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A Novel Human Mutation Disrupts Dendritic Morphology and Synaptic Transmission, and Causes ASD-Related Behaviors.
Stephenson JR, Wang X, Perfitt TL, Parrish WP, Shonesy BC, Marks CR, Mortlock DP, Nakagawa T, Sutcliffe JS, Colbran RJ
(2017) J Neurosci 37: 2216-2233
MeSH Terms: Animals, Autism Spectrum Disorder, Brain, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cells, Cultured, Cycloheximide, Dendrites, Disease Models, Animal, Embryo, Mammalian, Excitatory Postsynaptic Potentials, Exploratory Behavior, Female, Gene Expression Regulation, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Rats, Rats, Sprague-Dawley, Receptors, AMPA, Receptors, N-Methyl-D-Aspartate, Sialoglycoproteins, Synaptic Transmission
Show Abstract · Added February 2, 2017
Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes. Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.
Copyright © 2017 the authors 0270-6474/17/372217-18$15.00/0.
1 Communities
2 Members
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25 MeSH Terms
Quantifying heterogeneity and dynamics of clonal fitness in response to perturbation.
Frick PL, Paudel BB, Tyson DR, Quaranta V
(2015) J Cell Physiol 230: 1403-12
MeSH Terms: Cell Culture Techniques, Cell Line, Cell Proliferation, Clone Cells, Cycloheximide, Gene Expression Regulation, Genetic Fitness, Humans, Protein Synthesis Inhibitors
Show Abstract · Added February 19, 2015
The dynamics of heterogeneous clonal lineages within a cell population, in aggregate, shape both normal and pathological biological processes. Studies of clonality typically relate the fitness of clones to their relative abundance, thus requiring long-term experiments and limiting conclusions about the heterogeneity of clonal fitness in response to perturbation. We present, for the first time, a method that enables a dynamic, global picture of clonal fitness within a mammalian cell population. This novel assay allows facile comparison of the structure of clonal fitness in a cell population across many perturbations. By utilizing high-throughput imaging, our methodology provides ample statistical power to define clonal fitness dynamically and to visualize the structure of perturbation-induced clonal fitness within a cell population. We envision that this technique will be a powerful tool to investigate heterogeneity in biological processes involving cell proliferation, including development and drug response.
© 2015 Wiley Periodicals, Inc.
2 Communities
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9 MeSH Terms
Derivation and experimental comparison of cell-division probability densities.
Leander R, Allen EJ, Garbett SP, Tyson DR, Quaranta V
(2014) J Theor Biol 359: 129-35
MeSH Terms: Antineoplastic Agents, Cell Count, Cell Cycle, Cell Division, Cell Proliferation, Cycloheximide, Dimethyl Sulfoxide, Erlotinib Hydrochloride, Humans, Models, Theoretical, Neoplasms, Probability, Quinazolines, Stochastic Processes
Show Abstract · Added February 19, 2015
Experiments have shown that, even in a homogeneous population of cells, the distribution of division times is highly variable. In addition, a homogeneous population of cells will exhibit a heterogeneous response to drug therapy. We present a simple stochastic model of the cell cycle as a multistep stochastic process. The model, which is based on our conception of the cell cycle checkpoint, is used to derive an analytical expression for the distribution of cell cycle times. We demonstrate that this distribution provides an accurate representation of cell cycle time variability and show how the model relates drug-induced changes in basic biological parameters to variability in response to drug treatment.
Copyright © 2014 Elsevier Ltd. All rights reserved.
2 Communities
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14 MeSH Terms
GABAA receptor biogenesis is impaired by the γ2 subunit febrile seizure-associated mutation, GABRG2(R177G).
Todd E, Gurba KN, Botzolakis EJ, Stanic AK, Macdonald RL
(2014) Neurobiol Dis 69: 215-24
MeSH Terms: Cell Membrane, Conserved Sequence, Cycloheximide, Endoplasmic Reticulum, Endoplasmic Reticulum-Associated Degradation, Glycosylation, HEK293 Cells, Humans, Models, Molecular, Mutation, Missense, Patch-Clamp Techniques, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Synthesis Inhibitors, Protein Transport, Receptors, GABA-A, Seizures, Febrile, gamma-Aminobutyric Acid
Show Abstract · Added January 24, 2015
A missense mutation in the GABAA receptor γ2L subunit, R177G, was reported in a family with complex febrile seizures (FS). To gain insight into the mechanistic basis for these genetic seizures, we explored how the R177G mutation altered the properties of recombinant α1β2γ2L GABAA receptors expressed in HEK293T cells. Using a combination of electrophysiology, flow cytometry, and immunoblotting, we found that the R177G mutation decreased GABA-evoked whole-cell current amplitudes by decreasing cell surface expression of α1β2γ2L receptors. This loss of receptor surface expression resulted from endoplasmic reticulum (ER) retention of mutant γ2L(R177G) subunits, which unlike wild-type γ2L subunits, were degraded by ER-associated degradation (ERAD). Interestingly, when compared to the condition of homozygous γ2L(R177G) subunit expression, disproportionately low levels of γ2L(R177G) subunits reached the cell surface with heterozygous expression, indicating that wild-type γ2L subunits possessed a competitive advantage over mutant γ2L(R177G) subunits for receptor assembly and/or forward trafficking. Inhibiting protein synthesis with cycloheximide demonstrated that the R177G mutation primarily decreased the stability of an intracellular pool of unassembled γ2L subunits, suggesting that the mutant γ2L(R177G) subunits competed poorly with wild-type γ2L subunits due to impaired subunit folding and/or oligomerization. Molecular modeling confirmed that the R177G mutation could disrupt intrasubunit salt bridges, thereby destabilizing secondary and tertiary structure of γ2L(R177G) subunits. These findings support an emerging body of literature implicating defects in GABAA receptor biogenesis in the pathogenesis of genetic epilepsies (GEs) and FS.
Copyright © 2014. Published by Elsevier Inc.
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18 MeSH Terms
The sodium channel accessory subunit Navβ1 regulates neuronal excitability through modulation of repolarizing voltage-gated K⁺ channels.
Marionneau C, Carrasquillo Y, Norris AJ, Townsend RR, Isom LL, Link AJ, Nerbonne JM
(2012) J Neurosci 32: 5716-27
MeSH Terms: Analysis of Variance, Animals, Bacterial Proteins, Biophysics, Biotinylation, Cell Line, Transformed, Cerebral Cortex, Cycloheximide, Electric Stimulation, Endocytosis, Gene Expression Regulation, Green Fluorescent Proteins, Humans, Immunoprecipitation, Luminescent Proteins, Mass Spectrometry, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurons, Patch-Clamp Techniques, Protein Synthesis Inhibitors, Proteomics, RNA, Small Interfering, Receptors, Transferrin, Shal Potassium Channels, Sodium Channels, Transfection, Voltage-Gated Sodium Channel beta-1 Subunit
Show Abstract · Added February 20, 2015
The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.
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29 MeSH Terms
Proteolytic instability and the action of nonclassical transcriptional activators.
Wang X, Muratani M, Tansey WP, Ptashne M
(2010) Curr Biol 20: 868-71
MeSH Terms: Binding Sites, Cycloheximide, Mediator Complex, Polymerase Chain Reaction, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Transcriptional Activation, beta-Galactosidase
Show Abstract · Added March 10, 2014
Several transcriptional activators, called "classical" because each bears a natural acidic activating region attached to a DNA binding domain, are proteolytically unstable in yeast, and it has been suggested that this instability is required for transcriptional activation. Here we test the generality of that proposal by examining a set of activators (called "nonclassical") that lack activating regions. These activators (e.g., LexA-Gal11) comprise a LexA DNA binding domain fused to a component of the Mediator and are believed to insert the latter into the Mediator and recruit it (and, indirectly, other components required for transcription) to a gene bearing LexA sites. We find that three, and only three, Mediator subunits, all from its tail domain, work as activators when fused to LexA. All three are unstable, and for the case analyzed in detail, stabilization decreases activity. Thus, to the extent tested, both classical and nonclassical activators work most efficiently when proteolytically unstable.
(c) 2010 Elsevier Ltd. All rights reserved.
0 Communities
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9 MeSH Terms
KCNQ1/KCNE1 assembly, co-translation not required.
Vanoye CG, Welch RC, Tian C, Sanders CR, George AL
(2010) Channels (Austin) 4: 108-14
MeSH Terms: Animals, Brefeldin A, Cell Membrane, Cycloheximide, Endoplasmic Reticulum, Humans, Ion Channel Gating, KCNQ1 Potassium Channel, Kinetics, Membrane Potentials, Potassium, Potassium Channels, Voltage-Gated, Protein Synthesis Inhibitors, Protein Transport, Recombinant Proteins, Xenopus laevis
Show Abstract · Added November 21, 2018
Voltage-gated potassium channels are often assembled with accessory proteins that increase their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (K(V)) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some K(V) channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (I(Ks)). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of I(Ks). Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent I(Ks) expression following prKCNE1 injection. By contrast, incubation with brefeldin A prevented KCNQ1 modulation by prKCNE1. Moreover, injection of the trafficking-deficient KCNE1-L51H reduced KCNQ1 currents. Together, these observations indicate that while assembly of KCNE1 with KCNQ1 does not require co-translation, functional KCNQ1-prKCNE1 channels assemble early in the secretory pathway and reach the plasma membrane via vesicular trafficking.
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MeSH Terms
Androgen receptor-mediated repression of novel target genes.
Prescott J, Jariwala U, Jia L, Cogan JP, Barski A, Pregizer S, Shen HC, Arasheben A, Neilson JJ, Frenkel B, Coetzee GA
(2007) Prostate 67: 1371-83
MeSH Terms: Androgen Antagonists, Anilides, Cell Line, Tumor, Cycloheximide, Dihydrotestosterone, Gene Expression Regulation, Neoplastic, Humans, Male, Nitriles, Prostatic Neoplasms, Protein Synthesis Inhibitors, RNA, Messenger, RNA, Small Interfering, Receptor, Muscarinic M1, Receptors, Androgen, Receptors, Muscarinic, Reverse Transcriptase Polymerase Chain Reaction, Tosyl Compounds, Transfection
Show Abstract · Added September 23, 2013
BACKGROUND - The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis.
METHODS - ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments.
RESULTS - We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions.
CONCLUSIONS - Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.
2007 Wiley-Liss, Inc
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19 MeSH Terms
RhoB plays an essential role in CXCR2 sorting decisions.
Neel NF, Lapierre LA, Goldenring JR, Richmond A
(2007) J Cell Sci 120: 1559-71
MeSH Terms: Actins, Bridged Bicyclo Compounds, Heterocyclic, Cell Line, Chemotaxis, Cycloheximide, Cytochalasin B, Endosomes, Guanosine Triphosphate, Humans, Interleukin-8, Lysosome-Associated Membrane Glycoproteins, Lysosomes, Models, Biological, Mutation, Protein Transport, RNA, Small Interfering, Receptor, IGF Type 2, Receptors, Interleukin-8B, Receptors, Transferrin, Thiazolidines, Transfection, rab GTP-Binding Proteins, rab4 GTP-Binding Proteins, rhoB GTP-Binding Protein
Show Abstract · Added May 30, 2013
The CXCR2 chemokine receptor is a G-protein-coupled receptor that undergoes clathrin-mediated endocytosis upon ligand binding. The trafficking of CXCR2 is crucial for cells to maintain a proper chemotactic response. The mechanisms that regulate the recycling/degradation sorting decision are unknown. In this study, we used dominant-negative (T19N) and GTPase-deficient activated (Q63L) RhoB mutants, as well as RhoB small interfering RNA (siRNA) to investigate the role of RhoB in CXCR2 trafficking. Expression of either of the RhoB mutants or transfection of RhoB siRNA impaired CXCR2-mediated chemotaxis. Expression of RhoB T19N and transfection of RhoB siRNA impaired sorting of CXCR2 to the lysosome after 3 hours of CXCL8 stimulation and impaired CXCL8-induced CXCR2 degradation. In cells expressing the RhoB Q63L mutant, CXCR2 recycling through the Rab11a recycling compartment was impaired after 30 minutes of CXCL8 stimulation as was CXCL8-induced CXCR2 degradation. For cells expressing activated RhoB, CXCR2 colocalized with Rab4, a marker for the rapid recycling pathway, and with the mannose-6-phosphate receptor, which traffics between the trans-Golgi network and endosomes. These data suggest that CXCR2 recycles through alternative pathways. We conclude that oscillation of RhoB GTPase activity is essential for appropriate sorting decisions, and for directing CXCR2 degradation and recycling--events that are required for optimal chemotaxis.
2 Communities
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24 MeSH Terms
Progressive and inhibitory cell cycle proteins act simultaneously to regulate neurotrophin-mediated proliferation and maturation of neuronal precursors.
Simpson PJ, Moon C, Kleman AM, Connolly E, Ronnett GV
(2007) Cell Cycle 6: 1077-89
MeSH Terms: Animals, Brain-Derived Neurotrophic Factor, Cell Cycle Proteins, Cell Differentiation, Cell Proliferation, Cells, Cultured, Cyclin D1, Cyclin-Dependent Kinase 4, Cycloheximide, MAP Kinase Signaling System, Natriuretic Peptide, C-Type, Nerve Growth Factor, Olfactory Receptor Neurons, Proteasome Endopeptidase Complex, Proteasome Inhibitors, Rats, Stem Cells
Show Abstract · Added August 14, 2014
Neuronal stem cell expansion and differentiation is a process involving stages of proliferation and maturation governed by the sequential and combinatorial exposure of cells to extrinsic factors. The olfactory epithelium is an excellent model to investigate regulation of this process, as it undergoes neuronal replacement post-natally. We have shown that the neurotrophins NGF and BDNF sequentially promote proliferation of developing olfactory sensory neuronal precursors, although their kinetics of proliferation and cell fate outcomes differ. Interestingly, CNP inhibits this neurotrophin-induced proliferation and promotes the maturation of these precursors to their next developmental stage. Here, we investigate the mechanisms behind these actions. Both NGF and BDNF increase the expression of cyclin D1 and cyclin-dependent kinase 4 (cdk4), with temporal expression patterns that parallel the proliferation kinetics of their cellular targets. The timing of cyclin D1 expression reflects differences in the need for transcription and translation in early and late stage precursors. CNP inhibits neurotrophin-induced cyclin D1 expression, and induces the expression of different profiles of inhibitory cell cycle proteins, which are neurotrophin-specific and correlate with the attainment of different maturational cell fates. Inhibition of protein degradation reverses the effects of neurotrophins and CNP on cyclin D1 and inhibitor expression levels, respectively. These results suggest a model for cell cycle regulation that involves the simultaneous expression of progressive and inhibitory cell cycle regulatory proteins in response to both proliferation and differentiation agents, followed by selective degradation of these proteins, providing a mechanism for rapid and exquisite control of the cell cycle.
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17 MeSH Terms