Rajiv P. Sharma - US grants

From the animal literature, we know that chronic neuroleptic treatment attenuates the heightened dopamine activity seen with acute treatment, a phenomenon usually described as 'tolerance'. However, a review of longitudinal studies in schizophrenic patients, examining the response of the major dopamine metabolite homovanillic acid (HVA) to chronic neuroleptic treatment, suggests two patterns of metabolic response. The first pattern is sometimes characterized by an acute increase in HVA, but followed more generally by a gradual decrease of the HVA level to within or below the range of the pretreatment value. The second pattern is best characterized by continued heightened production of HVA during chronic treatment. Further, the first pattern is usually associated with a good clinical outcome, while the second pattern with a poor outcome. Concomitantly, there is also evidence to suggest an abnormality in serotonergic functioning in schizophrenic patients. Thus, the reported therapeutic effects of various serotonergic agonists and antagonists, the antidopaminergic side effects of high potency serotonergic antidepressants, and the efficacy of clozapine indicate a significant serotonergic involvement both in the pathophysiology of schizophrenia as well as the modulation of dopaminergic activity. We hypothesize that the inability of a subsample of schizophrenic patients to reduce their dopamine activity with chronic neuroleptic treatment (i.e., nontolerance) indicates the dysfunctioning of an internal regulatory system providing long loop feedback inhibition that may include nondopaminergic neurotransmitter systems such as serotonin. The tolerance/nontolerance paradigm may identify a biochemically homogeneous subgroup of patients who have abnormalities in serotonergic activity. We will identify a subgroup of patients who demonstrate 'nontolerance' to treatment with steady state plasma levels of haloperidol for four weeks by measuring CSF and plasma HVA. We will compare these patients with those that show the expected 'tolerance' pattern, on differences in clinical response, and several peripheral and central measures of serotonergic activity. We will compare platelet 5-HT2 receptors, platelet 5-HT content, and the endocrine (prolactin), metabolic (plasma HVA), and behavioral response to fenfluramine.

From the animal literature, we know that chronic neuroleptic treatment attenuates the heightened dopamine activity seen with acute treatment, a phenomenon usually described as 'tolerance'. However, a review of longitudinal studies in schizophrenic patients, examining the response of the major dopamine metabolite homovanillic acid (HVA) to chronic neuroleptic treatment, suggests two patterns of metabolic response. The first pattern is sometimes characterized by an acute increase in HVA, but followed more generally by a gradual decrease of the HVA level to within or below the range of the pretreatment value. The second pattern is best characterized by continued heightened production of HVA during chronic treatment. Further, the first pattern is usually associated with a good clinical outcome, while the second pattern with a poor outcome. Concomitantly, there is also evidence to suggest an abnormality in serotonergic functioning in schizophrenic patients. Thus, the reported therapeutic effects of various serotonergic agonists and antagonists, the antidopaminergic side effects of high potency serotonergic antidepressants, and the efficacy of clozapine indicate a significant serotonergic involvement both in the pathophysiology of schizophrenia as well as the modulation of dopaminergic activity. We hypothesize that the inability of a subsample of schizophrenic patients to reduce their dopamine activity with chronic neuroleptic treatment (i.e., nontolerance) indicates the dysfunctioning of an internal regulatory system providing long loop feedback inhibition that may include nondopaminergic neurotransmitter systems such as serotonin. The tolerance/nontolerance paradigm may identify a biochemically homogeneous subgroup of patients who have abnormalities in serotonergic activity. We will identify a subgroup of patients who demonstrate 'nontolerance' to treatment with steady state plasma levels of haloperidol for four weeks by measuring CSF and plasma HVA. We will compare these patients with those that show the expected 'tolerance' pattern, on differences in clinical response, and several peripheral and central measures of serotonergic activity. We will compare platelet 5-HT2 receptors, platelet 5-HT content, and the endocrine (prolactin), metabolic (plasma HVA), and behavioral response to fenfluramine.

From the animal literature, we know that chronic neuroleptic treatment attenuates the heightened dopamine activity seen with acute treatment, a phenomenon usually described as 'tolerance'. However, a review of longitudinal studies in schizophrenic patients, examining the response of the major dopamine metabolite homovanillic acid (HVA) to chronic neuroleptic treatment, suggests two patterns of metabolic response. The first pattern is sometimes characterized by an acute increase in HVA, but followed more generally by a gradual decrease of the HVA level to within or below the range of the pretreatment value. The second pattern is best characterized by continued heightened production of HVA during chronic treatment. Further, the first pattern is usually associated with a good clinical outcome, while the second pattern with a poor outcome. Concomitantly, there is also evidence to suggest an abnormality in serotonergic functioning in schizophrenic patients. Thus, the reported therapeutic effects of various serotonergic agonists and antagonists, the antidopaminergic side effects of high potency serotonergic antidepressants, and the efficacy of clozapine indicate a significant serotonergic involvement both in the pathophysiology of schizophrenia as well as the modulation of dopaminergic activity. We hypothesize that the inability of a subsample of schizophrenic patients to reduce their dopamine activity with chronic neuroleptic treatment (i.e., nontolerance) indicates the dysfunctioning of an internal regulatory system providing long loop feedback inhibition that may include nondopaminergic neurotransmitter systems such as serotonin. The tolerance/nontolerance paradigm may identify a biochemically homogeneous subgroup of patients who have abnormalities in serotonergic activity. We will identify a subgroup of patients who demonstrate 'nontolerance' to treatment with steady state plasma levels of haloperidol for four weeks by measuring CSF and plasma HVA. We will compare these patients with those that show the expected 'tolerance' pattern, on differences in clinical response, and several peripheral and central measures of serotonergic activity. We will compare platelet 5-HT2 receptors, platelet 5-HT content, and the endocrine (prolactin), metabolic (plasma HVA), and behavioral response to fenfluramine.

DESCRIPTION (provided by applicant): The GABA intemeuron plays a central role in integrating cortical output and is emerging as a major locus of schizophrenia molecular pathology. Reelin (RELN), GAD67 and DNA methylating enzyme (DNMT1) are all expressed in GABA interneurons, and are each abnormally regulated in schizophrenic brain. Further, DNMT1 is a critical component of the epigenetic regulatory cascade, now shown to play an essential role in neuronal gene expression. We have earlier reported on the effects of epigenetic modifiers (DNA methylation, chromatin structure, and methylated-DNA binding proteins) on the levels of RELN and GAD67 expression. This career development award will extend these observations using primary neuronal cultures (PNC) to describe variations in epigenetic modifications of the RELN and GAD67 promoters resulting from pharmacological treatment and membrane depolarization. Supporting the use of PNCs, we and others have observed El4 mouse neuron cultures to be predominantly GABAergic in phenotype (co-expressing RELN, GAD and DNMT1). Our first objective, using these homogenous neuronal cultures, will be to analyze DNA methylation, local chromatin structure and promoter DNA-protein interactions in response to pharmacological treatments with known epigenetic effects, specifically L-methionine, a hypermethylating stimulus, and histone deactylase (HDAC) inhibitors such as valproic acid. Supporting this first objective is our earlier demonstration that L-methionine down regulates RELN and GAD67 mRNA expression as a consequence of epigenetic modifications along their promoters, and this is reversed by the application of valproic acid. Our second objective will be to examine the effects of membrane depolarization on epigenetic modification of depolarization inducible promoters within these GABAergic neurons. We will apply antisense oligonucleotide strategies to down regulate epigenetic repressor proteins such as DNMT1 and MeCP2 to dissect their contribution to depolarization induced gene transcription. Supporting this second objective, we have now shown that antisense induced down regulation of DNMT1 mRNA and protein will result in significant increases in RELN mRNA expression. Also, we have preliminary data indicating robust changes in GAD67 and DNMT1 mRNA in response to depolarization. With this KO1 career development award, the candidate seeks training in focused methods in the molecular study of epigenetic gene regulation. Because epigenetic mechanisms are modifiable by drug interventions, this work has immediate relevance to the development of novel therapeutics.

DESCRIPTION (provided by applicant): Chromatin is a complex of histone proteins and genomic DNA, and serves to transduce the impact of environmental events on the transcriptome by either restricting or permitting the attachment of DNA binding proteins to the regulatory sequences of a gene. The H3K9 histone bimodal switch is pivotal in assembling either 'restrictive' (DimH3K9) or 'permissive' (AceH3K9) chromatin to gene-rich regions of the genome. The central hypothesis for this proposal is that, in schizophrenia, chromatin equilibrates towards a more restrictive state. This has direct clinical consequences because genomic DNA sequestered with restrictive chromatin, is inefficiently transcribed, may explain the less than optimal clinical response to synaptic psychopharmacology, and also because histone covalent modifications can be targeted with small molecule pharmacology. The specific aims in-toto will investigate the hypotheses: a) the H3K9 switch is differentially in the restrictive state (DimH3K9) in schizophrenia patients in both blood and brain; b) measurement of peripheral 'blood levels' of H3K9 in living patients will identify a homogeneous sample with lower levels of the permissive state (AceH3K9) that will respond preferentially to a clinical HDAC inhibitor such as valproic acid. Chromatin structure and function will be examined in both, postmortem brain samples from the Harvard brain collection, and circulating blood mononuclear cells (PBMC) from living patients obtained from three diagnostic groups; i) normals, ii) schizophrenia (both first episode and chronic), iii) bipolar disorder. In the postmortem brain, we will apply genome-wide molecular techniques (ChIP-seq) to survey and analyze the distribution of DimH3K9. Data from the ChIP-seq experiments on postmortem brain will reveal gene networks that are entrained by this restrictive chromatin and have repressed mRNA transcription. Accordingly, mRNA output from DimH3K9 repressed networks will be verified and compared across diagnostic groups using qRT-PCR. In living patients, we will quantify 'blood levels' of both DimH3K9 and AceH3K9 from nuclear extracts of blood mononuclear cells; those subjects with the lowest levels of AceH3K9 (and thereby possessing higher levels of restrictive chromatin) will be randomized to a controlled trial with the HDAC inhibitor valproic acid (with gabapentin as anticonvulsant control) to explore the therapeutic potential of medications capable of 'relaxing' chromatin. At the end of this project, we would have conducted a survey of chromatin assemblies in two major mental disorders and be positioned to answer several questions relating to epigenetic gene regulation in a clinical population. Equally important we begin the translation of published findings from cell, animal and post-mortem brain investigations into living clinical populations in an area with great theoretical and therapeutic implications. PUBLIC HEALTH RELEVANCE: Chromatin is the platform on which the genetic code is regulated and interpreted, and consequently plays an extremely important role in the functioning of any cell including the brain neuron. This study will extend exciting findings from the basic science laboratory that strongly implicates chromatin abnormalities in the schizophrenia disease process, and attempt to study these mechanisms in living patients. If schizophrenia is associated with abnormalities in chromatin organization, this new approach will open the door to a whole new type of psychiatric medication, i.e., drugs that relax chromatin structure and allow a more efficient working of the genetic code.