where an individual protein may bear mutations that initiate a disease process, usually the coordinated dysregulation of multiple downstream genes and proteins results in the disease phenotype. Methods for analysis and comparison of the expression of multiple genes and proteins from normal and disease tissue are required to establish the set of genes that are coordinately regulated in the disease process. An added complication to this analysis is that individual diseased cells are often mixed in a heterogeneous population of cell types making it difficult to analyze the diseased cells independently of other cells. In addition, there is a paucity of well-characterized human tissue for molecular analysis of human disease states. In order to address these issues, methods have been developed in the last several years that allow the analysis of complex mRNA populations from discreet amounts of tissue, down to single cells and subregions of single cells. Additionally these methods can be applied to fixed human tissue specimens making it possible to analyze the mRNA compliment of pathological tissue samples. This presentation will high-light the methods utilized for analysis of complex mRNA populations using schizophrenia and Alzheimer’s Disease as examples. Problems associated with analysis of microarray data sets for multigenic diseases and the necessary confirmatory experiments will be discussed.
193. GENES REGULATED BY
ANTIDEPRESSANT TREATMENTS
R.C. Thompson (1) and J.F. Lo´pez (1,2)
(1) Department of Psychiatry, (2) Mental Health Research Institute, The University of Michigan, Ann Arbor, MI 48109-0512
Treatment strategies for depression have employed a host of pharmaco-logical agents known to alter multiple neurotransmitter and neuropeptide systems. These agents include lithium, tricyclic antidepressants (TCAs), and specific serotonin receptor inhibitors (SSRI). Further, the clinical efficacy of antidepressants requires a long treatment prior to the onset of improved symptoms. While these agents are targeted to specific neuro-transmitter systems (e.g., serotonin), the beneficial actions of these antidepressants appear to be the result of direct and/or indirect regulation of other neurotransmitter/neuropeptide systems and/or signal transduc-tion mechanisms. Additransduc-tionally, the time course of these effects is consistent with the hypothesis that the benefits of antidepressant treat-ments are the result of gene regulatory events.
Across the past several years, molecular biology technologies have developed that permit investigators to evaluate the gene expression patterns of thousands of genes. These advances represent a significant leap forward in our ability to evaluate total biological systems where subtle changes in the activity or amounts of multiple molecules can lead to complex physiological responses. One such molecular strategy is represented by gene arrays. Using gene arrays we have identified rodent genes whose activity is altered by chronic antidepressant administration. These antidepressant-regulated genes fall into many biochemical catego-ries including transcription factors, growth factors, and metabolic en-zymes as well as cell surface proteins. The genes regulated by antide-pressants appear to be regulated in a brain region specific fashion. We will present our array data and our strategy for refining lists of genes involved in antidepressant action within these specific brain regions.
194. ABNORMAL REGULATION OF
EXPRESSED GENES IN HUMAN BRAIN
DISEASES REVEALED USING cDNA
MICROARRAYS
J. Pevsner
Dept. of Neuroscience, Kennedy Krieger Institute and Johns Hopkins School of Medicine, 707 N. Broadway, Baltimore MD 21205 For many disorders of the human brain the primary genetic defects are not known. Additionally, a variety of secondary changes in gene expression may occur in these diseases as the brain compensates for the disruption of some pathway perturbed by the primary gene defect(s). For human disorders that affect cognition or other higher mental functions, animal models may be inadequate because they cannot accurately represent the pathological changes. Human brain biopsy material is not available except in extreme cases involving invasive surgery. Thus, an important approach to studying human brain disorders is to analyze postmortem brain samples. We have measured gene expression in brain samples from patients with autistic disorder (n 512) and a related pervasive developmental disorder, Rett Syndrome (n59) as well as age-, gender-, and regionally-matched controls. Rett Syndrome is caused by mutations in the gene encoding MeCP2, a transcriptional repressor, and the neuropathology may result from inappropriate over-expression of genes in brain. We measured the expression levels of up to 20,000 genes using the Atlas (CLONTECH Laboratories), GeneFilters (Research Genetics) and Micromax (NEN Life Sciences) and UniGEM V (Incyte Pharmaceuticals) cDNA microarrays. We performed control experiments in normal human brain samples to measure differences in expression profiles based upon factors such as age at death, gender, brain region, postmortem interval, and comparison of gene expression in brain to astrocytes and to fibroblasts. In Rett Syndrome, we found a consistent up-regulation of glial and other genes using multiple microarray tech-niques. These changes were confirmed by PCR, Western blotting, and by protein microsequencing of postmortem Rett Syndrome brains.
Application of Sensitive Differential Display
Methodologies to Study Gene Expression in
Neuropsychiatric Research: Promise and Pitfalls
Friday, May 12, 2:30 PM–5:00 PM
Location: Regency B
Chair: Husseini K. Manji
Co-Chair: L. Trevor Young
195. CAN DIFFERENTIAL DISPLAY MAKE
A DIFFERENCE IN BIOLOGICAL
PSYCHIATRY?
M.I. Strakhova, P. Skolnick
Lilly Research Laboratories, Eli Lilly and Company
The past decade has been marked by a dramatic increase in the availability of techniques to identify and clone genes that can be differentially expressed by drug treatments and pathologies. While there are remarkable success stories, it is becoming more and more apparent that each of these techniques has its limitations. Given that these methods (e.g., differential display, RNA fingerprinting, suppression subtractive
Friday Abstracts BIOL PSYCHIATRY 59S
hybridization, microarrays) are labor-intensive and potentially time-consuming, it is important to understand their limitations. For example, using a differential display procedure (which is probably the most sensitive among the above mentioned methods) we detected less than a 2-fold change in the expression of mRNA encoding cytochrome b following chronic, but not acute treatment with imipramine (N.-Y. Hung, M. Strakhova, R.T. Layer and P. Skolnick,J. Mol. Neurosci., 1997;9: 167–176) based on analysis of Northern blots. This effect was restricted to cerebral cortex and was not observed following chronic treatment with non-antidepressant drugs (e.g., haloperidol and morphine). However, differential display is biased toward highly abundant RNA species and may be unable to detect differences in rare messages. Conversely, methods like suppression subtractive hybridization are capable of detect-ing changes in rare transcripts but lack the sensitivity of differential display and related techniques. Moreover, both the functional and morphological organization of the central nervous system presents complexities that may not be encountered in other disciplines. Advan-tages and disadvanAdvan-tages of various approaches and their applicability to research in biological psychiatry will be discussed.
196. MOOD STABILIZERS INCREASE
NEUROGENESIS AND NEURITE
OUTGROWTH
G. Chen, G.J. Moore, and H.K. Manji
Laboratory of Molecular Pathophysiology, Dept. Psych. & Behav. Neurosci. WSU School of Medicine, Detroit, MI
It has become increasingly appreciated that the long term treatment of MDI involves the strategic regulation of gene expression in critical neuronal circuits. We have undertaken an extensive series of studies using differential display to identify genes regulated by both Li and VPA. These are two structurally highly dissimilar agents; although they likely do not exert their therapeutic effects by precisely the same mechanisms, identifying the genes which are regulated in concert by these two agents, when administered in a therapeutically relevant paradigm, may provide important leads about the molecular mechanisms underlying mood stabilization. Several novel candidates for the therapeutic actions of mood stabilizers have been identified, including an mRNA binding protein, which increases the levels of a kinase known to play a critical role in cytoskeletal remodeling. We have also found that lithium produces a marked increase in the expression of the neuroprotective protein bcl-2 in frontal cortex, hippocampus and striatum. Accompanying these effects, lithium not only robustly protects neurons from a variety of insults, but also increases neurogenesis in the dentate gyrus of adult rodents. Consistent with such neurotrophic effects, lithium also increases the levels of NAA (N-acetylaspaprtate, a marker of neuronal viability) in the brains of patients with MDI. We have also found that VPA robustly increases neurite outgrowth in cultured human neuroblastoma cells. Taken together, our data suggest that chronic lithium and VPA bring about persistent morphological changes in the brain, effects, which may play a major role in their long term therapeutic effects.
Supported by NIMH, Stanley Foundation, NARSAD and Joseph Young Sr. Awards
197. REGULATION OF MOOD STABILIZER
REGULATED GENES
J.F. Wang, X.J. Sun, and L.T. Young
McMaster University, Department of Psychiatry and Behavioural Neurosciences, Hamilton, Ontario, Canada L8N 3Z5
Mood stabilizers such as lithium and valproate are highly effective treatments for bipolar disorder (BD). Despite many years of investiga-tion, the mechanism of their therapeutic action is not yet clear. Recently, an increasing body of evidence has demonstrated effects of both drugs on signal transduction pathways. These signaling molecules trigger changes in gene expression which are thought to contribute to the effects of chronic treatment with these drugs. Differential display PCR and cDNA microarray were used to identify genes regulated by chronic treatment with lithium and valproate in rat cerebral cortex and rat C6 glioma cells. The expression of a variety of genes were altered by chronic treatment with lithium including: CNPase II, c-jun, M-ras, TGF-beta type II receptor, IGF-I-R alpha and presenilin-1 gene expression, suggesting novel targets for lithium that may be relevant to its mechanism of action. We also found that chronic treatment with valproate increased both mRNA and protein levels of 78-kilodalton glucose-regulated protein (GRP78). Since GRP78 performs molecular chaperone activities, partic-ipates in protein trafficking, and binds Ca21in the endoplasmic reticulum
as well as protecting cells from the deleterious effects of damaged proteins, the present findings suggest that valproate treatment may regulate one or more of these processes. A number of novel cDNAs have also been identified which are being characterized and studied further with 59RACE-PCR and library screening. These results may further our understanding of the mechanism of action of mood stabilizers, and identify new targets for genetic studies and therapeutic strategies in BD. This work was supported by a grant from the Stanley Foundation (L.T.Y) and a grant from the Canadian Psychiatric Research Foundation (J.F.W.)
198. PROGRAMS OF GENE EXPRESSION IN
HUMAN FIBROBLASTS FROM PATIENTS
WITH MAJOR DEPRESSION
S.P. Rossby, S. Liang, D.H. Manier, R.C. Shelton
and F. Sulser
Departments of Psychiatry and Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-2647
Results from our laboratory have provided compelling evidence that human fibroblasts, a nonneuronal tissue, provide a relevant model of signal transduction in affective disorders: (1) they express neuronal genes encoding aminergic receptors, G proteins, and the effector enzymes that produce second messengers and protein kinases; (2) transfected with the luciferase transporter gene pAD neo2-C12-BGL fibroblasts illustrate the amplification mechanism of signal transduction mediated by the beta adrenoceptor i.e., the cyclic AMP-PKA-CREB cascade; (3) fibroblasts from patients with major depression show a blunted beta adrenoceptor mediated PKA response and a significant reduction in nuclear CREB-P versus fibroblasts from normal control subjects; (4) in addition to their effects on receptor mediated signal transduction cascades in fibroblasts, antidepressants affect the cytoplasmic-nuclear trafficking of transcription factors (e.g. GR). Previously we have hypothesized that transcription factors modify programs of gene expression, the ultimate effect of agonist-receptor activation. Presently we are utilizing the Differential Display technology (developed by Liang and Pardee in 1992) to compare the simultaneous expression of approximately 10,000 genes in fibroblasts
60S BIOL PSYCHIATRY Friday Abstracts
