Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

DNAzyme-Based Lithium-Selective Imaging Reveals Higher Lithium Accumulation in Bipolar Disorder Patient-Derived Neurons.

Lithium has been a drug for bipolar disorders (BD) for over 70 years; however, its usage has been limited by its narrow therapeutic window (between 0.6 and 1.2 mM). Understanding the cellular distribution of lithium ions (Li+) in patient cells will offer deep insight into this limitation, but selective imaging of Li+ in living cells under biomedically relevant concentration ranges has not been achieved. Herein, we report in vitro selection and development of a Li+-specific DNAzyme fluorescent sensor with >100-fold selectivity over other biorelevant metal ions. This sensor allows comparative Li+ visualization in HeLa cells, human neuronal progenitor cells (NPCs), and neurons derived from BD patients and healthy controls. Strikingly, we detected enhanced accumulation of Li+ in cells derived from BD patients compared with healthy controls in differentiated neurons but not NPCs. These results establish the DNAzyme-based sensor as a novel platform for biomedical research into BD and related areas using lithium drugs.

Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells.

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.

Induced pluripotent stem cells from human placental chorion for perinatal tissue engineering applications.

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly, we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4, SOX2, KLF4, and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology, immunocytochemistry, and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives, including neural stem cells, beating cardiomyocytes, and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications.

Emergence of a seizure phenotype in aged apolipoprotein epsilon 4 targeted replacement mice.

The apolipoprotein ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and is associated with earlier age of onset. The incidence of spontaneous seizures has been reported to be increased in sporadic AD as well as in the early onset autosomal dominant forms of AD. We now report the emergence of a seizure phenotype in aged apolipoprotein E4 (apoE4) targeted replacement (TR) mice but not in age-matched apoE2 TR or apoE3 TR mice. Tonic-clonic seizures developed spontaneously after 5 months of age in apoE4 TR mice and are triggered by mild stress. Female mice had increased seizure penetrance compared to male mice, but had slightly reduced overall seizure severity. The majority of seizures were characterized by head and neck jerks, but 25% of aged apoE4 TR mice had more severe tonic-clonic seizures which occasionally progressed to tonic extension and death. Aged apoE4 TR mice progressed through pentylenetetrazol-induced seizure stages more rapidly than did apoE3 TR and apoE2 TR mice. Electroencephalographic (EEG) recordings revealed more frequent bursts of synchronous theta activity in the hippocampus of apoE4 TR mice than in apoE2 TR or apoE3 TR mice. Cortical EEG recordings also revealed sharp spikes and other abnormalities in apoE4 TR mice. Taken together, these findings demonstrate the emergence of an age-dependent seizure phenotype in old apoE4 TR mice in the absence of human amyloid-ß peptide (Aß) overexpression, suggesting increased central nervous system neural network excitability.

Human APOE isoform-dependent effects on brain beta-amyloid levels in PDAPP transgenic mice.

To investigate the role of human apolipoprotein E (apoE) on Abeta deposition in vivo, we crossed PDAPP mice lacking mouse Apoe to targeted replacement mice expressing human apoE (PDAPP/TRE2, PDAPP/TRE3, or PDAPP/TRE4). We then measured the levels of apoE protein and Abeta peptides in plasma, CSF, and brain homogenates in these mice at different ages. We also quantified the amount of brain Abeta and amyloid burden in 18-month-old mice. In young PDAPP/TRE4 mice that were analyzed at an age before brain Abeta deposition, we observed a significant decrease in the levels of apoE in CSF and brain when compared with age-matched mice expressing either human E2 or E3. The brain levels of Abeta42 in PDAPP/TRE4 mice were substantially elevated even at this very early time point. In older PDAPP/TRE4 mice, the levels of insoluble apoE protein increased in parallel to the dramatic rise in brain Abeta burden, and the majority of apoE was associated with Abeta. In TRE4 only mice, we also observed a significant decrease in the level of apoE in brain homogenates. Since the relative level of apoE mRNA was equivalent in PDAPP/TRE and TRE only mice, it appears that post-translational mechanisms influence the levels of apoE protein in brain (E4 < E3 << E2), resulting in early and dramatic apoE isoform-dependent effects on brain Abeta levels (E4 >> E3 > E2) that increase with age. Therapeutic strategies aimed at increasing the soluble levels of apoE protein, regardless of isoform, may effectively prevent and (or) treat Alzheimer's disease.

Macrophage-mediated degradation of beta-amyloid via an apolipoprotein E isoform-dependent mechanism.

Recent studies suggest that bone marrow-derived macrophages can effectively reduce beta-amyloid (Abeta) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Abeta, we cultured murine macrophages on top of Abeta plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP(717V>F) mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Abeta in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Abeta degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Abeta than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Abeta was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Abeta-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Abeta degradation by macrophages. These apoE isoform-dependent effects of macrophages on Abeta degradation suggest a novel "peripheral" mechanism for Abeta clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimer's disease.

Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products.

Dietary fish oil containing omega 3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the omega 6 fatty acid arachidonic acid (AA) and the omega 3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA(3) is about equipotent to TxA(2) at the TP alpha receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of omega 3 fatty acids such as EPA.

Regulation of intracellular cyclooxygenase levels by gene transcription and protein degradation.

Cyclooxygenases-1 and -2 (COX-1 and -2) catalyze the committed step in prostaglandin formation. Each isozyme subserves different biological functions. This is, at least in part, a consequence of differences in patterns of COX-1 and COX-2 expression. COX-1 is induced during development, and COX-1 mRNA and COX-1 protein are very stable. These latter properties can explain why COX-1 protein levels usually remain constant in those cells that express this isozyme. COX-2 is usually expressed inducibly in association with cell replication or differentiation. Both COX-2 mRNA and COX-2 protein have short half-lives relative to those of COX-1. Therefore, COX-2 protein is typically present for only a few hours after its synthesis. Here we review and develop the concepts that (a) COX-2 gene transcription can involve at least six different cis-acting promoter elements interacting with trans-acting factors generated by multiple, different signaling pathways, (b) the relative contribution of each cis-acting COX-2 promoter element depends on the cell type, the stimulus and the time following the stimulus and (c) a unique 27 amino acid instability element located just upstream of the C-terminus of COX-2 targets this isoform to the ER-associated degradation system and proteolysis by the cytosolic 26S proteasome.

Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry.

Glycerophospholipids (GPL) in animal tissues are composed of a large array of molecular species that mainly differ in the fatty acyl composition. In order to further understand the roles of GPL at the molecular level, it is necessary to have comprehensive, accurate accounts of the molecular makeup for these molecules in animal tissues. However, this task was difficult simply because the conventional technologies of profiling GPL species depended heavily on technical skill for accuracy and reliability and were extremely labor-intensive. In recent years, tandem mass spectrometry (MS/MS) proved to be a highly reliable and sensitive technology for profiling small molecules, including GPL, in biological samples. In this study, we used this technology to perform simultaneous comparative analyses for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) in the same lipid preparations of liver, lung, kidney, heart, pancreas, stomach, small intestine, spleen, skeleton muscle and brain of an adult rat. We produced molecular profiles of these 4 GPL classes in these 10 different tissues that are highly reproducible between different scans of the same sample and between samples from different animals. It is intriguing that each tissue was found to possess a unique signature of GPL profile that may be used to identify unknown tissues. More importantly, these profiles may also set reference points for studying changes of GPL metabolism in different physiological and pathological conditions.

An intronic enhancer regulates cyclooxygenase-1 gene expression.

To identify cis-elements regulating PMA-induced prostaglandin H synthase-1 (PGHS-1) gene expression in the human megakaryoblast cell line, MEG-01, we performed promoter reporter assays with a luciferase reporter vector containing the -2030/-22 region of the human PGHS-1 gene. PMA treatment for 24 h increased PGHS-1 promoter activity by twofold. Mutagenesis studies of the promoter revealed a single Sp1 site essential for PMA-inducible transcription. Insertion of a highly conserved 100 bp sequence cloned from intron 8 into the -2030/-22 reporter plasmid enhanced PMA-dependent transcription 10-fold. Mutation of either a consensus AP-1 site within intron 8 or the Sp1 site in the promoter reduced PMA-induced activity by 80-100%. Gel shift assays using the intron 8 AP-1 sequence demonstrated the formation of an AP-1-specific DNA-protein complex. Our results suggest that inducible PGHS-1 gene expression involves the coordinate functioning of a Sp1 site in the promoter and an AP-1 site in intron 8.

Polyploid formation via chromosome duplication induced by CTP:phosphocholine cytidylyltransferase deficiency and Bcl-2 overexpression: identification of two novel endogenous factors.

Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CTalpha prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CTalpha as a negative regulator of polyploid formation.

Spontaneous regression of advanced cancer: identification of a unique genetically determined, age-dependent trait in mice.

We have established and studied a colony of mice with a unique trait of host resistance to both ascites and solid cancers induced by transplantable cells. One dramatic manifestation of this trait is age-dependent spontaneous regression of advanced cancers. This powerful resistance segregates as a single-locus dominant trait, is independent of tumor burden, and is effective against cell lines from multiple types of cancer. During spontaneous regression or immediately after exposure, cancer cells provoke a massive infiltration of host leukocytes, which form aggregates and rosettes with tumor cells. The cytolytic destruction of cancer cells by innate leukocytes is rapid and specific without apparent damage to normal cells. The mice are healthy and cancer-free and have a normal life span. These observations suggest a previously unrecognized mechanism of immune surveillance, which may have potential for therapy or prevention of cancer.

Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells.

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.

Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model.

We have previously shown that chronic treatment with the monoclonal antibody m266, which is specific for amyloid beta-peptide (Abeta), increases plasma concentrations of Abeta and reduces Abeta burden in the PDAPP transgenic mouse model of Alzheimer's disease (AD). We now report that administration of m266 to PDAPP mice can rapidly reverse memory deficits in both an object recognition task and a holeboard learning and memory task, but without altering brain Abeta burden. We also found that an Abeta/antibody complex was present in both the plasma and the cerebrospinal fluid of m266-treated mice. Our data indicate that passive immunization with this anti-Abeta monoclonal antibody can very rapidly reverse memory impairment in certain learning and memory tasks in the PDAPP mouse model of AD, owing perhaps to enhanced peripheral clearance and (or) sequestration of a soluble brain Abeta species.

Disruption of choline methyl group donation for phosphatidylethanolamine methylation in hepatocarcinoma cells.

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.