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Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.
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Enfermedad de Alzheimer , Glicoproteínas de Membrana , Microglía , Esclerosis Múltiple , Receptores Inmunológicos , Animales , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Glicoproteínas de Membrana/agonistas , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Femenino , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Transgénicos , Anticuerpos/farmacología , Humanos , Péptidos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMEN
INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
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TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.
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Habénula , Animales , Ansiedad/genética , Neuronas Colinérgicas , Ratones , Ratones Endogámicos C57BLRESUMEN
Ion channels have recently emerged as stable biomarkers and anticancer targets particularly when the applications of the currently available therapeutic regimens are limited, as in case of osteosarcoma, a malignant bone tumor. Here, we evaluated the expression of TTYH2, a presumably calcium-activated chloride channel, in a human osteosarcoma cell line U2OS. We used small-interfering RNA (siRNA)-mediated gene silencing to demonstrate the downregulation in the expression of TTYH2 that resulted in the decrease in the invasion and migration, but not proliferation, of U2OS cells. The expression levels of Slug and ZEB1, the transcription factors involved in epithelial-mesenchymal transition, significantly reduced after TTYH2 silencing. Based on these results, we suggest that TTYH2 may serve as a novel target for the treatment of osteosarcoma.
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Neoplasias Óseas/genética , Neoplasias Óseas/patología , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Osteosarcoma/genética , Osteosarcoma/patología , Regulación hacia Arriba/genética , Línea Celular Tumoral , Silenciador del Gen , Humanos , Proteínas de la Membrana/metabolismo , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismoRESUMEN
Two-pore domain K⺠(K2P) channels play essential roles in regulating resting membrane potential and cellular excitability. Although TWIK-1 (TWIK-tandem of pore domains in a weak inward rectifying K⺠channel) was the first identified member of the K2P channel family, it is only in recent years that the physiological roles of TWIK-1 have been studied in depth. A series of reports suggest that TWIK-1 may underlie diverse functions, such as intrinsic excitability of neurons, astrocytic passive conductance, and astrocytic glutamate release, as a homodimer or heterodimer with other K2P isotypes. Here, we summarize expression patterns and newly identified functions of TWIK-1 in the brain.
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Encéfalo/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Multimerización de Proteína , Animales , Astrocitos/metabolismo , Encefalopatías/metabolismo , Encefalopatías/patología , Humanos , Neuronas/metabolismoRESUMEN
Anoctamin-1 (ANO1) is a Ca(2+)-activated chloride channel (CaCC) that plays important physiological roles in normal and cancerous tissues. However, the plasma membrane trafficking mechanisms of ANO1 remain poorly characterized. In yeast two-hybrid screening experiments, we observed direct interactions of ANO1 with ß-COP, which is a subunit of Coat Protein Complex I (COPI). This interaction was then confirmed using several in vitro and in vivo binding assays. Moreover, the cotransfection of ß-COP with ANO1 into HEK293T cells led to decreased the surface expression and the channel activity of ANO1. Accordingly, endogenous ANO1 was associated with ß-COP in U251 glioblastoma cells, and silencing of ß-COP enhanced surface expression and whole-cell currents of ANO1 in these cells. Taken together, these data suggest that ß-COP negatively regulates ANO1 surface expression.
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Canales de Cloruro/metabolismo , Proteína Coatómero/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Anoctamina-1 , Transporte Biológico , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteína Coatómero/análisis , Glioblastoma/metabolismo , Células HEK293 , HumanosRESUMEN
DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-ß-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.
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Antibióticos Antineoplásicos/farmacología , Apoptosis/fisiología , Proteínas de Ciclo Celular/deficiencia , Senescencia Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Doxorrubicina/farmacología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Supresoras de Tumor/deficiencia , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Senescencia Celular/fisiología , Reparación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Fibroblastos , Fase G2/efectos de los fármacos , Fase G2/fisiología , Humanos , Morfolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pironas/farmacología , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas Supresoras de Tumor/antagonistas & inhibidoresRESUMEN
Heterozygous deletions in the ANKS1B gene cause ANKS1B neurodevelopmental syndrome (ANDS), a rare genetic disease characterized by autism spectrum disorder (ASD), attention deficit/hyperactivity disorder, and speech and motor deficits. The ANKS1B gene encodes for AIDA-1, a protein that is enriched at neuronal synapses and regulates synaptic plasticity. Here we report an unexpected role for oligodendroglial deficits in ANDS pathophysiology. We show that Anks1b-deficient mouse models display deficits in oligodendrocyte maturation, myelination, and Rac1 function, and recapitulate white matter abnormalities observed in ANDS patients. Selective loss of Anks1b from the oligodendrocyte lineage, but not from neuronal populations, leads to deficits in social preference and sensory reactivity previously observed in a brain-wide Anks1b haploinsufficiency model. Furthermore, we find that clemastine, an antihistamine shown to increase oligodendrocyte precursor cell maturation and central nervous system myelination, rescues deficits in social preference in 7-month-old Anks1b-deficient mice. Our work shows that deficits in social behaviors present in ANDS may originate from abnormal Rac1 activity within oligodendrocytes.
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Trastorno del Espectro Autista , Animales , Humanos , Lactante , Ratones , Trastorno del Espectro Autista/genética , Péptidos y Proteínas de Señalización Intracelular , Neuronas , Oligodendroglía , Conducta SocialRESUMEN
Sevoflurane is a safe and well-known inhaled anesthetic. Given that sevoflurane can be delivered to developing fetuses through the mother, it is critical to determine whether this agent affects fetal neurodevelopment. Recent research has sought to determine whether sevoflurane affects fetal brain development when the mother is exposed during the second to third trimester of pregnancy, considered to be the crucial period for the development of nervous system. However, even though the first trimester is a critical period for fetal organogenesis and the most susceptible time to teratogen exposure, research regarding the effects of sevoflurane on organogenesis, especially on brain development, is insufficient. In the present study, human embryonic stem cells (hESC)-derived cerebral organoids were exposed to sevoflurane during the time corresponding to the first trimester to investigate the effect of early sevoflurane exposure on fetal brain development, specifically the processes of neuronal differentiation and maturation. Organoid size exposed to the intermediate concentration of sevoflurane did not differ from control, immunofluorescence demonstrated that sevoflurane temporarily decreased the size of SOX2 + /N-cad + ventricular zone structures only during the mid-time point, and upregulated expression of TUJ1 and MAP2 only during the early time point. However, all markers returned to normal levels, and organoids formed normal cortical structures at the late time point. Our results suggest that maternal sevoflurane exposure during the first trimester of pregnancy can cause abnormal neuronal differentiation in the fetal brain. However, considering the recovery observed in later periods, sevoflurane exposure might not have lasting impacts on fetal brain development.
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Anestésicos por Inhalación , Embarazo , Femenino , Humanos , Sevoflurano/toxicidad , Anestésicos por Inhalación/toxicidad , Encéfalo/metabolismo , Feto , OrganoidesRESUMEN
Pancreatic cancer is a devastating disease and is highly resistant to anticancer drugs because of its complex microenvironment. Cancer-associated fibroblasts (CAFs) are an important source of extracellular matrix (ECM) components, which alter the physical and chemical properties of pancreatic tissue, thus impairing effective intratumoral drug delivery and resulting in resistance to conventional chemotherapy. The objective of this study was to develop a new cancer organoid model, including a fibrous tumor microenvironment (TME) using CAFs. The CAF-integrated pancreatic cancer organoid (CIPCO) model developed in this study histologically mimicked human pancreatic cancer and included ECM production by CAFs. The cancer cell-CAF interaction in the CIPCO promoted epithelial-mesenchymal transition of cancer cells, which was reversed by CAF inhibition using all-trans retinoic acid. Deposition of newly synthesized collagen I in the CIPCO disturbed the delivery of gemcitabine to cancer cells, and treatment with collagenase increased the cytotoxic effect of gemcitabine. This model may lead to the development of next-generation cancer organoid models recapitulating the fibrous TME.
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Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary AMPA receptor subunits that regulate both the trafficking and gating properties of AMPA receptors, and different TARP isoforms display distinct expression patterns in brain. Here, we compared the effects of four TARP isoforms on the kinetics of AMPA receptor currents. Each isoform slowed the deactivation of GluR1 currents, but the slowing was greatest with gamma-4 and gamma-8. Isoform-specific differences in desensitization were also observed that correlated with effects on deactivation. TARP isoforms also differentially modulated responses to trains of glutamate applications designed to mimic high-frequency presynaptic firing. Importantly, whereas both stargazin and gamma-4 rescued excitatory synaptic transmission in cerebellar granule cells from stargazer mice, the decay of miniature EPSCs was 2-fold slower in neurons expressing gamma-4. The results show that heterogeneity in the composition of AMPA receptor/TARP complexes contributes to synapse-specific differences in EPSC decays and frequency-dependent modulation of neurotransmission.
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Proteínas Nucleares/fisiología , Receptores AMPA/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Canales de Calcio/genética , Canales de Calcio/fisiología , Línea Celular , Cerebelo/citología , Cerebelo/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Activación del Canal Iónico/fisiología , Isomerismo , Cinética , Ratones , Neuronas/fisiología , Proteínas Nucleares/química , Técnicas de Placa-Clamp , Receptores AMPA/agonistas , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: The pathogenesis of osteonecrosis of the femoral head (ONFH) has been implicated in hypofibrinolysis and blood supply interruption. Previous studies have demonstrated that decreased fibrinolytic activity due to elevated plasminogen activator inhibitor-1 (PAI-1) levels correlates with ONFH pathogenesis. The -675 4G/5G single nucleotide polymorphism (SNP rs1799889) in the PAI-1 gene promoter is associated with PAI-1 plasma level. We investigated whether rs1799889 and two other SNPs of the PAI-1 gene (rs2227631, -844 G/A in the promoter; rs11178, +10700 C/T in the 3'UTR) are associated with increased ONFH risk. METHODS: Three SNPs in PAI-1 were genotyped in 206 ONFH patients and 251 control subjects, using direct sequencing and a TaqMan® 5' allelic discrimination assay. We performed association analysis for genotyped SNPs and haplotypes with ONFH. RESULTS: The 4G allele of rs1799889, A allele of rs2227631, and C allele of rs11178 were significantly associated with increased ONFH risk (p = 0.03, p = 0.003, and p = 0.002, respectively). When we divided the population according to gender, an association between the three SNPs and increased risk of ONFH was found only in men. In another subgroup analysis based on the etiology of ONFH, rs2227631 (A allele) and rs11178 (C allele) in the idiopathic subgroup (p = 0.007 and p = 0.021) and rs1799889 (4G allele) and rs11178 (C allele) in the alcohol-induced subgroup (p = 0.042 and p = 0.015) were associated with increased risk of ONFH. In addition, a certain haplotype (A-4G-C) of PAI-1 was also significantly associated with ONFH (p < 0.001). CONCLUSION: Our findings demonstrated that three SNPs (rs1799889, rs2227631, and rs11178) of the PAI-1 gene were associated with ONFH risk. This study also suggests that PAI-1 SNPs may play an important role in ONFH.
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Trastornos de la Coagulación Sanguínea Heredados/genética , Necrosis de la Cabeza Femoral/genética , Predisposición Genética a la Enfermedad/genética , Inhibidor 1 de Activador Plasminogénico/genética , Polimorfismo Genético/genética , Adulto , Trastornos de la Coagulación Sanguínea Heredados/epidemiología , Trastornos de la Coagulación Sanguínea Heredados/patología , Estudios de Casos y Controles , Femenino , Necrosis de la Cabeza Femoral/epidemiología , Necrosis de la Cabeza Femoral/patología , Predisposición Genética a la Enfermedad/epidemiología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Chemotherapy-induced cognitive impairment (CICI) is often reported as a neurotoxic side effect of chemotherapy. Although CICI has emerged as a significant medical problem, meaningful treatments are not currently available due to a lack of mechanistic understanding underlying CICI pathophysiology. Using the platinum-based chemotherapy cisplatin as a model for CICI, we show here that cisplatin suppresses nicotinamide adenine dinucleotide (NAD+) levels in the adult female mouse brain in vivo and in human cortical neurons derived from induced pluripotent stem cells in vitro. Increasing NAD+ levels through nicotinamide mononucleotide (NMN) administration prevented cisplatin-induced abnormalities in neural progenitor proliferation, neuronal morphogenesis, and cognitive function without affecting tumor growth and antitumor efficacy of cisplatin. Mechanistically, cisplatin inhibited expression of the NAD+ biosynthesis rate-limiting enzyme nicotinamide phosphoribosyl transferase (Nampt). Selective restoration of Nampt expression in adult-born neurons was sufficient to prevent cisplatin-induced defects in dendrite morphogenesis and memory function. Taken together, our findings suggest that aberrant Nampt-mediated NAD+ metabolic pathways may be a key contributor in cisplatin-induced neurogenic impairments, thus causally leading to memory dysfunction. Therefore, increasing NAD+ levels could represent a promising and safe therapeutic strategy for cisplatin-related neurotoxicity. SIGNIFICANCE: Increasing NAD+ through NMN supplementation offers a potential therapeutic strategy to safely prevent cisplatin-induced cognitive impairments, thus providing hope for improved quality of life in cancer survivors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3727/F1.large.jpg.
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Neoplasias de la Mama/tratamiento farmacológico , Cisplatino/toxicidad , Disfunción Cognitiva/prevención & control , Fármacos Neuroprotectores/farmacología , Mononucleótido de Nicotinamida/farmacología , Animales , Antineoplásicos/toxicidad , Apoptosis , Neoplasias de la Mama/patología , Proliferación Celular , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/patología , Femenino , Humanos , Ratones , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ANO1, a Ca2+-activated chloride channel, is highly expressed in glioblastoma cells and its surface expression is involved in their migration and invasion. However, the regulation of ANO1 surface expression in glioblastoma cells is largely unknown. In this study, we found that Ca2+/Calmodulin-dependent protein kinase II (CaMKII) ß specifically enhances the surface expression and channel activity of ANO1 in U251 glioblastoma cells. When KN-93, a CaMKII inhibitor, was used to treat U251 cells, the surface expression and channel activity of ANO1 were significantly reduced. Only CaMKIIß, among the four CaMKII isoforms, increased the surface expression and channel activity of ANO1 in a heterologous expression system. Additionally, gene silencing of CaMKIIß suppressed the surface expression and channel activity of ANO1 in U251 cells. Moreover, gene silencing of CaMKIIß or ANO1 prominently reduced the migration and invasion of U251 and U87 MG glioblastoma cells. We thus conclude that CaMKIIß plays a specific role in the surface expression of ANO1 and in the ANO1-mediated tumorigenic properties of glioblastoma cells, such as migration and invasion.
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Anoctamina-1/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Anoctamina-1/genética , Transporte Biológico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/genética , Humanos , Proteínas de Neoplasias/genéticaRESUMEN
Impulsivity is defined as a predisposition toward rapid, unplanned reactions in response to internal or external stimuli, often yielding negative consequences. Accordingly, impulsivity is considered a significant risk factor for developing addictive behaviors. The hippocampus is involved in regulating behavioral adaptability and learned behaviors. Consequently, abnormal hippocampal function has been demonstrated to contribute to impulsive and addictive behaviors. Furthermore, differential reinforcement of low rates of behavior (DRL) has shown that the hippocampus is implicated in reward acquisition and impulsivity in humans and rodent models. We have previously shown that impulsive behavior potentiates hippocampal neuroblast proliferation. However, the fate of these precursor cells produced during impulsive reward seeking remains unknown. Here, we demonstrate that DRL-mediated impulsive reward seeking with the 2-choice reaction time task (2-CRTT) increases the number of BrdU labeled cells in the dentate gyrus region of the hippocampus. Importantly, our results also show a significant increase in BrdU+ and NeuN+ colocalized mature newborn neurons in mice exhibiting impulsivity compared to non-impulsive control mice. These results suggest that operant reward seeking during unpredictable schedules of reinforcement contributes to adult hippocampal neurogenesis.
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Hipocampo/fisiología , Conducta Impulsiva/fisiología , Neurogénesis/fisiología , Neuronas/fisiología , Animales , Conducta Animal/fisiología , Proliferación Celular/fisiología , Conducta de Elección/fisiología , Condicionamiento Operante/fisiología , Hipocampo/citología , Masculino , Ratones , Neuronas/citología , Tiempo de Reacción/fisiología , RecompensaRESUMEN
14-3-3γ plays diverse roles in different aspects of cellular processes. Especially in the brain where 14-3-3γ is enriched, it has been reported to be involved in neurological and psychiatric diseases (e.g. Williams-Beuren syndrome and Creutzfeldt-Jakob disease). However, behavioral abnormalities related to 14-3-3γ deficiency are largely unknown. Here, by using 14-3-3γ deficient mice, we found that homozygous knockout mice were prenatally lethal, and heterozygous mice showed developmental delay relative to wild-type littermate mice. In addition, in behavioral analyses, we found that 14-3-3γ heterozygote mice display hyperactive and depressive-like behavior along with more sensitive responses to acute stress than littermate control mice. These results suggest that 14-3-3γ levels may be involved in the developmental manifestation of related neuropsychiatric diseases. In addition, 14-3-3γ heterozygote mice may be a potential model to study the molecular pathophysiology of neuropsychiatric symptoms.
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Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat-containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression profiling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.
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Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Técnicas de Placa-Clamp , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
PURPOSE: Transient global ischemia arising in human due to cardiac arrest causes selective, delayed neuronal death in hippocampal CA1 and cognitive impairment. Growth arrest and DNA-damage-inducible protein 45 beta (Gadd45b) is a wellknown molecule in both DNA damage-related pathogenesis and therapies. Emerging evidence suggests that Gadd45b is an anti-apoptotic factor in nonneuronal cells and is an intrinsic neuroprotective molecule in neurons. However, the mechanism of Gadd45b pathway is not fully examined in neurodegeneration associated with global ischemia. METHODS: Rats were subjected to transient global ischemia by the 4-vessel occlusion or sham operation. The animals were sacrificed at 24 hours, 48 hours, and 7 days after ischemia. The hippocampal CA1 was microdissected and processed to examine mRNA and protein level. To assess neuronal death, tissue sections were cut and processed for Fluoro-Jade and Nissl staining. RESULTS: Here we show that ischemic insults increase abundance of Gadd45b and brain-derived neurotrophic factor, a known target of Gadd45 mediated demethylation, in selectively-vulnerable hippocampal CA1 neurons. We further show that knockdown of Gadd45b increases abundance of a pro-apoptotic Bcl-2 family member Bax while decreasing the antiapoptotic protein Bcl-2, which together promote neuronal death. CONCLUSION: These findings document a protective role of Gadd45b against neuronal insults associated with global ischemia and identify Gadd45b as a potential therapeutic target for the amelioration of hippocampal neurodegeneration.
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Wnt signaling is a well-known molecular pathway in age-related pathogenesis and therapy of disease. While prior studies have mainly focused on Wnt ligands or Wnt activators, the in vivo functions of naturally secreted Wnt inhibitors are not clear, especially in brain aging. Using BubR1H/H mice as a novel mouse model of accelerated aging, we report that genetic inhibition of sFRP3 restores the reduced body and brain size observed in BubR1H/H mice. Furthermore, sFRP3 inhibition ameliorates hypomyelination in the corpus callosum and rescues neural progenitor proliferation in the hippocampal dentate gyrus of BubR1H/H mice. Taken together, our study identifies sFRP3 as a new molecular factor that cooperates with BubR1 function to regulate brain development, myelination, and hippocampal neurogenesis.
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Proteínas de Ciclo Celular/metabolismo , Senescencia Celular , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Progeria/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Ciclo Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Mutantes , Proteínas Serina-Treonina Quinasas/genética , Vía de Señalización WntRESUMEN
Neurodevelopmental disorders, including autism spectrum disorder, have complex polygenic etiologies. Single-gene mutations in patients can help define genetic factors and molecular mechanisms underlying neurodevelopmental disorders. Here we describe individuals with monogenic heterozygous microdeletions in ANKS1B, a predicted risk gene for autism and neuropsychiatric diseases. Affected individuals present with a spectrum of neurodevelopmental phenotypes, including autism, attention-deficit hyperactivity disorder, and speech and motor deficits. Neurons generated from patient-derived induced pluripotent stem cells demonstrate loss of the ANKS1B-encoded protein AIDA-1, a brain-specific protein highly enriched at neuronal synapses. A transgenic mouse model of Anks1b haploinsufficiency recapitulates a range of patient phenotypes, including social deficits, hyperactivity, and sensorimotor dysfunction. Identification of the AIDA-1 interactome using quantitative proteomics reveals protein networks involved in synaptic function and the etiology of neurodevelopmental disorders. Our findings formalize a link between the synaptic protein AIDA-1 and a rare, previously undefined genetic disease we term ANKS1B haploinsufficiency syndrome.