Advancing Drug Discovery for Schizophrenia

Journal Articles

Schahram Akbarian

Akbarian S, Huang H. Epigenetic regulation in human brain-focus on histone lysine methylation. Biol. Psychiatry 2009;65(3):198-203.

John A. Allen

Allen JA, Halverson-Tamboli RA, Rasenick MM. Lipid raft microdomains and neurotransmitter signalling. Nat. Rev. Neurosci. 2007 Feb;8(2):128-40.

Walsh T, McClellan JM, McCarthy SE, et al. Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science 2008 Apr 25;320(5875):539-43.

Brian Campbell

Albuquerque EX, Pereira EFR, Alkondon M, Rogers SW. Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol. Rev. 2009;89(1):73-120.

Chess AC, Bucci DJ. Increased concentration of cerebral kynurenic acid alters stimulus processing and conditioned responding. Behav. Brain Res. 2006;170(2):326-332.

Jentsch JD, Roth RH. The neuropsychopharmacology of phencyclidine: from NMDA receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 1999;20(3):201-225.

Susan George

Gur RE. Neuropsychiatric aspects of schizophrenia. CNS Neurosci. Ther. 2011 Feb;17(1):45-51.

Perreault ML, O'Dowd BF, George SR. Dopamine receptor homooligomers and heterooligomers in schizophrenia. CNS Neurosci. Ther. 2011 Feb;17(1):52-7.

Jay Gingrich

Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 1983;301(5895):89-92.

Lambert SM, Masson P, Fisch H. The male biological clock. World J. Urol. 2006;24(6):611-617.

Malaspina D, Dalack G, Leitman D, et al. Low heart rate variability is not caused by typical neuroleptics in schizophrenia patients. CNS Spectr. 2002;7(1):53-57.

Martin SP. Diverging fertility among U.S. women who delay childbearing past age 30. Demography 2000;37(4):523-533.

Stephen Haggarty

Freyberg Z, Ferrando SJ, Javitch JA. Roles of the Akt/GSK-3 and Wnt signaling pathways in schizophrenia and antipsychotic drug action. Am. J. Psychiatry 2010;167(4):388-396.

Maria Karayiorgou

Karayiorgou M, Simon TJ, Gogos JA. 22q11.2 microdeletions: linking DNA structural variation to brain dysfunction and schizophrenia. Nat. Rev. Neurosci. 2010;11(6):402-416.

Amanda Law

Citri A, Yarden Y. EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 2006;7(7):505-516.

Law AJ, Kleinman JE, Weinberger DR, Weickert CS. Disease-associated intronic variants in the ErbB4 gene are related to altered ErbB4 splice-variant expression in the brain in schizophrenia. Hum. Mol. Genet. 2007;16(2):129-141.

Nicodemus KK, Law AJ, Radulescu E, et al. Biological validation of increased schizophrenia risk with NRG1, ERBB4, and AKT1 epistasis via functional neuroimaging in healthy controls. Arch. Gen. Psychiatry 2010;67(10):991-1001.

Silberberg G, Darvasi A, Pinkas-Kramarski R, Navon R. The involvement of ErbB4 with schizophrenia: association and expression studies. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2006;141B(2):142-148.

Tan H, Nicodemus KK, Chen Q, et al. Genetic variation in AKT1 is linked to dopamine-associated prefrontal cortical structure and function in humans. J. Clin. Invest. 2008;118(6):2200-2208.

Walsh T, McClellan JM, McCarthy SE, et al. Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science 2008;320(5875):539-543.

Jeffrey A. Lieberman

Glantz LA, Gilmore JH, Lieberman JA, Jarskog LF. Apoptotic mechanisms and the synaptic pathology of schizophrenia. Schizophr. Res. 2006;81(1):47-63.

Javitt DC. Glutamate and schizophrenia: phencyclidine, N-methyl-D-aspartate receptors, and dopamine–glutamate interactions. Int. Rev. Neurobiol. 2007;78:69-108.

Matute C, Melone M, Vallejo-Illarramendi A, Conti F. Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics. Glia 2005;49(3):451-455.

Molina V, Sanz J, Sarramea F, Benito C, Palomo T. Prefrontal atrophy in first episodes of schizophrenia associated with limbic metabolic hyperactivity. J. Psychiatr. Res. 2005;39(2):117-127.

Bita Moghaddam

Gattaz WF, and Busatto G, eds. Advances in Schizophrenia Research 2009. New York, NY: Springer Science+Business Media; 2010.

Gore CD, Bányai M, Gray PJ, Diwadkar V, Erdi P. Pathological effects of cortical architecture on working memory in schizophrenia. Pharmacopsychiatry 2010 May;43 Suppl 1:S92-7.

Gottesman II, Gould TD. The endophenotype concept in psychiatry: etymology and strategic intentions. Am. J. Psychiatry 2003 Apr;160(4):636-45.

Nakazawa K, Zsiros V, Jiang Z, Nakao K, Kolata S, Zhang S, Belforte JE. GABAergic interneuron origin of schizophrenia pathophysiology. Neuropharmacology 2011 Jan 26.

Novick D, Ascher-Svanum H, Zhu B, Brnabic A, Stauffer V, Peng X, Karagianis J, Perrin E. The number needed to treat for all-cause medication discontinuation in the treatment of schizophrenia: consistency across world geographies and study designs. Pharmacopsychiatry 2010 May;43(3):81-5.

Qi Z, Miller GW, Voit EO. Computational modeling of synaptic neurotransmission as a tool for assessing dopamine hypotheses of schizophrenia. Pharmacopsychiatry 2010 May;43 Suppl 1:S50-60.

Wolff-Menzler C, Hasan A, Malchow B, Falkai P, Wobrock T. Combination therapy in the treatment of schizophrenia. Pharmacopsychiatry 2010 Jun;43(4):122-9.

Eric J. Nestler

Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. The DISC locus in psychiatric illness. Mol. Psychiatry 2008;13(1):36-64.

O'Tuathaigh CMP, Desbonnet L, Moran PM, Kirby BP, Waddington JL. Molecular genetic models related to schizophrenia and psychotic illness: heuristics and challenges. Curr. Top. Behav. Neurosci. 2011;7:87-119.

Simunovic F, Yi M, Wang Y, et al. Gene expression profiling of substantia nigra dopamine neurons: further insights into Parkinson's disease pathology. Brain 2009;132(Pt 7):1795-1809.

Sklar P, Smoller JW, Fan J, et al. Whole-genome association study of bipolar disorder. Mol. Psychiatry 2008;13(6):558-569.

Akira Sawa

Rodríguez-Santiago B, Brunet A, Sobrino B, et al. Association of common copy number variants at the glutathione S-transferase genes and rare novel genomic changes with schizophrenia. Mol. Psychiatry 2010 Oct;15(10):1023-33.

Turetsky BI, Moberg PJ. An odor-specific threshold deficit implicates abnormal intracellular cyclic AMP signaling in schizophrenia. Am. J. Psychiatry 2009 Feb;166(2):226-33.

Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 2010 Feb 25;463(7284):1035-41.

Edward Scolnick

Tsuang MT, Lyons MJ, Faraone SV. Heterogeneity of schizophrenia: Conceptual models and analytic strategies. Br. J. Psychiatry 1990;156:17-26.

Patrick F. Sullivan

Levinson DF, Duan J, Oh S, et al. Copy Number Variants in Schizophrenia: Confirmation of Five Previous Findings and New Evidence for 3q29 Microdeletions and VIPR2 Duplications. Am. J. Psychiatry 2011;168(3):302-316.

Potkin SG, Macciardi F, Guffanti G, et al. Identifying gene regulatory networks in schizophrenia. Neuroimage 2010;53(3):839-847.

Purcell SM, Wray NR, Stone JL, et al. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 2009;460(7256):748-752.

Complete sequence and gene map of a human major histocompatibility complex. The MHC sequencing consortium. Nature 1999;401(6756):921-923.

Daniel Weinberger

Huffaker SJ, Chen J, Nicodemus KK, et al. A primate-specific, brain isoform of KCNH2 affects cortical physiology, cognition, neuronal repolarization and risk of schizophrenia. Nat. Med. 2009;15(5):509-518.

Sponsors

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The Company of Biologists, publishers of the journals Disease Models & Mechanisms, Journal of Cell Science, and The Journal of Experimental Biology

Grant Support

The project described is supported by Award Number R13MH085413 from the National Institute of Mental Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institutes of Health.

Schizophrenia is viewed by most experts as a neurodevelopmental mental disorder and recent studies of the disease have focused on the changes in brain activity that are observed in the hippocampus, frontal, and temporal lobes of schizophrenia patients. For most individuals with the illness, symptoms become apparent just after puberty during a critical period in young adulthood. As thought processes become disorganized and emotional responsiveness disintegrates, many patients suffer a significant reduction in quality of life. These patients can suffer from a variety of psychotic symptoms, including auditory, visual, olfactory, and gustatory hallucinations; paranoia; delusions; and halting or inarticulate speech. Because of the variability of symptoms, clinicians and scientists continue to debate whether the "schizophrenia" diagnosis describes one disorder or encapsulates a number of related diseases.

Currently, schizophrenia is primarily treated with antipsychotics. These drugs act on the dopamine receptor pathway to reduce psychotic symptoms such as delusions and hallucinations, but most fail to mitigate the emotional symptoms and cognitive impairments associated with the disease. In the 1950s, when the first generation of antipsychotics were discovered, these drugs (called the "typical antipsychotics") were referred to as "the major tranquilizers" because of their sedation effects. The atypical, or second-generation antipsychotics block dopamine receptors as did their first-generation counterparts, but some may block or partially block serotonin receptors as well. Although second-generation drugs are considered less likely to cause the neurological movement disorders (Parkinson's-type movement, rigidity, and tremors) that are associated with first-generation antipsychotics, some of the newer medications are thought to cause weight gain and metabolic abnormalities that raise serious health risks.

The New York Academy of Sciences conference Advancing Drug Discovery for Schizophrenia was held on March 9–11, 2011. The collaborative effort provided a forum for scientists and clinical experts—from academia, industry and government—and fostered vital progress in translational medicine by raising physician awareness of groundbreaking developments in basic research.

The purpose of this conference was to present and engender new avenues of exploration into developing treatments for schizophrenia. After highlighting the inadequacies of current pharmacotherapy, presenters focused on recent discoveries in genetics, epigenetics, and neuroscience and stressed the importance of building innovative partnerships between academia and industry. Such collaborations will undoubtedly lead to greater understanding of the basic mechanisms of disease and help to catalyze development of novel therapeutic targets.

Meeting organizer Stephen R. Marder of the Semel Institute for Neuroscience at the University of California, Los Angeles, lamented the fact that current therapies only attenuate certain aspects of disease symptoms. While current treatments may repress hallucinations and delusions somewhat, these drugs fail to cure the myriad schizophrenia-associated afflictions, and they ultimately fall short of getting to the heart of the disorder. Unfortunately, despite the need for better drugs, a number of pharmaceutical companies appear to be decreasing investment in R&D devoted to innovative treatment of schizophrenia, and many have abandoned the field altogether.

Marder urged researchers and drug developers to direct their focus away from developing traditional symptom-oriented treatments and toward the search for biomarkers that indicate risk for developing the disease. The latter approach includes the effort to identify mutations in key genes (called "risk genes") that lead to risk of developing schizophrenia. The search for a way to identify individuals well before they display full onset of the disease must be accompanied by the development of treatments that target the first decade of the illness. Ultimately, the goal is to provide preventive treatments so that those at risk do not develop the disease.

Speakers:
Patrick F. Sullivan, University of North Carolina at Chapel Hill
Edward M. Scolnick, The Broad Institute of MIT and Harvard University
Eric J. Nestler, Mount Sinai Medical Center

Highlights

• Collection of a larger number of patient genomes is predicted to identify all common mutations and reveal the polygenetic nature of schizophrenia/psychotic illness.
• Over the last 5 years the field has a new and clear intellectual approach — to identify the full genetic architecture of mental illness.
• New technologies are revealing underlying causes of schizophrenia, leading us away from symptom-based treatments.
• Pathognomonic data are essential for creating a relevant animal model of schizophrenia.
• Researchers grapple with the limitations of current animal models.

Banking on genome-wide association studies (GWAS)

In a concise introduction to his work, Patrick Sullivan of the University of North Carolina Chapel Hill rhetorically asked, "Why do GWAS [for schizophrenia]?" The goal, of course, is to identify a list of disease-associated gene variants that confer risk or protection. Genetic studies, unlike any other approach, offer researchers the mechanisms of causation, the initiating event rather than the end result. "The biological network interconnects at a central node, which is genetics," he explained. To target that central node, Sullivan's career has been focused on the primary cause of schizophrenia rather than on "the peripheral symptoms." This effort has been productive in the past couple of years—in fact, 8 to 15 genetic loci associated with risk for schizophrenia have been identified.

However, several GWA studies have identified markedly diverse sets of loci as important, thereby sparking heated debate in the field. Researchers are attempting to determine which of these genetic candidates confers direct risk of developing the disease. For example, using the International Schizophrenia Consortium (ISC) data set, a GWAS revealed common genetic variations in the major histocompatibility complex (MHC) locus associated with risk of schizophrenia. The MHC locus on Chromosome 6 spans about 4 Mb (4 million base pairs) and has been shown to be essential for immune system function. Studies have also identified a microRNA, miR-137, previously shown to be involved in adult neural stem cell proliferation, maturation, and epigenetic cross-talk. Notably, miR-137's predicted targets are genes already linked to schizophrenia. Experts hypothesize that schizophrenia represents a spectrum disorder and that it may arise from discrete mutations, duplications, or deletions in a number of different genes that result in common symptoms.

While it is not uncommon to find copy number variations (CNV) all over the genome, many of these variations remain silent. It is striking to find that hotspots for CNV appear to occur at specific loci in patients suffering from schizophrenia. Sullivan reviewed several studies demonstrating schizophrenia linked to CNV at specific loci, namely 1q21.1, 15q13.3, and 22q11.21 deletions, 16p11.2 duplications, and exonic NRXN1 deletions. However, these genome hotspots do not only confer risk of schizophrenia—they have also been linked to mental retardation, autism spectrum disorders, and epilepsy. Sullivan proposed two possible explanations, either the variant copy number(s) represent a risk of neuropsychiatric disease in general, or, more subtly, they represent a misdiagnosis of the disorder in certain individuals. He suggests that "going back" and blindly re-screening patient symptoms would eliminate the second of these influences, and would allow a narrower list of genetic culprits to emerge. Regardless of which explanation accounts for the variation in risk loci, GWAS have certainly revealed, what researchers have suspected for some time, that schizophrenia is a multi-gene disorder.

A schematic of Sullivan's protocol design.

Sullivan elegantly detailed how researchers are testing the Polygenic Model. Briefly, a previously identified list of risk alleles, in this case generated from samples from the International Schizophrenia Consortium (ISC) data set, or discovery sample, were compared to the individual genotypic and phenotypic data from additional sets, or target samples, in order to identify for each person in the target sample how many risk alleles share common genetic variants that are specifically associated with disease. Common polygenic variant risk alleles are plotted for schizophrenia and bipolar disorder and non-psychiatric data sets. What Sullivan and co-workers have found is that the ISC list was replicated in certain target groups: the risk alleles from the ISC discovery sample were also identified in the schizophrenia target samples and were not found in the control data sets. They also found overlap of risk alleles with bipolar disorder target samples. These results support the hypothesis that many loci are involved and that the two disorders (schizophrenia and bipolar disorder) may be closely associated.

Genome wide association studies have proven to be dependent on sample size. Therefore, Sullivan argues, "If we put more into it, we will get more out of it." By increasing the sample size to 100,000 with 50,000 in the schizophrenia group and 50,000 controls (current data include 15,000 schizophrenia cases), Sullivan predicts, perhaps 20 to 30 new risk loci will be discovered, and moreover, sufficient data will be generated for the identification of complete pathways that are disrupted in the disease.

New technologies continue to change the landscape

Edward Scolnick of the Broad Institute of MIT and Harvard University reviewed three relatively recent technological advances that are helping to unravel the genetics behind mental illness. First, since the sequencing of the human genome in 2001, several discoveries have been made that would not otherwise have been possible. As Sullivan also described, GWAS has helped elucidate the multiple genetic risk factors that contribute to mental illness. Second, Scolnick discussed how optogenetic methods, which rely on a keen understanding of the genes expressed in particular neurons, have added to the basic understanding of neurological circuits. (Karl Deisseroth and colleagues developed optogenetics as a noninvasive, real-time imaging technique used to probe brain circuits in living tissue.) Third, Scolnick described the discovery made by Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University that pluripotent stem cells could be induced from mouse embryonic and adult skin fibroblast cultures by adding only a few defined factors. This finding allows for the study of abnormalities in neural development, non-degenerative neurological diseases, and cell autonomous events underlying disease, which would not otherwise be possible because of the difficulty of obtaining fresh brain tissue.

Despite these major technological improvements, Scolnick noted, the discovery of mechanistically distinct drugs used to treat schizophrenia has not advanced since the 1950s. This was the most common complaint throughout the conference. The number of novel drugs used to treat mental illness has not fundamentally changed in 60 years, a number that stands in marked contrast to, for example, the rate of medicines developed to treat heart disease. Scolnick explained that this failure has been partially a result of a lack of clear understanding of the underlying biochemical pathways and of the basic molecular biology of mental illness. Affirming Sullivan's focus on genetics, Scolnick asserted that as has been demonstrated by advances in cancer therapeutics over the last 20 years, only in the process of identifying the underlying genetics of schizophrenia will the appropriate treatments come to light.

In the 1990s the single largest risk factor for developing schizophrenia was shown to be family history, and Scolnick maintained that we are now only limited by "dollars and time" before full elucidation of the genetic causes of schizophrenia is realized. Many challenges lie ahead in the next 5 to 10 years: scientists must create a complete analysis of genetic trait susceptibility and translate that into clinical applications.

To that end, the ISC has compiled a bank of population-based DNA samples of disease cases (half of the cases are individuals with schizophrenia and half are those with bipolar disorder) and control cases. This collection is projected to grow to 86,000 total cases in the next 2 years and will be available for both GWAS and sequencing studies.

Scolnick compiled a table showing the total sample number of cases in current and projected data collection contributing to the GWAS database. The pie charts indicate by color and abbreviation the contribution from each international consortium member.

Both Sullivan and Scolnick reason that the rate of identifying traits significantly associated with risk of developing schizophrenia is greatly limited by the sample size of GWAS studies and that as sample size increases, the identification of associated genetic regions will accelerate. This, they predict, will in turn lead to a revolution in the diagnosis of disease, from one that is clinical symptom-based to one that is DNA variation-based. Finally, and perhaps most importantly, they believe GWAS will provide the foundation for "discovering new biology"—through the identification of common CNVs, detailed molecular pathways will emerge, which will ultimately reveal specific targets and generate desperately needed drugs for those targets.

Why treatment for schizophrenia has not kept pace

The third keynote speaker, from the Mount Sinai Medical Center, Eric J. Nestler, offered what he termed "a high altitude perspective" of the field. He too lamented the lack of advancement in the treatment of psychosis. The problem, as Nestler put it, is that the field has been hampered by its lack of an objective diagnostic measure of the disease, a problem compounded by the sheer complexity of the brain itself and of its behavioral manifestations. While the Diagnostic and Statistical Manual of Mental Disorders (DSM) aims to provide a common language and standard criteria for the classification of mental illness, "it doesn't make scientific sense," Nestler argued. He explained, "the manual overstates the meaning of the words [the descriptive criteria] in the absence of quantifiable data." Nestler reasoned further that because we know so little about the underlying pathophysiology, we have not been able to make the appropriate animal models. Previously unavailable, current sequencing technology permits the measurement of genetic variants and therefore the quantification of disease, a process that is crucial for the generation of appropriate animal models.

There are, however, unique challenges in parsing psychiatric genetics. Even in cases where the genetic risks are identified, the diagnosis can vary. For example, the Disrupted-In-Schizophrenia-1 (DSC1) mutation is associated with several diseases: schizophrenia, bipolar disorder, and psychotic depression. Therefore, a gene variation can be implicated in multiple illnesses. However, as the collection of sequencing data expands, Nestler predicts the etiology of mental illness will become better understood.

Where animal models fall short

Although virtually all the presentations at the conference described data from model organisms, Nestler summarized three contributing limitations on the validity of current animal models of schizophrenia. First, without knowing the disease etiology, Nestler contends, we cannot easily generate relevant animal models. As previously discussed, known genes associated with psychiatric diseases (for example, the DISC1 locus) manifest in multiple syndromes in humans. Moreover, each disease is associated with more than one gene. For these reasons, models that use single genes or genes of very minimal effect are not likely to generate useful insight into schizophrenia (although they do tell us about neurobiology in general). Likewise, if schizophrenia is caused by multiple genetic and non-genetic factors, as is believed, animal models will have to be equally complex to prove their utility.

Second, Nestler explained that psychotic behaviors (including delusions and hallucinations) are uniquely measurable in humans, and therefore, many key features of mental illness are not accessible in animal models. However, as quantifiable biological variables are identified as pathognomonic (i.e., diagnostically definitive) for schizophrenia, in much the same way that selective death of substantia nigra dopamine neurons is pathognomonic for Parkinson's disease for example, those factors should be validated via animal models. Nestler's third validity limitation for animal models involves problems with testing schizophrenia drugs on model organisms. Pharmacological validity hinges on reproducing disease symptoms in animals. How, for example, would an anti-hallucinogenic drug effect be tested accurately in rodents given that it is impossible to tell if a rat is hallucinating? Although animal models inform general understanding of neurobiology, Nestler contends that with respect to human psychiatric syndromes, the results of such investigations must be interpreted with caution.

With those admonitions about animal modeling in mind, Nestler continued with a fascinating description of his group's work on chronic social defeat in rodents. In these studies, researchers expose an experimental mouse to an aggressor mouse for 5 minutes. The limited "face to face" encounter is repeated with a new aggressor daily for 10 days, but between the episodes of direct exposure, the experimental mouse is housed in the same cage as the aggressor mouse, though they are separated by a transparent wall. The team found that the experimental mouse exhibited profound social defeat characterized by decreased interest in sex and sucrose, anxiety-like symptoms, hyperactivity, disrupted circadian rhythms, metabolic syndrome, and social avoidance.

Nestler's work shows persistent social defeat induces persistent avoidance in mice. Figure on the left shows video tracking data from control and defeated mice in the absence (no target) and presence (target) of an aggressive target mouse placed in the interaction zone.

Social defeat is induced in an otherwise normal mouse with long lasting effects. Nestler and colleagues found that the social avoidance phenotype was reversed by chronic treatment with antidepressants but not by anxiolytics, which are used to treat anxiety. They also determined that the phenotype was pathological—affected mice avoided not just the aggressor mouse but all other mice as well. One-third of the experimental mice were actually resilient to social defeat and did not present social avoidance or most of the other pathological behaviors. Nestler's group went on to compare gene and chromatin expression profiles of susceptible versus resilient mice in two brain areas of interest, nucleus accumbens and ventral tegmental area (NAc and VTA, respectively), both of which play key roles in motivation and reward, among other aspects of behavior. From the gene arrays, they identified clear differences in the expression of particular mRNAs in the mice, depending on whether the mice were susceptible or resilient to social defeat, or on whether they were "naturally" resilient or treated with antidepressant medications.

Nestler's team investigated further by considering chromatin changes relative to gene expression data. They found an overlap between chromatin changes that occur naturally in resilient mice and those changes caused by treatment with antidepressants, suggesting that antidepressants work in part by inducing a kind of resilience. Perhaps novel human treatments will some day exploit a molecular pathway of resilience and promote those relevant changes in gene expression within a susceptible individual.

Nestler envisions a future where after a series of specific laboratory tests, genetic profiling, and brain imaging, a patient suffering from (or predicted to be at risk for) mental illness would receive a concise diagnosis with treatment targeting the particular pathophysiology involved in his or her illness.

Speakers:
Daniel R. Weinberger, National Institute of Mental Health
Maria Karayiorgou, Columbia University
Alessandro Guidotti, University of Illinois at Chicago
Jay A. Gingrich, Columbia University and the New York State Psychiatric Institute
Schahram Akbarian, University of Massachusetts Medical School

Highlights

• Variation in KCNH2, a voltage-dependent potassium channel, is a risk factor for schizophrenia.
• Copy number variations (CNVs) provide etiology-based animal models and inform novel therapeutic targets.
• Agents that affect chromatin remodeling provide an important tool in the next generation of antipsychotics.
• Advanced paternal age is associated with epigenetic alterations that are then passed on to offspring.
• Methyl-MAPS provide a novel, ultra high-throughput method to assess DNA methylation status genome-wide.
• 20 billion base pairs from cortical neurons have been sequenced so far for Neuronal Epigenome Map.

Moving clinical diagnosis beyond statistical association–gene-based targets

In a classically driven experimental paradigm, Daniel Weinberger, from the National Institute of Mental Health, and co-workers discovered a novel therapeutic target for psychosis. Looking at differentially expressed genes in brains of patients suffering from schizophrenia enabled Weinberger's research team to identify a risk factor for developing the disease, a variant in KCNH2 (isoform 3.1), which codes for a voltage-dependent potassium channel member in the human ether-a-go-go (ERG)-family. In meta-analysis of five independent clinical data sets, Weinberger's group identified a single-nucleotide polymorphism (SNP) variant in a small intronic region of KCNH2 that effects changes in gene expression and regulation and that is associated with schizophrenia. They found that KCHN2 3.1 is upregulated in schizophrenia sufferers' prefrontal and hippocampal cortices, and, based on several lines of evidence, they hypothesized that the brain-specific isoform 3.1 may be a target for antipsychotic drugs. When the team expressed KCHN2 3.1 in rodent cortical neurons they observed abnormal neuronal firing patterns, and were able to further hypothesize that these changes in neuronal excitability may underlie fundamental aspects of abnormal circuitry in schizophrenia. With a focus on etiology and pathophysiology, Weinberger's group has uncovered a potential new target for antipsychotic therapy.

In contrast to SNPs, which affect only one single nucleotide base, CNVs involve abnormal numbers of copies of sections of the genome, may range from one kilobase to several megabases in size, and reportedly account for over 10% of DNA variation in the human genome. Alternations in copy number within the cell, deletions or duplications of DNA, are not necessarily harmful, but CNVs in some locations on the genome have been associated with disease. Maria Karayiorgou, from the Center for Human Genetics at Columbia University Medical Center, presented a compelling story linking DNA structural variation to brain dysfunction with her extensive work on understanding the genetic causes of psychiatric illness.

Specific cases where schizophrenia is caused by a known genetic factor have been identified, though such cases are rare. Fifteen years ago when Karayiorgou identified variants at the q11.2 region of human chromosome 22 associated with disease, it was the first demonstration that sporadic deletions or duplications of chromosomal regions play a role in the etiology of schizophrenia. Her work suggests that genomic microdeletions in the chromosomal region 22q11.2 account for as many as 1%-2% of schizophrenia cases. Recently, etiology-based animal models have allowed for the identification of specific developmental, neuronal, and behavioral changes that may undergird psychotic illness, although such behavioral changes are of limited relevance to human behavioral analysis. In the next fifteen years Karayiorgou predicts that through the generation and characterization of transgenic mice (with deleted candidate genes, and tissue-specific and inducible knockouts), novel and effective therapeutic targets for a new generation of antipsychotic medication will emerge.

Epigenetic modifications in schizophrenia and bipolar disorder

With the ultimate goal of disease prevention and treatment, Alessandro Guidotti of the University of Illinois at Chicago presented his collaborative work on uncovering the biochemical mechanisms (DNA-methylation and chromatin structure modifications) that turn genes on and off in specific neurons. Guidotti and co-workers are particularly interested in the observation that patients suffering from schizophrenia and bipolar disorder show a down regulation of certain genes, including those that code for glutamic acid decarboxlase₆₇ (GAD67) and reelin glycoprotein in GABAergic neurons (neurons that transmit gamma-Aminobutyric acid) of the telencephalic region.

Recent findings suggest an epigenetic mechanism may be responsible for this down regulation. Studies show that DNA methyl transferase (DNMT) is increased in the brain of schizophrenia and bipolar disorder patients. During his talk, Guidotti suggested that the inhibitory action of DNMT on gene expression leads to hypermethylation of GABAergic gene promoters, and that such DNA methylation is associated with psychosis. Guidotti proposes a combinatory pharmacological strategy aimed at increasing transcription at GABAergic promoters. The first strategy is to facilitate chromatin remodeling to induce DNA demethylation. Inhibition of histone deacetylase with the (HDAC) inhibitor valproate (VPA), results in a chromatin state that is thought to block DNA methylation. This therapy would be used in conjunction with antipsychotics. In his work using animal models, combinatory treatments of clozapine (an antipsychotic) with VPA showed mutually reinforcing effects on GABAergic promoter demethylation. As data suggesting that DNA packaging is altered in patients with mental illness accumulate, Guidotti predicts that chromatin remodeling mechanisms will become an important target in the next generation of antipsychotics.

Hot topics in genetic and epigenetic approaches

In light of the current trend to delay childbearing until later in life and given the connection between maternal age and an increased risk of several disorders for the infant, Jay A. Gingrich's presentation was of great contemporary relevance. Gingrich, who is based at Columbia University, is interested in addressing global questions of how neural networks form, including how genetics, epigenetics, the environment, and to some extent individuals' perception of their experience, contribute to the development of neuropsychiatric disorders.

In 2002 Gingrich and colleagues focused on taking the epidemiological observations that suggested advanced paternal age (APA) is a risk factor for schizophrenia, and they translated these observations into a testable biological hypothesis. Using a mouse model, they set up a mating scheme wherein 12-month old males were mated to 3-month old females to produce old-father offspring (OFO). The OFO could be compared to young-father offspring (YFO) produced by pairing 3-month old males with 3-month old females. The fathers were then removed from the cage two weeks before the birth of offspring. Analysis of a series of behavioral tests (including exploratory behavior in an open-field, startle stimulus amplitude, startle inhibition, and reaction time) produced a Gaussian curve, which enabled the team to categorize the mice into two groups based on their performance in the tests.

High-performing offspring (h-YFO) and low-performing offspring (l-OFO) were chosen for further analysis because any difference between these two groups might provide insight into the influence of parental age on offspring behavior. Because spermatogonia continue to divide throughout life there is increased opportunity for genetic mutations to arise in these cells relative to female germ cells, which undergo several divisions during fetal development and are then quiescent until menarch. Thus, the researchers expected to see changes in the genetic and epigenetic content passed down from older fathers.

To test what information was different in the older sperm (assessed by what is passed on to offspring), Gingrich and collaborator Timothy Bestor, undertook a genome-wide assessment of DNA methylation fidelity in the brain tissue of h-YFO and l-OFO mice using Methyl-MAPS, a novel, unbiased ultra high-throughput method to assess DNA methylation status. While overall levels of methylation did not differ between the two groups of offspring, delving deeper into the data revealed significantly altered DNA methylation patterns around cytosine and guanine rich (CpG) islands, which are frequently near promoter regions of genes. Much literature has been focused on these "island shores," the areas nearby CpG islands, where aberrant DNA methylation has been found in human cancers. It is here at the island shores and also at intron/exon junctions where a significant difference between the offspring emerged, indicating potential effects on promoter activity and on alternative splicing. Moreover, preliminary data suggest these hotspots of altered DNA methylation may occur specifically in genes associated with schizophrenia and autism spectrum disorders. Behavioral abnormalities observed in APA offspring, Gingrich hypothesized, are possibly a result of specific differences in brain DNA methylation, and he concluded that age is associated with the accumulation of changes in gene expression in the paternal germline that are randomly passed on to offspring.

Pre-clinical studies have shown that several environmental factors, such as stress and drugs, lead to changes in the levels of particular RNA transcripts in the brain, and these changes can contribute to disease. It remains unclear whether the underlying molecular pathology involves changes in gene expression (i.e., transcription levels) or alternatively, alterations in mRNA processing. Using human postmortem brain tissue and pre-clinical models, Schahram Akbarian, from the University of Massachusetts Medical School, and colleagues are studying the distribution of histone modifications, particularly those associated with the sites of transcription initiation and with CpG rich sequences, in genome-wide analyses. Because trimethylated forms of histone 3 lysine (H3K4me3) appear to be relatively stable in postmortem tissue, H3K4me3 can be used as a marker of DNA accessible to transcription factors. Akbarian's work has focused on comparing prefrontal cortices (PFCs) of individuals diagnosed with schizophrenia or autism spectrum disorders to the PFCs of control subjects across a wide age range, and specifically on profiling histone methylation that would affect particular promoter regions to create a "neuronal epigenome map." In the last 2 years, Akbarian's group has developed a technique for separating out millions of neuronal nuclei from non-neural cortical tissue.

Epigenome profiling in human postmortem prefrontal cortex (PFC) neurons. Representative example of fluorescence-activated cell sorting (FACS) of chromatin from NeuN+ tagged neuronal nuclei. NeuN is a specific marker for mature neurons and is expressed in a majority of excitatory and inhibitory neurons. NeuN- samples are primarily non-neuronal cells (glia, microglia, and endothelium). DAPI is a nuclear marker. Scale bar,10um). Gel to the right shows in increasing concentration micrococcal nuclease (MNase) treatment of NeuN+ cells which induces double-strand breaks within the nucleosome. Cartoon depicts a single nucleosome with wrapped DNA in red, and the H3K4me3 tagged (an epigenetic marker found mostly at transcription start sites) with site-specific antibody. Center gel shows the purified material used in Chip-seq deep sequencing analysis. One such data set result is shown for 20kb of the genome, here depicts the CpG island distribution of H3K4trimethyl tag in the GAD1gene promoter region where the rise in signal is indicated by peaks (red) of NeuN+ tagged neuronal nuclei.

In a collaborative effort with the UMASS Brain Epigenome Project, his team has been successful in sequencing 20 billion base pairs from PFC neuronal-H3K4me3+ tagged nucleosomes, using a chromatin immunoprecipitation and deep sequencing (ChiP-seq) approach. Comparing, by Pearson correlation, disease and control cases, Akbarian found that H3K4me3 occupancy at transcriptional start sites did not differ overall between samples. That is to say, the loci generally methylated in chromatin from disease prefrontal neurons were also generally methylated in non-psychiatric control samples.

Yet subtle chromatin remodeling events were readily identified in the brains of those who had had autism spectrum disorder. The team identified hundreds of loci that were altered in clinical sample brains. After searching several genetic risk databases for overlap, they found, at least to some degree, evidence for overlap with their epigenetic risk map. Broadly, such risk genes included those regulating synaptic connectivity, social behaviors, and cognition. Such work supports the argument for deeper explorations into the myriad genetic and epigenetic risk factors evidently contributing to the etiology of psychosis.

Speakers:
Jeffrey A. Lieberman, Columbia University and the New York State Psychiatric Institute
P. Jeffrey Conn, Vanderbilt University Medical Center
Stephen J. Haggarty, Harvard Medical School, Massachusetts General Hospital, and the Broad Institute of MIT and Harvard

Highlights

• Changes in gray matter volume may contribute to schizophrenia.
• Brain-imaging improves on behavioral diagnostic markers for early diagnosis by revealing changes in cerebral blood volume.
• A high-throughput screen identifies diverse and highly selective positive allosteric modulators (PAMs) of mGluR5.
• A high-throughput small-molecule microarray screen identifies small molecule probes that selectively target discrete disrupted-in-schizophrenia-1 (DISC1) binding domains.

Prevention, prevention, prevention

On the second day of the conference, Jeffrey A. Lieberman of Columbia University and the New York State Psychiatric Institute opened the session with a comprehensive summary of the limitations of current schizophrenia treatments and offered his view on future genetic strategies for novel pharmacological intervention.

A lack of theoretical consensus is not the only roadblock for schizophrenia research. One of the overriding challenges in studying schizophrenia involves capturing the complexity and context of the disease in a model organism—nothing related to the brain happens in isolation. The connections between drugs and impacts can therefore be subtle, indirect, and often difficult to establish with certainty.

Medications, to-date approximately 30 drugs, used to treat psychosis are "variations on a theme," Lieberman notes. All of them affect dopamine-2 (D2) receptor modulation—they are thought to bind to and block dopamine receptors and to have an anti-psychotic effect. Additional targets have not been developed beyond this receptor. While in their mechanism of action all are similar, antipsychotics do differ in their side effects. Lieberman argues, we know that in some cases combinatorial antipsychotics do depress psychosis, may prevent recurrence, progression, and gray matter volume loss (although some literature contradicts this claim), but these drugs come with enormous costs. They fail to alleviate cognitive defects, refractory psychotic symptoms, and negative symptoms (including social withdrawal, diminished speech and movement, and problems with motivation).

Treatments remain based on trial and error rather than on definitive biomarkers, and clinicians are left with only subjective behavioral measurements to evaluate treatment efficacy. As the research community tries to advance the field from here, Lieberman lamented, "We have our work cut out for us." There certainly has been tremendous scientific effort put forth on all fronts, he admitted, and schizophrenia researchers now appreciate that for the myriad disease symptoms, no single compound and no single molecule can address every aspect of schizophrenia pathology. Instead, Lieberman contends, adjunctive therapies, which involve what he termed a rational platform whereby therapies used in patient-specific combinations, are the future of schizophrenia treatment.

The majority of conference presenters, Lieberman among them, also remarked that the therapeutic answers lie in prevention. Lieberman has observed that when patients are treated early, they often experience much better treatment outcomes, and he has, to this end, focused his career on early detection and identification. However as many presenters noted, using behavior-based identification of high-risk patients is problematic, as diagnoses typically involve a 35% true positive and a 65% false positive rate.

Over the past decade, researchers have extensively debated the hypothesis that decreases in gray matter volume are associated with psychosis, and many studies have linked excess apoptosis to the synaptic pathology of schizophrenia. In question are such fundamental issues as whether changes in gray matter volume constitute the cause or effect of the disease.

A better way to identify risk, Lieberman believes, is through a brain imaging-based approach. His group and others have focused on cellular malfunctions that appear before the severity of the illness sets in, and they have found that not all brain circuits are affected equally by disease progression. He and colleagues have hypothesized that the presence (and severity) of the disease may be indicated by gray matter (GM) reduction in the hippocampus and that initial abnormalities in blood flow occur in specific brain circuits. Looking at the functional rather than the structural results of these changes, his team and others have used positron emission tomography (PET), Blood-Oxygen-Level Dependence functional magnetic resonance imaging (BOLD-fMRI, which relies on the local ratio of oxyhemoglobin to deoxyhemoglobin to determine regional activity in the brain), and gadolinium-enhanced magnetic resonance (MR) imaging in order to measure cerebral blood volume (CBV) changes and metabolic abnormalities occurring in schizophrenia. These non-invasive imaging techniques measure brain metabolism and indicate what parts of the brain are active during specific activities.

In particular, their research has focused on a region of the hippocampus in individuals at an early stage of the disease. Scott Schobel and Scott Small, from Columbia University, discovered CBV differs in CA1 subset of hippocampal neurons in prodomal (pre- or early symptomatic) patients. After undertaking longitudinal studies (that track and measure changes within the same patients over time), Lieberman found that differences in hippocampal volume, shape, and metabolism correlated with risk. In other words, by comparing high-risk individuals with normal controls over time they found that changes in CBV in certain key brain regions correlated with psychosis and that those young people with abnormal cerebral blood flow went on to develop schizophrenia. These results provide evidence of excess blood flow in CA1 as a marker of a high-risk state for developing the disease.

In order to investigate further what specific genes are differentially expressed in patients compared to controls, researchers performed microarray analysis on tissue collected from selected brain regions based on the brain imaging results identifying key regions. Microarray analysis on postmortem CA1 tissue indicated the greatest difference in transcription level occurred in the glutamate dehydrogenase (GDH) gene. Altered levels of glutamine synthase and GDH, the key enzymes involved in glutamine metabolism, have been found in patients with schizophrenia. GDH affects glutamate signaling by regulating glutamate reuptake, and increased GDH enzymatic activity has been found in brain extracts from patients with schizophrenia. Approximately 20 years ago the glutamate metabolism hypothesis (that dysfunctional glutamate metabolism contributes to schizophrenia pathophysiology) began to gain a footing in the literature. Its acceptance by some researchers was in part based on the observation that the psychotomimetic agents phencyclidine (PCP) and ketamine induce psychotic symptoms and neurocognitive disturbances similar to those of schizophrenia, and that they do so by blocking neurotransmission at N-methyl-D-aspartate (NMDA)-type glutamate receptors.

This hypothesis is strongly supported by studies that identified increased expression of glutamate transporter GLT in the prefrontal cortex of patients with schizophrenia. Lieberman's work, along with others' work he reviewed at the conference, provides evidence that impairment of glutamate metabolism and the abnormal functioning of the glutamate-glutamine cycle contribute significantly to schizophrenia. Lieberman sees the future of effective drug design as one focused on tailoring treatments for the illness to each of its developmental stages. Through this approach he hopes that novel treatments will maintain or improve efficacy, involve few if any side effects, and target disease etiology rather than symptoms long after the disease manifests.

P. Jeffrey Conn of Vanderbilt University has been devoted to developing treatments that do just that. Through the investigation of specific cellular and molecular mechanisms involved in chemical and electrical signaling, he has focused on one specific target, the metabotropic glutamate receptor 5 (mGluR5). His group has been investigating a novel approach, based on extensive literature that indicates that increased NMDA glutamate receptor function may directly help in the treatment of schizophrenia and other central nervous system disorders.

As was addressed in Lieberman's presentation, the glutamate/ NMDA hypofunction hypothesis of schizophrenia is supported by observations that decreased levels of glutamate, as well as changes in several markers of glutamatergic function, occur in brain tissue of those diagnosed with schizophrenia. Conn and co-workers have successfully developed a high-throughput screen to identify diverse and highly selective positive allosteric modulators (PAMs) of mGluR5. Importantly, these potentiators of mGluR5 dramatically enhance the response to the endogenous agonist.

Cartoon depicts structure of metabotropic glutamate receptor 5 (mGluR5) G-protein-coupled receptor (GPCR) in the lipid bilayer. The two lower plots show allosteric modulators of mGluR and their concentration-response curves. The normalized fluorescence is plotted against the concentration of glutamate and shows that the 2 components tested potentiate the response to glutamate (generating a 10 fold leftward shift) in a dose-dependent manner.

Moreover, Conn's team has tested many of these compounds in animal models, and these in vivo studies have shown cognitive-enhancing effects in rodents. Interestingly, Conn's team has found that at least two of these PAM compounds are working by quite different allosteric means—mutagenesis and modeling studies show each binding to distinct sites. These studies might therefore result in several selective activators of different G-protein coupled receptors (GPCR) for treatment of schizophrenia. Through continued profiling of these novel compounds, Conn and colleagues are working at the forefront of translational medicine, elegantly demonstrating how basic science is applied to clinical studies.

Stephen J. Haggarty from Harvard Medical School and the Broad Institute argued that uncovering the etiology and pathophysiology of schizophrenia is critical to advancing therapy development. His team has been working on discovering small molecule therapeutics using a chemical genomic approach, specifically probing the DISC1/glycogen synthase kinase-3 (GSK-3) signaling pathway. Based on extensive evidence that DISC1 and GSK—one of its direct binding targets—are disrupted in schizophrenia, Haggarty's group has identified small-molecule probes that target known and novel components of the DISC1/GSK3 pathway. A regulator of neurogenesis and synaptic function, GSK has also been shown to be inhibited by lithium and anti-psychotics in vivo. Accordingly, Haggarty and colleagues used a high-throughput microarray screen in order to identify small molecule probes that selectively target discrete DISC1 binding domains. Their high-throughput methodology has enabled them to identify selective inhibitors of GSK3 and furthermore, has allowed them to begin to dissect the neurobiology of DISC1 variants. By targeting individual domains and functions of DISC1 in this way, they have been able to disrupt specific protein–protein interactions.

Convergence of molecular pathways and targets in bipolar disorder and schizophrenia: A schematic of the signaling pathways disrupted by treatments for schizophrenia and biopolar disorder.

Finally, using patient-specific human neural progenitors from skin biopsies, Haggarty's team has been able to model the pathogenesis and treatment of disease and to functionally assay small molecules (for example, selective inhibitors of GSK3) in an unprecedented high-throughput approach through automated quantitative microscopy. This innovative system of neural progenitors represents a remarkably accurate genetic model in which to study functional pathways in order to improve treatment of schizophrenia.

Speakers:
Amanda J. Law, National Institutes of Mental Health
Brian Campbell, Pfizer

Highlights

• ErbB4 variants that modulate alternative splicing are associated with risk of schizophrenia.
• Downstream modulators of NRG1-ErbB4 signaling introduce PIK3CD as a candidate drug target.
• Scientists characterize new class of KAT II inhibitors of kynurenic acid synthesis, increased in individuals with schizophrenia.

Target practice

The vast body of schizophrenia literature has so far supported the assertion that schizophrenia is a polygenic disorder. Whether risk loci converge to affect a common neural process or to encode proteins within the same or overlapping signaling pathways is not yet known, however. Through genetic linkage and association studies, researchers have identified a growing number of risk genes. One such researcher, Amanda J. Law from the NIH, focuses her work on the genetic basis of neurobiological processes that are altered in schizophrenia. One aspect of her research is centered on the epidermal growth factor (EGF)-family member Neuregulin (NRG1) and its tyrosine kinase receptor ErbB4. NRG1 is a ligand that binds to the ErbB4 receptor, and both play critical pleiotropic roles in neurodevelopment.

Through the genetic dissection of NRG1-ErbB4 signaling networks, Law and others have shown a genetic association of (common and rare) ErbB4 variants with schizophrenia. Law has found, by molecular cloning and cDNA sequencing in human brain tissue, that ErbB4 transcripts undergo complex splicing, generating multiple structurally distinct isoforms and that disease-associated variants in the ErbB4 gene are a result of intronic mutations that may alter splicing of the gene. One of the several structurally and functionally distinct ErbB4 isoforms Law's team has investigated is the cytoplasmic domain (CYT-1) isoform, which has a phosphotidylinositol 3-kinase (PI3K) binding site and is linked to risk for schizophrenia. Regulation of intracellular signaling downstream of ErbB4 CYT-1 was the focus of her presentation at this conference. Law stated that PI3-kinases have been linked to numerous cellular functions through their activation of serine/threonine protein kinase (AKT). PI3-kinases are heteromeric proteins encoded by a family of genes, including PIK3CD/PIK3R3. PI3-kinases affect biological processes ranging from cell growth, proliferation, differentiation, motility, and intracellular trafficking to cell survival. Moreover, studies have identified AKT, which is activated by PI3K, as a signaling intermediate downstream of dopamine (DA) receptor 2, the known target of current antipsychotic drugs.

Law detected an increased expression level of the CYT-1 isoform in the dorsolateral prefrontal cortex (DLFPC) of individuals with schizophrenia. Using several assays including human brain mRNA expression profiling, patient-derived lymphoblastoid cell cultures (LCLs), molecular genetics, and pharmacological studies in rodents, Law tested her hypothesis that the aberrant PI3K/AKT signaling represents a pathogenic consequence of schizophrenia-associated genetic variation in ErbB4 and that pharmacological manipulation of this pathway may represent a novel therapeutic avenue for the treatment of psychosis.

To search for possible functional mutations in ErbB4 that could be analyzed as candidates for PI3K-linked psychiatric problems, Law looked for direct links between genetic variation in the ErbB4 gene and disease. First, in patients with schizophrenia, Law and her team found that disease- and risk-associated polymorphism in ErbB4 predicts increased CYT-1 expression in human LCLs, similar to that seen in the brain. Second, levels of mRNA transcripts of the PI3K gene (PIK3CD/PIK3R3) complex is increased in LCLs of patients and predicted by variation in ErbB4. Third, by comparing mRNA expression in DLPFC to that in the LCLs, Law determined that changes to the levels of phosphotidylinositol 3-kinase (PIK3CD) transcripts are also predicted by ErbB4 genetic variation in human brain. Finally, in LCL cultures, Neuregulin-mediated chemotaxis (cell migration) and intracellular [PI(3,4,5)P3] production is associated with genetic variation in ErbB4 and impaired in schizophrenia. Neuregulin itself has been identified as a candidate gene that confers risk for schizophrenia; several studies have shown that neuregulin-induced stimulation of ErbB4 receptor plays a part in normal neurodevelopment and in expression of several neurotransmitter receptors. In fact, in animal studies, mutations in neuregulin-ErbB4 signaling lead to abnormal behaviors resembling certain features of schizophrenia.

A number of animal models of schizophrenia are used to study psychosis. Several pharmacological means are used to mimic psychosis and certain aspects of dopamine signaling, including treatment with amphetamine. Functionally, in human brain, Law has shown that antipsychotic drugs downregulate PIK3CD, and in proof-of-concept studies using a mouse model, her group has shown that a specific small molecule inhibitor of PIK3CD blocks the effects of amphetamine. Law's group has identified downstream modulators of NRG1-ErbB4 signaling and has introduced PIK3CD as a candidate drug target for future schizophrenia treatments.

New class of inhibitors

An impressive indication of the ongoing shift in the research agenda, from a symptom-based research to investigations seeking to elucidate the underlying molecular neuropathology of schizophrenia, was the assembly of leading researchers from both industry and academia.

Researcher Brian Campbell from Pfizer presented his team's work exploring a new mechanism of treatment based on the kynurenic acid (KYNA) hypothesis. Studies have shown a measured increase of KYNA in a subset of patients and have led to the hypothesis that this biologically active by-product of tryptophan (TRP) metabolism may contribute to cognitive defects of schizophrenia.

KYNA has been reported as an endogenous antagonist of NMDA receptors, and it may also interfere with nicotinic a7 receptor function. Several studies have noted excessive cigarette smoking by schizophrenic patients and have suggested this behavior may be a kind of self-medicating of certain symptoms associated with the disease. In postmortem tissue from schizophrenic individuals compared to controls, morphological alterations in nicotinic receptors were consistent with the molecular studies showing an association between genetic polymorphisms in nicotinic receptors and schizophrenia.

Hallucinogenic drugs, such as PCP and ketamine, produce schizophrenic symptoms and are thought to be antagonists of the NMDA receptor as KYNA is. Previous research that sought to target KYNA synthesis by inhibiting kynurenine aminotransferase II (KATII) was limited by a lack of inhibitor selectivity and potency, and by difficulty delivering the inhibitors to the right targets, among other issues.

Campbell's group presented their discovery and characterization of a new class of KAT II inhibitors that appear to have resolved these brain penetration and potency issues. In a dose-dependent manner, these novel inhibitors were able to rapidly decrease KYNA by as much as 80% with peripheral delivery in rodents. To study the inhibitors' effects more closely, the team profiled the consequences of lowering KAT in rats and primates and measured the drugs' effects on performance in memory, attention, and mood assays. The Pfizer team found that KAT II inhibitors do not interfere with antipsychotic agents and suggested they may be an effective adjunct therapy for treating cognitive defects and negative symptoms in some cases of schizophrenia.

Speakers:
Bita Moghaddam, University of Pittsburgh
Akira Sawa, Johns Hopkins University School of Medicine
Marc G. Caron, Duke University Medical Center
Susan R. George, University of Toronto
John A. Allen, University of North Carolina School of Medicine

Highlights

• Work in animals yields different conclusions from those derived from cellular assays.
• Olfactory neurons may be a good source for genomics studies of schizophrenia.
• Newly discovered D1-D2 heteromerization adds potential diversity to dopamine receptor structure and to related drug design.
• Lipid-raft signaling may be altered in neuronal disease.

Aberrant cellular "powerhouse" as early biomarker

The topics covered in this session continued to promote a transition that has been occurring in the field recently—a move away from a descriptive and behavior-based diagnostic approach, to one based on mechanistic understanding and cellular and systems modeling. In animal model design, this transition is of particular relevance as it is not possible to recapitulate all behavioral aspects of schizophrenia in a model organism, but it may be possible to accurately represent relevant genetic and molecular systems in such an organism. Bita Moghaddam of the University of Pittsburgh has set up functional endophenotypes for these organisms, thereby stepping further away from behavioral assessments of schizophrenia and towards heritable biomarkers of the disease.

Moghaddam began by introducing a model, the disinhibition hypothesis, that has been gaining popularity as a way to explain schizophrenic symptoms. This model posits that schizophrenia results from dysfunction of GABAergic transmission. The model begins with the premise that GABAergic signaling inhibits excitatory glutamatergic pyramidal neurons in the prefrontal cortex. Excitatory afferents stimulate both GABAergic and pyramidal neurons in the cortex but the higher level of activation of the former trumps the excitatory effects on the latter. According to the hypothesis, in schizophrenia reduction of GABAergic transmission results in a disinhibition of excitatory signaling by pyramidal neurons as well as reduced gamma oscillations. Since gamma oscillations are thought to transiently link the activity of distributed cells that are processing related information, information processing would be aberrant in patients with schizophrenia. The disinhibition model arose from early findings that, in patients, both the concentration of cortical GABA and the activity of the enzyme glutamic acid decarboxylase (GAD), which is involved in the rate-limiting step of GABA synthesis, are reduced.

Moghaddam reviewed the three functional endophenotypes associated with this model and noted that these biomarkers had been tested in single cells and in sliced animal brain preparations. First, a reduction in GABA interneuron activity—measured by reduced GAD67, one of GAD's isoforms—led to the activation of pyramidal cells. Second, NMDA receptor inhibition or serotonin receptor stimulation led to increased pyramidal cell activity and to reduced gamma oscillations measured in these pyramidal neurons.

In awake, freely moving animals, however, Moghaddam and co-workers did not observe these phenotypes. Rather, at doses that impair working memory and attention, an inverse agonist of the GABA_A receptor actually suppresses cortical activity and enhances gamma power, even though one would expect the inverse agonist to have the opposite effect on pyramidal cells than does the agonist (GABA).

Collectively, these new findings have contributed to a re-evaluation of an influential cellular phenotype-based hypothesis. Moghaddam's team is now focused on understanding dynamic interactions that occur between whole cortical regions and circuits. Her findings have led her to query fundamental metabolic abnormalities that may underlie cortical activity in schizophrenia. There is a great deal of literature linking mitochondrial dysfunction and pathology in mental illness. Mitochondrial mechanisms that affect excitatory and inhibitory synapses are yet another source on which researchers can draw to help elucidate the etiology of schizophrenia.

Translational neuroscience

In order to detect biomarkers through molecular profiling, functional assays, and drug screening scientists need samples of human neurons. To avoid the limitations of using autopsied brain tissue—confounds of long-term drug exposure, the effects of general substance abuse, missing neurodevelopmental pathology, and the impact of changes that occur to tissue postmortem—several labs have recently begun to use nasal biopsies from clinically diagnosed patients instead.

Based on the observation that abnormalities in odor identification exist in patients with schizophrenia, Akira Sawa, from Johns Hopkins University School of Medicine, and colleagues have performed genome-wide microarray analysis on self-renewing olfactory neurons and on induced pluripotent stem (iPS) cells derived from patients with schizophrenia. In olfactory immature neurons, Sawa has confirmed by gene expression analysis that neuronal markers are highly expressed in this homogeneous population of cells. Conversely, his expression analysis from iPS cells revealed a significantly heterogeneous molecular profile. Furthermore, cell passage frequency, the number of times cells in culture have been moved to a new dish and allowed to re-grow to confluence, affects iPS cellular identity. Based on recent work demonstrating direct conversion of mouse fibroblast to functional neurons Sawa and colleagues have also established human iN (induced neuronal) cells converted from skin fibroblasts.

In the past 3 years genome-wide histone modification information and mRNA expression analyses have revealed that immature olfactory neurons from schizophrenia patients differentially express genes associated with the disease. In particular, Sawa's functional analysis revealed that cytoskeleton-related proteins and cellular stress response genes are differentially expressed in olfactory neurons from patients with schizophrenia. His group's data substantiated recent literature claims identifying glutathione S-transferase theta 2 (GSTT2), an oxidative stress protein, as a marker of schizophrenia, and they found this marker to be decreased in both olfactory neurons and postmortem brain tissue. The next series of validations for these cellular abnormalities will be to compare brain imaging, clinical phenotypes, and metabolic and oxidative stress susceptibility across patient groups, to confirm that the biomarker gives a diagnosis consistent with other symptoms of schizophrenia.

Not your classical GPCR signal

For the past 20 years, much investigation has sought to determine precisely how antipsychotics work in the brain. The majority of (current) antipsychotic drugs have been found to exert their effects on the D2 (dopamine) receptor. Unfortunately, the cognitive defects of schizophrenia are all but resistant to these conventional antipsychotic treatments.

Although most presenters at this conference focused on biochemical underpinnings of schizophrenia rather than on symptom management, Marc G. Caron of Duke University presented an approach for attenuating the symptoms of the disease based on the assertion that antipsychotics interact with the D2R and mediate their physiological effects through at least 2 pathways, classical G protein-dependent as well as independent (β-arrestin 2-dependent) signaling pathways. A few years ago, Caron's group began focusing on the latter when they observed that β-arrestin-2 knockout mice had diminished locomotor response to D2R stimulation. The realization that antipsychotics can invoke G protein-independent signaling and G protein-dependent signaling simultaneously and through distinct effector systems (compared to classical signaling) presented to Caron's team an opportunity to specifically target β-arrestin-dependent pathway (part of G protein-independent signaling) for drug development.

More from dopamine

Of the five dopamine receptor subtypes, the most abundant are the D1 and D2. Localized in the basal ganglia and frontal cortex, D1 and D2 have been recently been observed to form heteromers and as such, to respond to antipsychotics differently compared to their respective homomers. The in vivo oligomerization of D1-D2 receptors was the focus of Susan R. George's presentation. George is a professor of pharmacology and toxicology at the University of Toronto. Using a behavioral, biochemical, and electrophysiological approach, her lab is investigating whether D1-D2 synergy (cooperation) occurs within one neuron or via neighboring neurons.

George hypothesized that if a dopamine D1-D2 heteromer exists it should have a novel intracellular signaling output (compared to a D1-D1 or D2-D2 homomer, respectively). To begin testing this hypothesis, her team cotransfected a cell line with D1-D2 and treated the cells with agonists to these receptors to co-activate both receptors. This simultaneous stimulation generated a dose-dependent calcium signal. Interestingly, the generated calcium signal was dependent on co-activation of D1-D2, as treating with either agonist alone did not generate signal. Moreover, dopamine itself stimulated a calcium signal in D1-D2 cells, but did not elicit a signal in cells containing each receptor alone. Finally, the team found that the calcium signal was inhibited by using either D1 or D2 antagonists.

George is particularly interested in determining how this calcium signaling mechanism is generated, whether it occurs via activation of the cAMP-dependent pathway (Gs/PKA), inhibition of cAMP production (Gi/P13K/PKC), or activation of phospholipase C (Gq/PLC). Blockers of PLC were found to abolish the D1-D2 calcium signal, whereas inhibitors of PKA, AC, or PKC did not. Therefore George contends that D1-D2 agonists mediate intracellular calcium release in the PLC pathway through Gq, and they showed that this complex could be co-immunoprecipitated from cells and rat striatum. In order to verify that these receptors were in fact cooperating in the same complex they developed highly selective tools to visualize each receptor alone.

They then generated a series of ultra-specific antibodies that demonstrated colocalization of D1-D2 in cultured striatal neurons and in rat cortices. In addition, confocal imaging with fluorescence resonance energy transfer (FRET) on endogenous D1R and D2R, in several brain regions, showed both receptors in the same cell approximately 50Å-60Å apart. The highest densities of D1-D2 co-expressing neurons were measured in the shell and core of the nucleus accumbus and in the globus pallidus, both areas that are functionally disrupted in schizophrenia.

The discovery of D1-D2 heteromerization adds a new dimension to our view of dopamine receptor structure. George concluded that pharmacologically and functionally new approaches to therapeutic drug design must be cognizant of these receptor heteromers as they will undoubtedly be beneficial for the future of schizophrenia treatment.

What makes up half the human brain (in dry weight)?

The final presentation at the conference offered a unique perspective on specialized submicroscopic structures of lipid and cytoskeleton, so called "lipid rafts" and proposed a mechanism by which they may be altered in psychiatric diseases. Neural transporters and receptors have been isolated from lipid rafts, which suggests a deeper role for these structures in neurotransmitter signaling. In particular, these membrane "microdomains" are thought to be involved in endocytosis and trafficking during neuronal signaling.

The lipid raft signaling hypothesis proposes that these microdomains spatially organize signaling molecules at the membrane, perhaps in complexes, either to promote kinetically favorable interactions that are necessary for signal transduction or to inhibit interactions by separating signaling molecules and thereby dampen signaling responses. John A. Allen from Brian Roth's laboratory at the University of North Carolina, presented his work on one key component of the membrane scaffolding machinery, Caveolin-1 (encoded by the CAV1 gene) in a mouse knockout (Cav-1 KO) paradigm.

In 2008, a rare structural copy number variant of CAV1 was identified as a gene associated with risk for schizophrenia. Previous work had demonstrated a structural role for the Cav-1 protein in scaffolding serotonin and dopamine receptors. Allen tested in an animal model the hypothesis that loss of Caveolin-1 might be involved in psychotic behavior. Comparing Cav-1 KO animals to wild-type animals, he measured the distribution and number of serotonin receptors, the activity of receptor-mediated signaling, and behavioral responses to PCP, antipsychotics, and serotonin receptor agonists. With autoradiographic localization measurements in prefrontal cortex, Allen found that the Cav-1 KO appeared to disrupt neither the distribution nor the number of serotonin receptors. However, levels of serotonin-receptor mediated calcium signaling were reduced in Cav-1 KO cortical neurons compared to their wild-type counterparts. Allen found that Cav-1 KO mice had increased sensitivity to PCP in locomotion and paired-pulse inhibition (PPI) studies and that clozapine (an atypical drug) was less effective as an antipsychotic in the KO mice. Allen therefore concluded that caveolin-1 facilitates serotonin functioning in vivo and is required for the action of atypical antipsychotics in mice.