Bradley Watmuff

A Targeted NKX2.1 Human Embryonic Stem Cell Reporter Line Enables Identification of Human Basal Forebrain Derivatives

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1‐GFP+ cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1‐GFP+ progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1‐GFP+ cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1‐GFP+ cells gave rise to γ‐aminobutyric acid‐, tyrosine hydroxylase‐, and somatostatin‐expressing neurons as well as to platelet‐derived growth factor receptor α‐positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1+ progenitors and demonstrate the utility of NKX2.1GFP/w hESCs for investigating human forebrain development and neuronal differentiation. STEM CELLS 2011;29:462–473

In Vitro Maturation of Dopaminergic Neurons Derived from Mouse Embryonic Stem Cells: Implications for Transplantation

The obvious motor symptoms of Parkinsons disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca2+ and Cl− imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl−]i progressively dropped in tyrosine hydroxylase positive (TH+) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH+ neurons responded to GABA (30 µM) with reductions in intracellular Cl− ([Cl−]i); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl−]i. As [Cl−]i reduced, the ability of GABA (30 µM) to elevate intracellular Ca2+ ([Ca2+]i) did also. At day 13 of differentiation a three hour exposure to GABA (30 µM) or L-glutamate (30 µM) increased the number of midbrain dopaminergic (TH+ and Pitx3+) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.

Disease signatures for schizophrenia and bipolar disorder using patient-derived induced pluripotent stem cells.

Schizophrenia and bipolar disorder are complex psychiatric disorders that present unique challenges in the study of disease biology. There are no objective biological phenotypes for these disorders, which are characterized by complex genetics and prominent roles for gene-environment interactions. The study of the neurobiology underlying these severe psychiatric disorders has been hindered by the lack of access to the tissue of interest - neurons from patients. The advent of reprogramming methods that enable generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and peripheral blood mononuclear cells has opened possibilities for new approaches to study relevant disease biology using iPSC-derived neurons. While early studies with patient iPSCs have led to promising and intriguing leads, significant hurdles remain in our attempts to capture the complexity of these disorders in vitro. We present here an overview of studies to date of schizophrenia and bipolar disorder using iPSC-derived neuronal cells and discuss potential future directions that can result in the identification of robust and valid cellular phenotypes that in turn can lay the groundwork for meaningful clinical advances.

Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling

PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3eGFP/w neurons exhibited spontaneous Ca2+ transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min-1, respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3eGFP/w neurons began to respond to more than one neurotransmitter. MPP+ and tumor necrosis factor (TNF), but not prostaglandin E2, caused death of the ∼50% of PITX3eGFP/w neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3eGFP/w neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.

Unbiased Metabolite Profiling of Schizophrenia Fibroblasts under Stressful Perturbations Reveals Dysregulation of Plasmalogens and Phosphatidylcholines

We undertook an unbiased metabolite profiling of fibroblasts from schizophrenia patients and healthy controls to identify metabolites and pathways that are dysregulated in disease, seeking to gain new insights into the disease biology of schizophrenia and to discover potential disease-related biomarkers. We measured polar and nonpolar metabolites in the fibroblasts under normal conditions and under two stressful physiological perturbations: growth in low-glucose media and exposure to the steroid hormone dexamethasone. We found that metabolites that were significantly different between schizophrenia and control subjects showed separation of the two groups by partial least-squares discriminant analysis methods. This separation between schizophrenia and healthy controls was more robust with metabolites identified under the perturbation conditions. The most significant individual metabolite differences were also found in the perturbation experiments. Metabolites that were significantly different between schizophrenia and healthy controls included a number of plasmalogens and phosphatidylcholines. We present these results in the context of previous reports of metabolic profiling of brain tissue and plasma in schizophrenia. These results show the applicability of metabolite profiling under stressful perturbations to reveal cellular pathways that may be involved in disease biology.

Lysine Deacetylation by HDAC6 Regulates the Kinase Activity of AKT in Human Neural Progenitor Cells

The AKT family of serine-threonine kinases functions downstream of phosphatidylinositol 3-kinase (PI3K) to transmit signals by direct phosphorylation of a number of targets, including the mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β), and β-catenin. AKT binds to phosphatidylinositol (3,4,5)-triphosphate (PIP3) generated by PI3K activation, which results in its membrane localization and subsequent activation through phosphorylation by phosphoinositide-dependent protein kinase 1 (PDK1). Together, the PI3K-AKT signaling pathway plays pivotal roles in many cellular systems, including in the central nervous system where it governs both neurodevelopment and neuroplasticity. Recently, lysine residues (Lys14 and Lys20) on AKT, located within its pleckstrin homology (PH) domain that binds to membrane-bound PIP3, have been found to be acetylated under certain cellular contexts in various cancer cell lines. These acetylation modifications are removed by the enzymatic action of the class III lysine deacetylases, SIRT1 and SIRT2, of the sirtuin family. The extent to which reversible acetylation regulates AKT function in other cell types remains poorly understood. We report here that AKT kinase activity is modulated by a class IIb lysine deacetylase, histone deacetylase 6 (HDAC6), in human neural progenitor cells (NPCs). We find that HDAC6 and AKT physically interact with each other in the neuronal cells, and in the presence of selective HDAC6 inhibition, AKT is acetylated at Lys163 and Lys377 located in the kinase domain, two novel sites distinct from the acetylation sites in the PH-domain modulated by the sirtuins. Measurement of the functional effect of HDAC6 inhibition on AKT revealed decreased binding to PIP3, a correlated decrease in AKT kinase activity, decreased phosphorylation of Ser552 on β-catenin, and modulation of neuronal differentiation trajectories. Taken together, our studies implicate the deacetylase activity of HDAC6 as a novel regulator of AKT signaling and point to novel mechanisms for regulating AKT activity with small-molecule inhibitors of HDAC6 currently under clinical development.

Perturbational Profiling of Metabolites in Patient Fibroblasts Implicates α-Aminoadipate as a Potential Biomarker for Bipolar Disorder

Many studies suggest the presence of aberrations in cellular metabolism in bipolar disorder. We studied the metabolome in bipolar disorder to gain insight into cellular pathways that may be dysregulated in bipolar disorder and to discover evidence of novel biomarkers. We measured polar and nonpolar metabolites in fibroblasts from subjects with bipolar I disorder and matched healthy control subjects, under normal conditions and with two physiologic perturbations: low-glucose media and exposure to the stress-mediating hormone dexamethasone. Metabolites that were significantly different between bipolar and control subjects showed distinct separation by principal components analysis methods. The most statistically significant findings were observed in the perturbation experiments. The metabolite with the lowest p value in both the low-glucose and dexamethasone experiments was α-aminoadipate, whose intracellular level was consistently lower in bipolar subjects. Our study implicates α-aminoadipate as a possible biomarker in bipolar disorder that manifests under cellular stress. This is an intriguing finding given the known role of α-aminoadipate in the modulation of kynurenic acid in the brain, especially as abnormal kynurenic acid levels have been implicated in bipolar disorder.

S193. EX VIVO SIGNATURE OF PSYCHOSIS AND TREATMENT RESPONSE IN PATIENT-DERIVED NEURONS

Abstract Background Postmortem studies in schizophrenia show well-replicated neuronal differences in the prefrontal cortex (PFC), specifically showing lower dendritic spine density in upper-layer cortical pyramidal neurons. Animal models that recapitulate features of psychosis also show lower dendritic spine density and synapse number in the PFC, with a more pronounced effect in upper-layer cortical neurons. Furthermore, the decrease in dendritic spines and synapses in animal models have been shown to be reversible with antipsychotic treatment. Results from postmortem brains, animal models and in vitro rodent cultures provide a strong impetus to test the hypothesis that dendritic spine biology plays an important role in the biology of schizophrenia and in mediating the effects of antipsychotic medications. Methods To extend these findings, we studied cortical neurons generated from subjects with schizophrenia. We reprogrammed induced pluripotent stem cells (iPSCs) from human subjects with schizophrenia and from matched healthy controls. We differentiated human iPSCs along the forebrain lineage to generate mature cortical neurons. We developed a robust experimental approach to delineate and quantify spines in the dendrites as well as methods to outline and measure the spines in order to classify the different spine types. We also developed methodology for functional characterization of individual neurons using calcium imaging in the cortical neuron cultures. Results We found that cortical neurons generated from the iPSCs of schizophrenia patients had a lower density of dendritic spines when compared to cortical neurons generated from the iPSCs of healthy control subjects. We also delineated the different composition of spine types in cortical neurons from schizophrenia patients when compared to those from healthy control subjects. In cortical neurons from schizophrenia subjects, we found that clozapine exposure in vitro leads to a robust increase in dendritic spine density. Discussion We found that cortical neurons from iPSCs of schizophrenia subjects recapitulate the dendritic spine differences reported in postmortem brains of schizophrenia subjects. Moreover, we found that human cortical neurons from schizophrenia subjects show increased dendritic spine density when exposed in vitro to clozapine. The ability to delineate cellular features related to disease biology in iPSC-derived neurons opens the door to understand the pathophysiology of schizophrenia and lay the foundations for the development of novel therapeutics.

Increased microglial synapse elimination in patient-specific models of schizophrenia

Schizophrenia patients display decreased synaptic density in postmortem studies, suggesting aberrant microglial synapse elimination during neurodevelopment. Here, we use cellular reprogramming to create patient-specific in vitro models of microglia-mediated synapse engulfment that demonstrate increased synapse elimination in schizophrenia-derived models compared to healthy controls. We show that excessive synaptic pruning in schizophrenia reflects abnormalities in microglia-like cells as well as synaptic structures. Further, we find that schizophrenia risk-associated variants within the complement component 4 locus contribute to the increased uptake in schizophrenia models. Finally, we demonstrate that the antibiotic minocycline reduces microglia-mediated synapse uptake and show that minocycline treatment for acne is associated with a reduction in incident schizophrenia risk compared to other treatments in a cohort of more than 9,000 young adults drawn from health records. Specific pharmacological interventions targeting excessive pruning merit further study for their capacity to delay or prevent the onset of schizophrenia in high-risk individuals.

Stem cell-derived neurons in the development of targeted treatment for schizophrenia and bipolar disorder

The recent advent of induced pluripotent stem cells has enabled the study of patient-specific and disease-related neurons in vitro and has facilitated new directions of inquiry into disease mechanisms. With these approaches, we now have the possibility of correlating ex vivo cellular phenotypes with individual patient response to treatment and/or side effects, which makes targeted treatments for schizophrenia and bipolar disorder a distinct prospect in the coming years. Here, we briefly review the current state of stem cell-based models and explore studies that are providing new insights into the disease biology of schizophrenia and bipolar disorder, which are laying the foundations for the development of novel targeted therapies.