Patient-Derived Stem Cells for Phosphoproteomic Profiling Neuropsychopathology


Principal Investigator: Dieter A Wolf
Abstract: DESCRIPTION (provided by applicant): ): Bipolar disorder (BPD) is a neuropsychiatric condition defined by a lifetime of relapsing &remitting manic &depressive episodes. This mood disorder has been shown to have strong genetic linkage with familial predisposition. A major impediment to proper diagnosis has been such confounders as the prevalence of substance &alcohol abuse/dependence among those meeting criteria for BPD. Substance abuse, intoxication &withdrawal may elicit mood episodes that recapitulate bipolar phenotypes. Typically, at least 6-12 months of abstinence from substances is required to diagnose a mood episode as underlying BPD. Clinical practice often resorts to early psychotropic medication because of potentially extreme irritability &impulsive suicidal actions. Lithium has been the standard of treatment for BPD, but, because of its side effects, anti-convulsants (including valproic acid, lamotrigine, topiramate &carbamazepine) &atypical anti-psychotics have also been prescribed. The underlying etiology &mechanisms of disease therapy are poorly understood {Rosenberg, 2007 #160}. Adequate laboratory (including animal) models have been difficult to establish {Fornito, 2009 #161}. Microarray analyses have been performed on post-mortem brain samples &compared with controls &patients with schizophrenia, but no clear distinction between neural subtypes, glia, &surrounding vascular cells has been evident (Kim &Webster, 2008). There is no consensus on gene expression patterns linked to BPD &, hence, very little insight into underlying molecular mechanisms. Protein kinase pathways may be altered. Lithium alters MEK &ERK phosphorylation {Pardo, 2003 #162}, decreases CREB phosphorylation &activity of CaM kinase IV {Tardito, 2007 #163} in rat hippocampal neurons. Lithium &valproate may reduce phosphorylation of rat GluR1 {Du, 2008 #164}. Lithium correlates with reduced phosphorylation of the NMDA receptor subunit NR2B in rat cortical neurons {Hashimoto, 2002 #159}. Studies in rat brains have also suggested that lithium administration reduces translocation of Protein Kinase C from the cytosol to the cell membrane {Hahn, 1999 #158}, &inhibits GSK3 activity in mice (Catapano &Manji, 2008). Given the diversity of implicated kinase pathways in BPD and limited by obstacles in studying human neuropsychiatric diseases, we sought to develop a representative, predictive model system to explore regulation of protein phosphorylation in neural cells that most faithfully recapitulates underlying defects of actual human BPD. Recent advances have allowed the conversion of patient-specific human fibroblasts to human induced pluripotent stem cells (hIPSCs), cells which become sufficiently dedifferentiated to a primordial developmental stage that they now emulate human embryonic stem cells (hESCs) in terms of their ability to give rise to the 3 primitive embryonic germ layers and following various differentiation protocols, to more mature, tissue- and organ-specific cell types, including those in the neural lineage. Our group not only has the ability to generate hIPSCs from fibroblasts from normal individuals &from those carrying difficult-to-model diseases, but has actually done so for some neurogenetic/neuropsychiatric entities, e.g., Rett Syndrome, &has differentiated them to neural lineages. We propose to generate hIPSCs from a well-defined subset of BPD patients (i.e., the lithium-responsive subpopulation) in order to begin more faithfully modeling BPD from a rigorous molecular mechanistic perspective using material derived from actual patients. We will include control cell lines from unaffected patients as well as patients with neurologic or psychiatric disorders that are not BPD. At the core of beginning to understand the molecular basis of BPD - and an aspect for which hIPSCs would be particularly well-suited &informative - is a better understanding of changes in the expression of key proteins, particularly their phosphorylation state, at the level of the proteome &phosphoproteome. Our team has been particularly adept at using phosphoproteomic analysis of hESCs (the 1st such analysis in the field) to identify key (including novel) drugable signal transduction pathways that influence neural differentiation. We believe we can apply a similar approach to hIPSCs from BPD patients. In other words, we hypothesize that identification of proteomic &phosphoproteomic differences between hIPSC-derived neurons from BPD vs. unaffected controls or controls with other neuropsychiatric disorders will help elucidate pivotal, diagnostic, and potentially drugable molecular mechanisms underlying BPD. Multidimensional liquid chromatography (MDLC) tandem mass spectrometry (MS/MS) is a powerful tool for analysis of proteomes, phosphoproteomes, and is a strength of the Burnham Institute. MDLC-MS/MS will be used to analyze the proteomes and phosphoproteomes of normal and patient-derived hIPSCs and their neural derivatives. We will use refined bioinformatic tools (another Burnham strength) to glean critical differences among the cell types. We will quantify total cellular proteins, with a particular focus on site- specific phophorylation. This large-scale analysis will likely yield a number of candidate molecular differences between control &BPD cells, any of which might suggest improved methods of diagnosis &treatment. While we will be able to perform follow-up analyses on only a few proteins predicted to be key to control of pluripotency, neural differentiation and neural function, this valuable dataset will be made available to the broader research community so that complementary parallel studies may be launched on the basis of these proteomic &phosphoproteomic results. PUBLIC HEALTH RELEVANCE: Bipolar disorder (BPD) is a severe and prominent societal malady with poorly understood etiology. Proteomic and phosphoproteomic technology is a powerful analytical platform allowing unbiased identification of molecular profiles of healthy and diseased cells, e.g. those from normal and BPD patients. We propose to merge the application of induced pluripotent cells (iPSCs) from BPD patients with comprehensive proteomic/phosphoproteomic analyses of these iPSCs and their neural derivatives, to discover molecular underpinnings of the abnormalities in BPD patients, as well as potential targets for improved diagnosis and treatment.
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