The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSCderived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active nonphosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2:CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the "lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknownmight reveal otherwise inscrutable intracellular pathogenic pathways. have proven valuable for studying the molecular pathology of monogenic diseases, one of the technique's greatest challenges has been to offer similar insights into the molecular pathogenesis of polygenic, multifactorial disorders for which the underlying pathophysiology is unknown. The struggle has been to go beyond phenotypic description to discerning underlying molecular mechanisms. Neuropsychiatric illnesses are a prototype for such complex conditions (1-3). They are difficult to model not only because of the likelihood of polygenic influences, but also because of the subjectivity with which these diseases must often be diagnosed, the empirical fashion with which drugs are prescribed, and the heterogeneity of patient response. Of such maladies, bipolar disorder
Lesch-Nyhan disease (LND) is an X-linked genetic disorder caused by mutations of the hypoxanthine guanine phosphoribosyltransferase (HPRT) purine biosynthesis gene and characterized by aberrant purine metabolism, deficient basal ganglia dopamine levels, dystonia, and severe neurobehavioral manifestations, including compulsive self-injurious behavior. Although available evidence has identified important roles for purinergic signaling in brain development, the mechanisms linking HPRT deficiency, purinergic pathways, and neural dysfunction of LND are poorly understood. In these studies aimed at characterizing purinergic signaling in HPRT deficiency, we used a lentivirus vector stably expressing an shRNA targeted to the HPRT gene to produce HPRT-deficient human CVB induced pluripotent stem cells and human HUES11 embryonic stem cells. Both CVB and HUES11 cells show >99% HPRT knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA and protein down-regulation by HPRT knockdown is refractory to activation by the P2Y1 receptor agonist ATP and shows aberrant purinergic signaling, as reflected by marked deficiency of the transcription factor pCREB and constitutive activation of the MAP kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells also demonstrate marked reduction of phosphorylated β-catenin. These results indicate that the housekeeping gene HPRT regulates purinergic signaling in pluripotent human stem cells, and that this regulation occurs at least partly through aberrant P2Y1-mediated expression and signaling. We propose that such mechanisms may play a role in the neuropathology of HPRT-deficiency LND and may point to potential molecular targets for modulation of this intractable neurological phenotype.neurodevelopment | neurotransmitters C omplete genetic deficiency of the enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) causes LeschNyhan disease (LND), an intractable neurobehavioral disorder characterized by hyperuricemia, cognitive impairment, dystonia with spasticity choreoathetosis and compulsive self-mutilation (1). The enzyme HPRT plays a major role in salvage purine biosynthesis by catalyzing the conversion of hypoxanthine and guanine to the nucleotides inosine monophosphate (IMP) and guanosine monophosphate (1, 2). HPRT deficiency leads to the hallmark biochemical defect in LND: greatly increased de novo purine synthesis, with resulting elevated levels of oxypurines and tissue accumulation of uric acid, gout, and life-threatening nephrolithiasis (3, 4). Although treatment with the xanthine oxidase inhibitor allopurinol effectively prevents hyperuricemia and renal damage in LND and thereby markedly extends the lifespan of patients, neither allopurinol nor any other treatment produces lasting improvement in neurological manifestations (5, 6).PET scanning and postmortem studies of brains from patients with LND have demonstrated decreased levels of dopamine, dopamine biosynthetic enzymes, and dopamine uptake in the basal ganglia (...
Cancer chemopreventive activity of sulforaphane has been predominantly associated with its ability to induce phase II detoxification enzymes. In the present study, novel targets of sulforaphane were identified and characterized using a proteomics approach. Two-dimensional gel electrophoresis and mass spectrometry were used to produce protein profiles of human colon adenocarcinoma Caco-2 cells treated with 5 Mmol/L sulforaphane for 48 h and control cells (0.05% DMSO). Gel comparisons showed the down-regulation to undetectable level of the serotonin receptor 5-HT 3 after sulforaphane treatment. In addition, Aldo-keto reductase and D-dopachrome decarboxylase were also differentially expressed in control and treated cell extracts. To elucidate two-dimensional gel findings, the neurotransmitter receptors 5-HT 3A , 5-HT 1A , 5-HT 2C , and the serotonin reuptake transporter were analyzed using Western blotting. Results showed a decrease of neurotransmitter receptors in a dose-dependent manner after sulforaphane treatment. Moreover, after exposure of Caco-2 cells to sulforaphane, nicotinic acetylcholine receptor protein level was increased. These findings suggested a potential effect of sulforaphane on serotonin release. Activation of neurotransmitter receptors followed by initiation of cyclic AMP signaling might be crucial events in colon carcinoma progression. Thus, the effect of sulforaphane may help to elucidate signaling pathways serotonin-mediated in colon cancer and lead to development of potential novel therapeutic agents. [Cancer Res 2008;68(13):5487-91]
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