Fouad Lemtiri-Chlieh

Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader–Willi syndromes

Angelman syndrome (AS) and Prader–Willi syndrome (PWS) are neurodevelopmental disorders of genomic imprinting. AS results from loss of function of the ubiquitin protein ligase E3A (UBE3A) gene, whereas the genetic defect in PWS is unknown. Although induced pluripotent stem cells (iPSCs) provide invaluable models of human disease, nuclear reprogramming could limit the usefulness of iPSCs from patients who have AS and PWS should the genomic imprint marks be disturbed by the epigenetic reprogramming process. Our iPSCs derived from patients with AS and PWS show no evidence of DNA methylation imprint erasure at the cis-acting PSW imprinting center. Importantly, we find that, as in normal brain, imprinting of UBE3A is established during neuronal differentiation of AS iPSCs, with the paternal UBE3A allele repressed concomitant with up-regulation of the UBE3A antisense transcript. These iPSC models of genomic imprinting disorders will facilitate investigation of the AS and PWS disease processes and allow study of the developmental timing and mechanism of UBE3A repression in human neurons.

Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

BDNF Evokes Release of Endogenous Cannabinoids at Layer 2/3 Inhibitory Synapses in the Neocortex

The neurotrophin brain-derived neurotrophic factor (BDNF) is a potent regulator of inhibitory synaptic transmission, although the locus of this effect and the underlying mechanisms are controversial. We explored a potential interaction between BDNF and endogenous cannabinoid (endocannabinoid) signaling because activation of type 1 cannabinoid (CB1) receptors potently regulates γ-aminobutyric acid (GABA) release and both trkB tyrosine kinase receptors and CB1 receptors are highly expressed at synapses in neocortical layer 2/3. Here, we found that the effects of BDNF at inhibitory cortical synapses are mediated by the release of endocannabinoids acting retrogradely at presynaptic CB1 receptors. Specifically, acute application of BDNF rapidly reduced the amplitude of inhibitory postsynaptic currents (IPSCs) via postsynaptic trkB receptor activation because intracellular delivery of the tyrosine kinase inhibitor K252a completely blocked the BDNF effect. Although triggered by postsynaptic trkB activation, BDNF exposure decreased presynaptic release probability, as evidenced by increases in the paired-pulse ratio and coefficient of variation of evoked responses. In addition, BDNF decreased the frequency but not the amplitude of action potential-independent miniature IPSCs and BDNF did not alter the postsynaptic response to locally applied GABA. These results suggest that BDNF induces the release of a retrograde messenger from the postsynaptic cell that regulates presynaptic neurotransmitter release. Consistent with a role for endocannabinoids as the retrograde signal, the effect of BDNF on IPSCs was blocked by CB1 receptor antagonists and was occluded by a cannabinoid receptor agonist. Furthermore, inhibiting endocannabinoid synthesis or transport also disrupted the BDNF effect, implicating postsynaptic endocannabinoid release triggered by BDNF.

Kalirin-7 is necessary for normal NMDA receptor-dependent synaptic plasticity

BackgroundDendritic spines represent the postsynaptic component of the vast majority of excitatory synapses present in the mammalian forebrain. The ability of spines to rapidly alter their shape, size, number and receptor content in response to stimulation is considered to be of paramount importance during the development of synaptic plasticity. Indeed, long-term potentiation (LTP), widely believed to be a cellular correlate of learning and memory, has been repeatedly shown to induce both spine enlargement and the formation of new dendritic spines. In our studies, we focus on Kalirin-7 (Kal7), a Rho GDP/GTP exchange factor (Rho-GEF) localized to the postsynaptic density that plays a crucial role in the development and maintenance of dendritic spines both in vitro and in vivo. Previous studies have shown that mice lacking Kal7 (Kal7KO) have decreased dendritic spine density in the hippocampus as well as focal hippocampal-dependent learning impairments.ResultsWe have performed a detailed electrophysiological characterization of the role of Kal7 in hippocampal synaptic plasticity. We show that loss of Kal7 results in impaired NMDA receptor-dependent LTP and long-term depression, whereas a NMDA receptor-independent form of LTP is shown to be normal in the absence of Kal7.ConclusionsThese results indicate that Kal7 is an essential and selective modulator of NMDA receptor-dependent synaptic plasticity in the hippocampus.

Cyclic Nucleotides and Nucleotide Cyclases in Plant Stress Responses

The cyclic nucleotides monophosphates (cNMP) and in particular adenosine 3’,5’-cyclic monophosphate (cAMP) and guanosine 3’,5’-cyclic monophosphate (cGMP) are cyclic catalytic products of adenosine 5-triphosphate (ATP) and guanosine 5’-triphosphate (GTP) respectively. These cNMPs are universal second messengers with key roles in many and diverse physiological responses and processes in prokaryotes, and in both higher and lower eukaryotes. Cyclic AMP is arguably one of the most extensively studied second messengers in animals, lower eukaryotes and bacteria where it has critical roles in signaling the metabolic status. In bacteria, cAMP is involved in the positive regulation of the lac operon where in an environment of a low glucose, cAMP accumulates and binds to the allosteric site of the cAMP receptor protein (CRP), a transcription activator protein. Once the CRP is in its active configuration, it binds to a cis-element upstream of the lac promoter and activates transcription. At high glucose concentrations, cAMP concentration decreases and CRP disengages from the lac operon promoter (Meiklejohn & Gralla, 1985). Cyclic AMP signaling is also critical for many aspects of the development of the slime mold Dictyostelium discoideum that grows unicellularly, but develops as multicellular organism (Kimmel & Firtel, 2004; McMains et al., 2008). Cyclic AMP has a role in chemotaxis and cAMP oscillations act as intracellular feedback loops in the transcriptional regulation of many regulatory pathways. Post aggregation, cAMP-dependent signals mediate cell sorting, pattern formation, and morphogenetic changes and cAMP receptor affinity can control wave dynamics, geometry and morphogenesis (Dormann et al., 2001). In animals, cAMP can be produced e.g. by Gs-coupled activation of adenylate cyclases (ACs) which triggers a signal cascade that includes the modification of Ca2+ channels, the phosphorylation of target proteins including enzymes involved in regulating glycogen metabolism and eventually the cAMP-dependent activation of the transcription factor, cAMP response element-binding (CREB) (Bolwell, 1995; Karin & Smeal, 1992). Cyclic AMP also plays a role in excitation-

Direct Modulation of the Guard Cell Outward-Rectifying Potassium Channel (GORK) by Abscisic Acid

Abscisic acid (ABA) induces turgor loss and hence stomatal closure by promoting rapid net K+ efflux from guard cells (GCs) through outward-rectifying K+ (K+out) channels (Schroeder et al., 1987; Blatt, 1990). The mechanisms of ABA signaling in GCs are detailed elsewhere (see Munemasa et al., 2015; Weiner et al., 2010; Pandey et al., 2007). Briefly, ABA binds to the PYR/PYL/RCARs, a family of soluble steroidogenic acute regulatory-related lipid transfer (START) proteins, and, in turn, inactivates the downstream PP2C (type 2C protein phosphatase), leading to the activation of SnRK2.6 (SNF1 [sucrose non-fermenting-1-related protein kinase]/OST1 [open stomata 1]) protein kinases.

Functional characterisation of an intron retaining K+ transporter of barley reveals intron‐mediated alternate splicing

Intron retention in transcripts and the presence of 5 and 3 splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K(+) transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K(+) uptake restored growth in medium containing hygromycin in the presence of different concentrations of K(+) and mediated hypersensitivity to Na(+) . HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K(+) and Na(+) concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

Characterization of Heterologously Expressed Transporter Genes by Patch- and Voltage-Clamp Methods: Application to Cyclic Nucleotide-Dependent Responses

The application of patch- and voltage-clamp methods to study ion transport can be limited by many -hurdles: the size of the cells to be patched and/or stabbed, the subcellular localization of the molecule of interest, and its density of expression that could be too low even in their own native environment. Functional expression of genes using recombinant DNA technology not only overcomes those hurdles but also affords additional and elegant investigations such as single-point mutation studies and subunit -associations/regulations. In this chapter, we give a step-by-step description of two electrophysiological methods, patch clamp and two-electrode voltage clamp (TEVC), that are routinely used in combination with heterologous gene expression to assist researchers interested in the identification and characterization of ion transporters. We describe how to (1) obtain and maintain the cells suitable for the use with each of the above-mentioned methods (i.e., HEK-293 cells and yeast spheroplasts to use with the patch-clamp methodology and Xenopus laevis oocytes with TEVC), (2) transfect/inject them with the gene of interest, and (3) record ion transport activities.