scholarly journals Age-Dependent and Sleep/Seizure-Induced Pathomechanisms of Autosomal Dominant Sleep-Related Hypermotor Epilepsy

2020 ◽  
Vol 21 (21) ◽  
pp. 8142 ◽  
Author(s):  
Kouji Fukuyama ◽  
Motohiro Okada
Keyword(s):  
Plasma Membrane ◽  
Autosomal Dominant ◽  
Loss Of Function ◽  
Glutamatergic Transmission ◽  
Wild Type ◽  
Cx43 Expression ◽  
Nicotine Administration ◽  
Extracellular Signal ◽  
Age Dependent ◽  
Before And After

The loss-of-function S284L-mutant α4 subunit of the nicotinic acetylcholine receptor (nAChR) is considered to contribute to the pathomechanism of autosomal dominant sleep-related hypermotor epilepsy (ADSHE); however, the age-dependent and sleep-related pathomechanisms of ADSHE remain to be clarified. To explore the age-dependent and sleep-induced pathomechanism of ADSHE, the present study determined the glutamatergic transmission abnormalities associated with α4β2-nAChR and the astroglial hemichannel in the hyperdirect and corticostriatal pathways of ADSHE model transgenic rats (S286L-TG) bearing the rat S286L-mutant Chrna4 gene corresponding to the human S284L-mutant CHRNA4 gene of ADSHE, using multiprobe microdialysis and capillary immunoblotting analyses. This study could not detect glutamatergic transmission in the corticostriatal pathway from the orbitofrontal cortex (OFC) to the striatum. Before ADSHE onset (four weeks of age), functional abnormalities of glutamatergic transmission compared to the wild-type in the cortical hyperdirect pathway, from OFC to the subthalamic nucleus (STN) in S286L-TG, could not be detected. Conversely, after ADSHE onset (eight weeks of age), glutamatergic transmission in the hyperdirect pathway of S286L-TG was enhanced compared to the wild-type. Notably, enhanced glutamatergic transmission of S286L-TG was revealed by hemichannel activation in the OFC. Expression of connexin43 (Cx43) in the OFC of S286L-TG was upregulated after ADSHE onset but was almost equal to the wild-type prior to ADSHE onset. Differences in the expression of phosphorylated protein kinase B (pAkt) before ADSHE onset between the wild-type and S286L-TG were not observed; however, after ADSHE onset, pAkt was upregulated in S286L-TG. Conversely, the expression of phosphorylated extracellular signal-regulated kinase (pErk) was already upregulated before ADSHE onset compared to the wild-type. Both before and after ADSHE onset, subchronic nicotine administration decreased and did not affect the both expression of Cx43 and pErk of respective wild-type and S286L-TG, whereas the pAkt expression of both the wild-type and S286L-TG was increased by nicotine. Cx43 expression in the plasma membrane of the primary cultured astrocytes of the wild-type was increased by elevation of the extracellular K+ level (higher than 10 mM), and the increase in Cx43 expression in the plasma membrane required pErk functions. These observations indicate that a combination of functional abnormalities, GABAergic disinhibition, and upregulated pErk induced by the loss-of-function S286L-mutant α4β2-nAChR contribute to the age-dependent and sleep-induced pathomechanism of ADSHE via the upregulation/hyperactivation of the Cx43 hemichannels.

scholarly journals Upregulated Connexin 43 Induced by Loss-of-Functional S284L-Mutant α4 Subunit of Nicotinic ACh Receptor Contributes to Pathomechanisms of Autosomal Dominant Sleep-Related Hypermotor Epilepsy

2020 ◽  
Vol 13 (4) ◽  
pp. 58 ◽  
Author(s):  
Kouji Fukuyama ◽  
Masashi Fukuzawa ◽  
Ruri Okubo ◽  
Motohiro Okada
Keyword(s):  
Connexin 43 ◽  
Autosomal Dominant ◽  
Glutamatergic Transmission ◽  
Wild Type ◽  
Thalamic Nuclei ◽  
Cx43 Expression ◽  
Motor Pathway ◽  
Nicotine Administration ◽  
Cultured Astrocytes ◽  
Primary Cultured Astrocytes

To study the pathomechanism and pathophysiology of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), this study determined functional abnormalities of glutamatergic transmission in the thalamocortical motor pathway, from the reticular thalamic nucleus (RTN), motor thalamic nuclei (MoTN) tosecondary motor cortex (M2C) associated with the S286L-mutant α4β2-nicotinic acetylcholine receptor (nAChR) and the connexin43 (Cx43) hemichannel of transgenic rats bearing the rat S286L-mutant Chrna4 gene (S286L-TG), which corresponds to the human S284L-mutant CHRNA4 gene using multiprobe microdialysis, primary cultured astrocytes and a Simple Western system. Expression of Cx43 in the M2C plasma membrane fraction of S286L-TG was upregulated compared with wild-type rats. Subchronic nicotine administration decreased Cx43 expression of wild-type, but did not affect that of S286L-TG; however, zonisamide (ZNS) decreased Cx43 in both wild-type and S286L-TG. Primary cultured astrocytes of wild-type were not affected by subchronic administration of nicotine but was decreased by ZNS. Upregulated Cx43 enhanced glutamatergic transmission during both resting and hyperexcitable stages in S286L-TG. Furthermore, activation of glutamatergic transmission associated with upregulated Cx43 reinforced the prolonged Cx43 hemichannel activation. Subchronic administration of therapeutic-relevant doses of ZNS compensated the upregulation of Cx43 and prolonged reinforced activation of Cx43 hemichannel induced by physiological hyperexcitability during the non-rapid eye movement phase of sleep. The present results support the primary pathomechanisms and secondary pathophysiology of ADSHE seizures of patients with S284L-mutation.

scholarly journals Calcium Signaling Regulates Trafficking of Familial Hypocalciuric Hypercalcemia (FHH) Mutants of the Calcium Sensing Receptor

2012 ◽  
Vol 26 (12) ◽  
pp. 2081-2091 ◽  
Author(s):  
Michael P. Grant ◽  
Ann Stepanchick ◽  
Gerda E. Breitwieser
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
State Level ◽  
Membrane Targeting ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Sensing Receptor ◽  
Dynamic Changes ◽  
Calcium Sensing

Abstract Calcium-sensing receptors (CaSRs) regulate systemic Ca2+ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2+ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2+, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2+ signaling, which were substantially rescued by coexpression with wild-type CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2+ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSRΔ868 robustly rescued trafficking and Ca2+ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2+ response when extracellular Ca2+ is elevated and argues that Ca2+ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane.

scholarly journals Genotype-Phenotype Relationship in Patients with Mutations in Thyroid Hormone Transporter MCT8

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2184-2190 ◽  
Author(s):  
Jurgen Jansen ◽  
Edith C. H. Friesema ◽  
Monique H. A. Kester ◽  
Charles E. Schwartz ◽  
Theo J. Visser
Keyword(s):  
Plasma Membrane ◽  
Thyroid Hormone ◽  
Protein Expression ◽  
Elevated Serum ◽  
Monocarboxylate Transporter ◽  
Psychomotor Development ◽  
Substrate Affinity ◽  
Loss Of Function ◽  
Wild Type ◽  
Rt Pcr

Loss-of-function mutations in thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to severe X-linked psychomotor retardation and elevated serum T3 levels. Most patients, for example those with mutations V235M, S448X, insI189, or delF230, cannot stand, walk, or speak. Patients with mutations L434W, L568P, and S194F, however, walk independently and/or develop some dysarthric speech. To study the relationship between mutation and phenotype, we transfected JEG3 and COS1 cells with wild-type or mutant MCT8. Expression and function of the transporter were studied by analyzing T3 and T4 uptake, T3 metabolism (by cotransfected type 3 deiodinase), Western blotting, affinity labeling with N-bromoacetyl-T3, immunocytochemistry, and quantitative RT-PCR. Wild-type MCT8 increased T3 uptake and metabolism about 5-fold compared with empty vector controls. Mutants V235M, S448X, insI189, and delF230 did not significantly increase transport. However, S194F, L568P, and L434W showed about 20, 23, and 37% of wild-type activity. RT-PCR did not show significant differences in mRNA expression between wild-type and mutant MCT8. Immunocytochemistry detected the nonfunctional mutants V235M, insI189, and delF230 mostly in the cytoplasm, whereas mutants with residual function were expressed at the plasma membrane. Mutants S194F and L434W showed high protein expression but low affinity for N-bromoacetyl-T3; L568P was detected in low amounts but showed relatively high affinity. Mutations in MCT8 cause loss of function through reduced protein expression, impaired trafficking to the plasma membrane, or reduced substrate affinity. Mutants L434W, L568P, and S194F showed significant residual transport capacity, which may underlie the more advanced psychomotor development observed in patients with these mutations.

scholarly journals Upregulated and Hyperactivated Thalamic Connexin 43 Plays Important Roles in Pathomechanisms of Cognitive Impairment and Seizure of Autosomal Dominant Sleep-Related Hypermotor Epilepsy with S284L-Mutant α4 Subunit of Nicotinic ACh Receptor

2020 ◽  
Vol 13 (5) ◽  
pp. 99 ◽  
Author(s):  
Kouji Fukuyama ◽  
Masashi Fukuzawa ◽  
Motohiro Okada
Keyword(s):  
Connexin 43 ◽  
Cognitive Deficit ◽  
Autosomal Dominant ◽  
Thalamic Nucleus ◽  
Glutamate Release ◽  
Glutamatergic Transmission ◽  
Transgenic Rats ◽  
Cx43 Expression ◽  
Mediodorsal Thalamic Nucleus ◽  
Thalamocortical Pathway

To understand the pathomechanism and pathophysiology of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), we studied functional abnormalities of glutamatergic transmission in thalamocortical pathway from reticular thalamic nucleus (RTN), mediodorsal thalamic nucleus (MDTN) to orbitofrontal cortex (OFC) associated with S286L-mutant α4β2-nicotinic acetylcholine receptor (nAChR), and connexin43 (Cx43) hemichannel of transgenic rats bearing rat S286L-mutant Chrna4 gene (S286L-TG), corresponding to the human S284L-mutant CHRNA4 gene using simple Western analysis and multiprobe microdialysis. Cx43 expression in the thalamic plasma membrane fraction of S286L-TG was upregulated compared with that of wild-type. Subchronic administrations of therapeutic-relevant doses of zonisamide (ZNS) and carbamazepine (CBZ) decreased and did not affect Cx43 expression of S286L-TG, respectively. Upregulated Cx43 enhanced glutamatergic transmission during both resting and hyperexcitable stages in S286L-TG. Furthermore, activation of GABAergic transmission RTN–MDTN pathway conversely enhanced, but not inhibited, l-glutamate release in the MDTN via upregulated/activated Cx43. Local administration of therapeutic-relevant concentration of ZNS and CBZ acutely supressed and did not affect glutamatergic transmission in the thalamocortical pathway, respectively. These results suggest that pathomechanisms of ADSHE seizure and its cognitive deficit comorbidity, as well as pathophysiology of CBZ-resistant/ZNS-sensitive ADSHE seizures of patients with S284L-mutation.

scholarly journals Distinct Effects of Escitalopram and Vortioxetine on Astroglial L-Glutamate Release Associated with Connexin43

2021 ◽  
Vol 22 (18) ◽  
pp. 10013
Author(s):  
Takashi Shiroyama ◽  
Kouji Fukuyama ◽  
Motohiro Okada
Keyword(s):  
Plasma Membrane ◽  
Partial Agonist ◽  
Glutamate Release ◽  
Receptor Subtypes ◽  
Direct Effects ◽  
Inhibitory Effects ◽  
Cx43 Expression ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Free Environment

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.

scholarly journals Generation of a humanized Aβ expressing mouse demonstrating aspects of Alzheimer’s disease-like pathology

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David Baglietto-Vargas ◽  
Stefania Forner ◽  
Lena Cai ◽  
Alessandra C. Martini ◽  
Laura Trujillo-Estrada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Autosomal Dominant ◽  
Late Onset ◽  
Periodic Acid ◽  
Wild Type ◽  
Periodic Acid Schiff ◽  
Human Counterpart ◽  
Age Dependent

AbstractThe majority of Alzheimer’s disease (AD) cases are late-onset and occur sporadically, however most mouse models of the disease harbor pathogenic mutations, rendering them better representations of familial autosomal-dominant forms of the disease. Here, we generated knock-in mice that express wildtype human Aβ under control of the mouse App locus. Remarkably, changing 3 amino acids in the mouse Aβ sequence to its wild-type human counterpart leads to age-dependent impairments in cognition and synaptic plasticity, brain volumetric changes, inflammatory alterations, the appearance of Periodic Acid-Schiff (PAS) granules and changes in gene expression. In addition, when exon 14 encoding the Aβ sequence was flanked by loxP sites we show that Cre-mediated excision of exon 14 ablates hAβ expression, rescues cognition and reduces the formation of PAS granules.

scholarly journals Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

2021 ◽  
Author(s):  
Marian Hruska-Plochan ◽  
Katharina M Hembach ◽  
Silvia Ronchi ◽  
Vera I Wiersma ◽  
Zuzanna Maniecka ◽  
...  
Keyword(s):  
Functional Networks ◽  
Loss Of Function ◽  
Wild Type ◽  
Cellular Models ◽  
Self Organized ◽  
Rna Targets ◽  
Age Dependent ◽  
Manual Selection ◽  
Selection Of ◽  
Human Specific

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies, which involve human-specific mechanisms that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors. Single-cell transcriptomics (scRNA-seq) and comparison to independent NSCs, showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks. Neuronal and glial maturation in iCoMoNSC-derived cultures was similar to that of cortical organoids. Overexpression of wild-type TDP-43 in a minority of iCoMoNSC-derived neurons led to progressive fragmentation and aggregation, resulting in loss of function and neurotoxicity. scRNA-seq revealed a novel set of misregulated RNA targets coinciding in both TDP-43 overexpressing neurons and patient brains exhibiting loss of nuclear TDP-43. The strongest misregulated target encoded for the synaptic protein NPTX2, which was consistently misaccumulated in ALS and FTLD patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby highlighting a new pathway of neurotoxicity.

scholarly journals Homozygote loss-of-function variants in the human COCH gene underlie hearing loss

2020 ◽  
Author(s):  
Nada Danial-Farran ◽  
Elena Chervinsky ◽  
Prathamesh Thangaraj Nadar-Ponniah ◽  
Eran Cohen Barak ◽  
Shahar Taiber ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Autosomal Dominant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Wild Type ◽  
Cos7 Cell ◽  
Gene Encoding ◽  
Negative Effect ◽  
Syndromic Hearing Loss

AbstractSince 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While an additional recessive variant in the COCH gene (DFNB110) has recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data has a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

scholarly journals SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

2016 ◽  
Vol 36 (20) ◽  
pp. 2612-2625 ◽  
Author(s):  
Elina Siljamäki ◽  
Daniel Abankwa
Keyword(s):  
Plasma Membrane ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Membrane Domains ◽  
Loss Of Function ◽  
Positive Effects ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Legius Syndrome ◽  
Novel Interactions

The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. TheSPRED1gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.

scholarly journals IRF4 haploinsufficiency in a family with Whipple’s disease

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Antoine Guérin ◽  
Gaspard Kerner ◽  
Nico Marr ◽  
Janet G Markle ◽  
Florence Fenollar ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Purifying Selection ◽  
Tropheryma Whipplei ◽  
Dominant Negative ◽  
Incomplete Penetrance ◽  
Whipple’S Disease ◽  
Loss Of Function ◽  
Whipple's Disease ◽  
Age Dependent ◽  
Chronic Carriage

Most humans are exposed to Tropheryma whipplei (Tw). Whipple’s disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance.

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