in 477 genes in rat prefrontal cortex (PFC), a brain area associated with cognitive dysfunction in schizophrenics. Real-time quantitative PCR (RTQ-PCR) confirmed a subset of these changes ranging from -59% to 255% (smallest confirmation: -19%). Subsequent time-course and dose-response studies using RTQ-PCR confirmed and extended the original microarray results. At the molecular level, genes altered by phencyclidine are related to diverse biological processes including stress, inflammatory response, growth and development, neural plasticity and signal transduction. Further analysis, aimed at assessing the relevance of our results to schizophrenia, revealed dysregulation of genes related to: (i) thalamocortical projections, (ii) neurotransmission and neuromodulation, (iii) thyroid hormone activity, (iv) oligodendrocyte linage, (v) brain lipid metabolism, (vi) sleep architecture and (viii) the velocardiofacial syndrome.

Evaluation of common gene expression patterns in the rat nervous system.

In the postgenomic era, integrating data obtained from array technologies (e.g., oligonucleotide microarrays) with published information on eukaryotic genomes is beginning to yield biomarkers and therapeutic targets that are key for the diagnosis and treatment of disease. Nevertheless, identifying and validating these drug targets has not been a trivial task. Although a plethora of bioinformatics tools and databases are available, major bottlenecks for this approach reside in the interpretation of vast amounts of data, its integration into biologically representative models, and ultimately the identification of pathophysiologically and therapeutically useful information. In the field of neuroscience, accomplishing these goals has been particularly challenging because of the complex nature of nerve tissue, the relatively small adaptive nature of induced-gene expression changes, as well as the polygenic etiology of most neuropsychiatric diseases. This report combines published data sets from multiple transcript profiling studies that used GeneChip microarrays to illustrate a postanalysis approach for the interpretation of data from neuroscience microarray studies. By defining common gene expression patterns triggered by diverse events (administration of psychoactive drugs and trauma) in different nerve tissues (telencephalic brain areas and spinal cord), we broaden the conclusions derived from each of the original studies. In addition, the evaluation of the identified overlapping gene lists provides a foundation for generating hypotheses relating alterations in specific sets of genes to common physiological processes. Our approach demonstrates the significance of interpreting transcript profiling data within the context of common pathways and mechanisms rather than specific to a given tissue or stimulus. We also highlight the use of gene expression patterns in predictive biology (e.g., in toxicogenomics) as well as the utility of combining data derived from multiple microarray studies that examine diverse biological events for a broader interpretation of data from a particular microarray study.

Group II metabotropic glutamate receptor modulation of DOI-induced c-fos mRNA and excitatory responses in the cerebral cortex.

Recent studies have demonstrated that the hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) enhances glutamatergic transmission in the prefrontal cortex. This increase can be suppressed by metabotropic glutamate2/3 (mGlu2/3) receptor activation. In addition to enhancing glutamatergic transmission, DOI increases cortical c-fos expression. We tested if a reduction in glutamate release produced by mGlu2/3 receptor activation attenuates DOI-induced c-fos expression in the cortex. Similar to previous studies, DOI produced a robust increase in c-fos mRNA throughout the cortex, including the prefrontal, frontoparietal, and somatosensory regions. Pretreatment with the mGlu2/3 agonist LY379268 attenuated the DOI-induced increase in the prefrontal cortex. This suppression was blocked by the mGlu2/3 antagonist LY341495. In contrast, the DOI-induced increase in c-fos mRNA in the frontoparietal and somatosensory cortex was unaffected by the mGlu2/3 agents. These findings suggest that Group II metabotropic glutamate receptor agonists are capable of modulating postsynaptic function preferentially in the limbic cortex under conditions of enhanced glutamate release.