scholarly journals Disrupted choline clearance and sustained acetylcholine release in vivo by a common choline transporter coding variant associated with poor attentional control in humans

2021 ◽  
Author(s):  
Eryn Donovan ◽  
Cassandra Avila ◽  
Vinay Parikh ◽  
Cristina Fenollar-Ferrer ◽  
Randy D. Blakely ◽  
...  
Keyword(s):  
Frontal Cortex ◽  
Conformational Changes ◽  
Cognitive Disorders ◽  
Cholinergic Neurons ◽  
Dominant Negative ◽  
Ach Release ◽  
Negative Effects ◽  
Choline Transport ◽  
Choline Transporter

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional capacity as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, using in vivo cortical choline biosensing, CHT-mediated choline transport, and ACh release. CHT-mediated clearance of choline in mice expressing one or two Val89 alleles was reduced by over 7-fold relative to wild type (WT) mice, suggesting dominant-negative effects. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. Consistent with this hypothesis, structural modeling revealed that Val89 may attenuate choline transport by changing the ability of choline to induce conformational changes of CHT that support normal transport rates. Our findings suggest that diminished, sustained cholinergic signaling capacity in the frontal cortex underlies perturbed attentional performance in individuals expressing CHT Val89. Our work supports the utility of the CHT Val89 mouse model as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.

Lack of dominant-negative effects of a truncated γc on retroviral-mediated gene correction of immunodeficient mice

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1618-1624 ◽  
Author(s):  
Makoto Otsu ◽  
Kazuo Sugamura ◽  
Fabio Candotti
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Dominant Negative ◽  
Interleukin 4 ◽  
Gene Correction ◽  
Negative Effects ◽  
Gamma Chain ◽  
Immunodeficient Mice

A recent clinical trial of gene therapy for X-linked severe combined immunodeficiency (XSCID) has shown that retroviral-mediated gene correction of bone marrow stem cells can lead to the development of normal immune function. These exciting results have been preceded by successful immune reconstitution in several XSCID mouse models, all carrying null mutations of the common gamma chain (γc). One question not formally addressed by these previous studies is that of possible dominant-negative effects of the endogenous mutant γc protein on the activity of the wild-type transferred gene product. The present work was therefore undertaken to study whether corrective gene transfer was applicable to an XSCID murine model with preserved expression of a truncated γc molecule (Δγc+-XSCID). Gene correction of Δγc+-XSCID mice resulted in the reconstitution of lymphoid development, and preferential repopulation of lymphoid organs by gene-corrected cells demonstrated the selective advantage of γc-expressing cells in vivo. Newly developed B cells showed normalization of lipopolysaccharide-mediated proliferation and interleukin-4 (IL-4)–induced immunoglobulin G1 isotype switching. Splenic T cells and thymocytes of treated animals proliferated normally to mitogens and responded to the addition of IL-2, IL-4, and IL-7, indicating functional reconstitution of γc-sharing receptors. Repopulated thymi showed a clear increase of CD4−/CD8− and CD8+fractions, both dramatically reduced in untreated Δγc+-XSCID mice. These improvements were associated with the restoration of Bcl-2 expression levels and enhanced cell survival. These data indicate that residual expression of the endogenous truncated γc did not lead to dominant-negative effects in this murine model and suggest that patient selection may not be strictly necessary for gene therapy of XSCID.

scholarly journals Novel Transcription Coactivator Complex Containing Activating Signal Cointegrator 1

2002 ◽  
Vol 22 (14) ◽  
pp. 5203-5211 ◽  
Author(s):  
Dong-Ju Jung ◽  
Hee-Sook Sung ◽  
Young-Wha Goo ◽  
Hyun Mi Lee ◽  
Ok Ku Park ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Serum Response Factor ◽  
Ectopic Expression ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Mutant Form ◽  
Negative Effects ◽  
Direct Binding ◽  
Serum Response

ABSTRACT Human activating signal cointegrator 1 (hASC-1) was originally isolated as a transcriptional coactivator of nuclear receptors. Here we report that ASC-1 exists as a steady-state complex associated with three polypeptides, P200, P100, and P50, in HeLa nuclei; stimulates transactivation by serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor κB (NF-κB) through direct binding to SRF, c-Jun, p50, and p65; and relieves the previously described transrepression between nuclear receptors and either AP-1 or NF-κB. Interestingly, ectopic expression of Caenorhabditis elegans ASC-1 (ceASC-1), an ASC-1 homologue that binds P200 and P100, like hASC-1, while weakly interacting only with p65, in HeLa cells appears to replace endogenous hASC-1 from the hASC-1 complex and exerts potent dominant-negative effects on AP-1, NF-κB, and SRF transactivation. In addition, neutralization of endogenous P50 by single-cell microinjection of a P50 antibody inhibits AP-1 transactivation; the inhibition is relieved by coexpression of wild-type P50, but not of P50ΔKH, a mutant form that does not interact with P200. Overall, these results suggest that the endogenous hASC-1 complex appears to play an essential role in AP-1, SRF, and NF-κB transactivation and to mediate the transrepression between nuclear receptors and either AP-1 or NF-κB in vivo.

Pedunculopontine stimulation alters respiration and increases ACh release in the pontine reticular formation

1993 ◽  
Vol 264 (3) ◽  
pp. R544-R554 ◽  
Author(s):  
R. Lydic ◽  
H. A. Baghdoyan
Keyword(s):  
Respiratory Rate ◽  
Respiratory Depression ◽  
Reticular Formation ◽  
Time Course ◽  
Cholinergic Neurons ◽  
In Vivo Microdialysis ◽  
Pedunculopontine Tegmental Nucleus ◽  
Pontine Reticular Formation ◽  
Ach Release

The present study examined the hypothesis that cholinergic neurons in the pedunculopontine tegmental nucleus (PPT) can cause the release of acetylcholine (ACh) in the pontine reticular formation and contribute to respiratory depression. In vivo microdialysis of the gigantocellular tegmental field (FTG) was performed in 10 adult male cats while respiration was being measured. In four intact, unanesthetized cats these measurements were obtained during states of quiet wakefulness and during the rapid eye movement (REM) sleeplike state caused by FTG microinjections of carbachol. The results demonstrate a simultaneous time course of enhanced ACh release in the FTG and respiratory rate depression. In six barbiturate-anesthetized cats similar measurements were obtained while PPT regions containing NADPH-positive neurons were electrically stimulated. PPT stimulation caused increased ACh release in the FTG and caused respiratory rate depression. Together, these findings are consistent with the hypothesis of a causal relationship between ACh release in the FTG and respiratory depression.

scholarly journals SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Blood ◽  
2020 ◽  
Author(s):  
Christoph Schürch ◽  
Thorsten Schaefer ◽  
Joëlle Seraina Müller ◽  
Pauline Hanns ◽  
Marlon Arnone ◽  
...  
Keyword(s):  
De Novo ◽  
Dominant Negative ◽  
Severe Neutropenia ◽  
In Vivo Model ◽  
Factor X ◽  
Negative Effects ◽  
Congenital Neutropenia ◽  
Developmental Defects ◽  
Xbp1 Mrna

Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN), displaying symptoms overlapping with Shwachman-Diamond-Syndrome (SDS).1 Here, we investigate srp54 KO zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish are embryonically lethal and display, next to severe neutropenia, multi-systemic developmental defects. In contrast, srp54+/- zebrafish are viable, fertile and only show mild neutropenia. Interestingly, injection of human SRP54 mRNAs carrying mutations observed in patients (T115A, T117Δ and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the variable phenotypes observed in patients may be due to mutation-specific dominant negative effects on the functionality of the residual wildtype SRP54 protein. Consistently, overexpression of mutated SRP54 also induced neutropenia in wildtype fish and impaired granulocytic maturation of human promyelocytic HL-60 cells as well as of healthy cord-blood derived CD34+ HSPCs. Mechanistically, srp54 mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Vice-versa, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to SRP54 loss. The heterogenic phenotypes observed in patients, ranging from mild CN to SDS-like disease, may be due to different dominant negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.

scholarly journals Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2

2001 ◽  
Vol 21 (6) ◽  
pp. 2107-2117 ◽  
Author(s):  
Carmen L. de Hoog ◽  
Jackie A. Koehler ◽  
Marni D. Goldstein ◽  
Paul Taylor ◽  
Daniel Figeys ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpase ◽  
Dominant Negative ◽  
26S Proteasome ◽  
Guanine Nucleotide ◽  
Sequence Motifs ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange

ABSTRACT Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.

Ectopic expression of a Drosophila InsP3R channel mutant has dominant-negative effects in vivo

Cell Calcium ◽  
2006 ◽  
Vol 39 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Sonal Srikanth ◽  
Santanu Banerjee ◽  
Gaiti Hasan
Keyword(s):  
Ectopic Expression ◽  
Dominant Negative ◽  
Negative Effects

scholarly journals Syntheses and In-vitro Evaluation of Tetrahydroaminoacridine (THA) Based Analogues as High Affinity Choline Transporter (HAChT) Imaging Probe

2016 ◽  
Vol 17 (2) ◽  
pp. 97-102
Author(s):  
Mohammad Anwar Ul Azim ◽  
Takashi Kozaka ◽  
Izumi Uno ◽  
Daisuke Miwa ◽  
Yoji Kitamura ◽  
...  
Keyword(s):  
Binding Assay ◽  
Research Work ◽  
Cholinergic Neurons ◽  
Choline Uptake ◽  
Choline Transporter ◽  
High Affinity ◽  
20 Nm ◽  
High Affinity Choline Transporter

Introduction: In cholinergic neurons, high affinity choline uptake (HACU) by the high affinity choline transporter (HAChT) is a rate-limiting and regulatory step for the synthesis of Acetylcholine (Ach).Thus, HAChT appear to be a relatively specific presynaptic marker for cholinergic neurons in Alzheimer’s disease.Objectives: The principle objective of the study is to check the affinity of tetrahydroaminoacridine (THA) derivatives for HAChT. Another objective of the research work is to clarify whether the hemicholinium-3 (ChT inhibitor) and HACU enhancer molecules share the same binding sites or not.Materials and Methods: The inhibition activities of tacrine, the 2,3-dimethylfuran derivative of tacrine (DMTA) and their corresponding 2-oxo-1-pyrrolidineacetyl derivatives, namely PTAA and MKC-231 were measured by displacement of a typical HAChT antagonist [3H]HC-3 in rat cerebral membrane. The percentage of inhibition against the binding of [3H]HC-3 to HAChT were calculated using GraphPad Prism v4 software.Results: Hemicholinium-3 showed affinity for HAChT (IC50 = 20 nM) in the in vitro binding assay. A very insignificant inhibition activity (IC50 = 1000 nM) of Tacrine was revealed. The newly synthesized tacrine derivatives, DMTA and PTAA did not show any affinity for HAChT. Although MKC-231 was reported to enhance cholinergic activity at synaptic terminals, it did not show any affinity for the HAChT in [3H]HC-3 binding assay.Conclusion: In vitro [3H]HC-3 binding assay revealed no affinity of MKC-231, tacrine and its corresponding2-oxo-1-pyrrolidineacetate derivative towards HAChT. So, it is worthy to develop radiolabeled HC-3 derivatives with high affinity for HAChT, which can diffuse the BBB, to enable the in vivo investigation of HACU system.Bangladesh J. Nuclear Med. 17(2): 97-102, July 2014

Fentanyl and Morphine, but not Remifentanil, Inhibit Acetylcholine Release in Pontine Regions Modulating Arousal 

1999 ◽  
Vol 90 (4) ◽  
pp. 1070-1077 ◽  
Author(s):  
Steven Mortazavi ◽  
Janel Thompson ◽  
Helen A. Baghdoyan ◽  
Ralph Lydic
Keyword(s):  
Rem Sleep ◽  
Intravenous Infusion ◽  
Cholinergic Neurons ◽  
Body Region ◽  
Ach Release ◽  
Axon Terminals ◽  
Ringer’S Solution ◽  
Cholinergic Neurotransmission ◽  
Cholinergic Cell

Background Opioids inhibit the rapid eye movement (REM) phase of sleep and decrease acetylcholine (ACh) release in medial pontine reticular formation (mPRF) regions contributing to REM sleep generation. It is not known whether opioids decrease ACh release by acting on cholinergic cell bodies or on cholinergic axon terminals. This study used in vivo microdialysis to test the hypothesis that opioids decrease ACh levels at cholinergic neurons in the laterodorsal tegmental nuclei (LDT) and LDT axon terminals in the mPRF. Methods Nine male cats were anesthetized with halothane, and ACh levels within the mPRF or LDT were assayed using microdialysis and high-pressure liquid chromatography (HPLC). ACh levels were analyzed in response to dialysis of the mPRF and LDT with Ringer's solution (control), followed by dialysis with Ringer's solution containing morphine sulfate (MSO4) or naloxone. ACh in the mPRF also was measured during either dialysis delivery or intravenous infusion of remifentanil and during dialysis delivery of fentanyl. Results Compared with dialysis of Ringer's solution, microdialysis with MSO4 decreased ACh by 23% in the mPRF and by 30% in the LDT. This significant decrease in ACh was antagonized by naloxone. MSO4 and fentanyl each caused a dose-dependent decrease in mPRF ACh when delivered by dialysis. Remifentanil delivered by continuous intravenous infusion or by dialysis into the mPRF did not alter mPRF ACh. Conclusions Morphine inhibits ACh at the cholinergic cell body region (LDT) and the terminal field in the mPRF. ACh in the mPRF was not altered by remifentanil and was significantly decreased by fentanyl. Thus, MSO4 and fentanyl disrupt cholinergic neurotransmission in the LDT-mPRF network known to modulate REM sleep and cortical electroencephalographic activation. These data are consistent with the possibility that inhibition of pontine cholinergic neurotransmission contributes to arousal state disruption by opioids.

Basal forebrain acetylcholine release during REM sleep is significantly greater than during waking

2001 ◽  
Vol 280 (2) ◽  
pp. R598-R601 ◽  
Author(s):  
Jacqueline Vazquez ◽  
Helen A. Baghdoyan
Keyword(s):  
Cognitive Deficits ◽  
Rem Sleep ◽  
Basal Forebrain ◽  
Cholinergic Neurons ◽  
Nrem Sleep ◽  
In Vivo Microdialysis ◽  
Ach Release ◽  
Behavioral Arousal ◽  
Function Of Sleep

Cholinergic neurons of the basal forebrain supply the neocortex with ACh and play a major role in regulating behavioral arousal and cortical electroencephalographic activation. Cortical ACh release is greatest during waking and rapid eye movement (REM) sleep and reduced during non-REM (NREM) sleep. Loss of basal forebrain cholinergic neurons contributes to sleep disruption and to the cognitive deficits of many neurological disorders. ACh release within the basal forebrain previously has not been quantified during sleep. This study used in vivo microdialysis to test the hypothesis that basal forebrain ACh release varies as a function of sleep and waking. Cats were trained to sleep in a head-stable position, and dialysis samples were collected during polygraphically defined states of waking, NREM sleep, and REM sleep. Results from 22 experiments in four animals demonstrated that means ± SE ACh release (pmol/10 min) was greatest during REM sleep (0.77 ± 0.07), intermediate during waking (0.58 ± 0.03), and lowest during NREM sleep (0.34 ± 0.01). The finding that, during REM sleep, basal forebrain ACh release is significantly elevated over waking levels suggests a differential role for basal forebrain ACh during REM sleep and waking.

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