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Mouse Anti-Glyceraldehyde-3-PDH (GAPDH) Monoclonal antibody, Unconjugated

RRID:AB_2107445

Antibody ID

AB_2107445

Target Antigen

Glyceraldehyde-3-PDH (GAPDH) chicken/avian, feline, fish, human, mouse, other, rabbit, rat, xenopus, yeast, canine, human, mouse, rat

Proper Citation

(Millipore Cat# MAB374, RRID:AB_2107445)

Clonality

monoclonal antibody

Comments

seller recommendations: ELISA; Western Blot; Western Blotting,ELISA

Host Organism

mouse

Vendor

Millipore

Cat Num

MAB374

Publications that use this research resource

Changes in Corticotrope Gene Expression Upon Increased Expression of Peptidylglycine α-Amidating Monooxygenase.

  • Mains RE
  • Endocrinology
  • 2018 Jul 1

Literature context:


Abstract:

Throughout evolution, secretion has played an essential role in the ability of organisms and single cells to survive in the face of a changing environment. Peptidylglycine α-amidating monooxygenase (PAM) is an integral membrane monooxygenase, first identified for its role in the biosynthesis of neuroendocrine peptides released by the regulated secretory pathway. PAM was subsequently identified in Chlamydomonas reinhardtii, a unicellular green alga, where it plays an essential role in constitutive secretion and in ciliogenesis. Reduced expression of C. reinhardtii PAM resulted in significant changes in secretion and ciliogenesis. Hence, a screen was performed for transcripts and proteins whose expression responded to changes in PAM levels in a mammalian corticotrope tumor cell line. The goal was to identify genes not previously known to play a role in secretion. The screen identified transcription factors, peptidyl prolyl isomerases, endosomal/lysosomal proteins, and proteins involved in tissue-specific responses to glucose and amino acid availability that had not previously been recognized as relevant to the secretory pathway. Perhaps reflecting the dependence of PAM on molecular oxygen, many PAM-responsive genes are known to be hypoxia responsive. The data highlight the extent to which the performance of the secretory pathway may be integrated into a wide diversity of signaling pathways.

Funding information:
  • Medical Research Council - G0501957(United Kingdom)

Expression of BDNF and trkB in the hippocampus of a rat genetic model of vulnerability (Roman low-avoidance) and resistance (Roman high-avoidance) to stress-induced depression.

  • Serra MP
  • Brain Behav
  • 2018 Jul 9

Literature context:


Abstract:

INTRODUCTION: The selective breeding of Roman High- (RHA) and Low-Avoidance (RLA) rats for, respectively, rapid versus poor acquisition of the active avoidance response has generated two distinct phenotypes differing in many behavioral traits, including coping strategies to aversive conditions. Thus, RLA rats are considered as a genetic model of vulnerability to stress-induced depression whereas RHA rats are a model of resilience to that trait. Besides the monoamine hypothesis of depression, there is evidence that alterations in neuronal plasticity in the hippocampus and other brain areas are critically involved in the pathophysiology of mood disorders. MATERIALS AND METHODS: Western blot (WB) and immunohistochemistry were used to investigate the basal immunochemical occurrence of brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine-kinase receptor trkB in the dorsal and ventral hippocampus of adult RHA and RLA rats. RESULTS: WB analysis indicated that the optical density of BDNF- and trkB-positive bands in the dorsal hippocampus is, respectively, 48% and 25% lower in RLA versus RHA rats. Densitometric analysis of BDNF- and trkB-like immunoreactivity (LI) in brain sections showed that BDNF-LI is 24% to 34% lower in the different sectors of the Ammon's horn of RLA versus RHA rats, whereas line-related differences are observed in the dentate gyrus (DG) only in the ventral hippocampus. As for trkB-LI, significant differences are observed only in the dorsal hippocampus, where density is 23% lower in the DG of RLA versus RHA rats, while no differences across lines occur in the Ammon's horn. CONCLUSION: These findings support the hypothesis that a reduced BDNF/trkB signaling in the hippocampus of RLA versus RHA rats may contribute to their more pronounced vulnerability to stress-induced depression.

N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1.

  • Sanchez-Pupo RE
  • Int J Mol Sci
  • 2018 Jun 22

Literature context:


Abstract:

Pannexins (Panx1, 2, 3) are channel-forming glycoproteins expressed in mammalian tissues. We previously reported that N-glycosylation acts as a regulator of the localization and intermixing of Panx1 and Panx3, but its effects on Panx2 are currently unknown. Panx1 and Panx2 intermixing can regulate channel properties, and both pannexins have been implicated in neuronal cell death after ischemia. Our objectives were to validate the predicted N-glycosylation site of Panx2 and to study the effects of Panx2 glycosylation on localization and its capacity to interact with Panx1. We used site-directed mutagenesis, enzymatic de-glycosylation, cell-surface biotinylation, co-immunoprecipitation, and confocal microscopy. Our results showed that N86 is the only N-glycosylation site of Panx2. Panx2 and the N86Q mutant are predominantly localized to the endoplasmic reticulum (ER) and cis-Golgi matrix with limited cell surface localization was seen only in the presence of Panx1. The Panx2 N86Q mutant is glycosylation-deficient and tends to aggregate in the ER reducing its cell surface trafficking but it can still interact with Panx1. Our study indicates that N-glycosylation may be important for folding and trafficking of Panx2. We found that the un-glycosylated forms of Panx1 and 2 can readily interact, regulating their localization and potentially their channel function in cells where they are co-expressed.

Funding information:
  • NIAID NIH HHS - U54 AI057156(United States)

Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain.

  • Zhu H
  • Cell
  • 2018 Jun 14

Literature context:


Abstract:

Sunlight exposure is known to affect mood, learning, and cognition. However, the molecular and cellular mechanisms remain elusive. Here, we show that moderate UV exposure elevated blood urocanic acid (UCA), which then crossed the blood-brain barrier. Single-cell mass spectrometry and isotopic labeling revealed a novel intra-neuronal metabolic pathway converting UCA to glutamate (GLU) after UV exposure. This UV-triggered GLU synthesis promoted its packaging into synaptic vesicles and its release at glutamatergic terminals in the motor cortex and hippocampus. Related behaviors, like rotarod learning and object recognition memory, were enhanced after UV exposure. All UV-induced metabolic, electrophysiological, and behavioral effects could be reproduced by the intravenous injection of UCA and diminished by the application of inhibitor or short hairpin RNA (shRNA) against urocanase, an enzyme critical for the conversion of UCA to GLU. These findings reveal a new GLU biosynthetic pathway, which could contribute to some of the sunlight-induced neurobehavioral changes.

Funding information:
  • NIDCD NIH HHS - R01DC010154-01A2(United States)

The Temporal Dynamics of Arc Expression Regulate Cognitive Flexibility.

  • Wall MJ
  • Neuron
  • 2018 Jun 27

Literature context:


Abstract:

Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasome-dependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition.

Funding information:
  • NICHD NIH HHS - R21 HD065269(United States)
  • NIGMS NIH HHS - R25 GM109442()
  • NINDS NIH HHS - R00 NS076364()
  • NINDS NIH HHS - R01 NS085093()

A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity.

  • Percharde M
  • Cell
  • 2018 Jun 19

Literature context:


Abstract:

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.

Funding information:
  • NCI NIH HHS - P30 CA54174(United States)
  • NIGMS NIH HHS - R01 GM113014()
  • NIGMS NIH HHS - R01 GM123556()

Co-polymers of Actin and Tropomyosin Account for a Major Fraction of the Human Actin Cytoskeleton.

  • Meiring JCM
  • Curr. Biol.
  • 2018 Jun 22

Literature context:


Abstract:

Tropomyosin proteins form stable coiled-coil dimers that polymerize along the α-helical groove of actin filaments [1]. The actin cytoskeleton consists of both co-polymers of actin and tropomyosin and polymers of tropomyosin-free actin [2]. The fundamental distinction between these two types of filaments is that tropomyosin determines the functional capability of actin filaments in an isoform-dependent manner [3-9]. However, it is unknown what portion of actin filaments are associated with tropomyosin. To address this deficit, we have measured the relative distribution between these two filament populations by quantifying tropomyosin and actin levels in a variety of human cell types, including bone (U2OS); breast epithelial (MCF-10A); transformed breast epithelial (MCF-7); and primary (BJpar), immortalized (BJeH), and Ras-transformed (BJeLR) BJ fibroblasts [10]. Our measurements of tropomyosin and actin predict the saturation of the actin cytoskeleton, implying that tropomyosin binding must be inhibited in order to generate tropomyosin-free actin filaments. We find the majority of actin filaments to be associated with tropomyosin in four of the six cell lines tested and the portion of actin filaments associated with tropomyosin to decrease with transformation. We also discover that high-molecular-weight (HMW), unlike low-molecular-weight (LMW), tropomyosin isoforms are primarily co-polymerized with actin in untransformed cells. This differential partitioning of tropomyosins is not due to a lack of N-terminal acetylation of LMW tropomyosins, but it is, in part, explained by the susceptibility of soluble HMW tropomyosins to proteasomal degradation. We conclude that actin-tropomyosin co-polymers make up a major fraction of the human actin cytoskeleton.

Funding information:
  • Medical Research Council - G1000213(United Kingdom)

A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action.

  • Stoeber M
  • Neuron
  • 2018 Jun 6

Literature context:


Abstract:

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Funding information:
  • NIDA NIH HHS - R01 DA004443()
  • NIDDK NIH HHS - DK 59578(United States)

Early Nodal and Paranodal Disruption in Autoimmune Optic Neuritis.

  • Stojic A
  • J. Neuropathol. Exp. Neurol.
  • 2018 May 1

Literature context:


Abstract:

Disturbances in the nodes of Ranvier are an early phenomenon in many CNS disorders, including the autoimmune demyelinating disease multiple sclerosis (MS). Using an animal model of optic neuritis, a common early symptom of MS, we have investigated nodal and paranodal compartments in the optic nerve during disease progression. Both nodes and paranodes, as identified by immunohistochemistry against sodium channels (Nav) and Caspr, respectively, were observed to increase in length during the late induction phase of the disease, prior to onset of the demyelination and immune cell infiltration characteristic of optic neuritis. These changes were correlated with both axonal stress and microglial/macrophage activation, and were most apparent in the vicinity of the retrobulbar optic nerve head, the unmyelinated region of the optic nerve where retinal ganglion cell axons exit the retina. Using intravitreal glutamate injection as a model of a primary retinal insult, we demonstrate that this can induce similar nodal and paranodal changes. This may suggest that onset of neurodegeneration in the absence of demyelination, as reported in several studies into the nonaffected eyes of MS patients, may give rise to subtle disturbances in the axo-glial junction.

Funding information:
  • NIGMS NIH HHS - R01 GM0557226(United States)

Epigenetic Effects of an Adenosine Derivative in a Wistar Rat Model of Liver Cirrhosis.

  • Rodríguez-Aguilera JR
  • J. Cell. Biochem.
  • 2018 May 29

Literature context:


Abstract:

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.

RNA/Protein Discordant Expression of Fndc5 in Central Nervous System Is Likely to Be Mediated Through microRNAs.

  • Tanhaei S
  • DNA Cell Biol.
  • 2018 Apr 24

Literature context:


Abstract:

Fibronectin type III domain-containing 5 protein (Fndc5) is responsible for producing a secretory protein termed, "irisin." A modified expression of Fndc5 has been reported in different tissues during development, differentiation processes, as well as in metabolic events such as exercise. One of the important issues to be fixed is whether Fndc5 RNA level and protein content are concerted and modified hand in hand. Therefore, the aim of this study is to assess Fndc5 RNA and protein levels in various tissues of mouse and rat with emphasis on brain. Biopsies from various parts of neonatal and adult mouse and rat tissues were simultaneously assessed for transcript levels of Fndc5 and compared with the respective protein levels at the same time. Data indicated, unlike in muscle, no concerted fluctuations were observed for Fndc5 RNA and protein, especially in brain. Further look at four regions of brain (cerebellum, putamen, hippocampus, and cortex) revealed a similar discrepant expression. To hypothesize whether such discrepancy is arisen by miRNAs, we selected three main miRNAs, which were predicted to target Fndc5 and their expression levels were assessed in central nervous system (CNS) of mouse and hippocampus of rat. miRNA levels showed an antiparallel correlation with protein level of Fndc5, interpreting a putative role in regulating Fndc5 protein content in CNS. This phenomenon may represent the importance of governing Fndc5 content in neural cells, which seems to be crucial for neural function and differentiation.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/G017174/1(United Kingdom)

Repeat propofol anesthesia does not exacerbate plaque deposition or synapse loss in APP/PS1 Alzheimer's disease mice.

  • Woodhouse A
  • BMC Anesthesiol
  • 2018 Apr 25

Literature context:


Abstract:

BACKGROUND: There is increasing interest in whether anesthetic agents affect the risk or progression of Alzheimer's disease (AD). To mitigate many of the methodological issues encountered in human retrospective cohort studies we have used a transgenic model of AD to investigate the effect of propofol on AD pathology. METHODS: Six month-old amyloid precursor protein/presenilin 1 (APP/PS1) transgenic AD mice and control mice were exposed to 3 doses of propofol (200 mg/kg) or vehicle, delivered at monthly intervals. RESULTS: There was no difference in the extent of β-amyloid (Aβ) immunolabeled plaque deposition in APP/PS1 mice in vehicle versus propofol treatment groups. We also detected no difference in plaque-associated synapse loss in APP/PS1 mice following repeat propofol exposure relative to vehicle. Western blotting indicated that there was no difference in post-synaptic density protein 95, synaptophysin or glutamic acid decarboxylase 65/67 expression in control or APP/PS1 mice subjected to repeat propofol treatment relative to vehicle. CONCLUSIONS: These data suggest that repeat propofol anesthesia may not exacerbate plaque deposition or associated synapse loss in AD. Interestingly, this data also provides some of the first evidence suggesting that repeat propofol exposure in adult wild-type mice does not result in robust long-term alterations in the levels of key excitatory and inhibitory synaptic markers.

Funding information:
  • Alzheimer's Australia Dementia Research Foundation - N/A()
  • JO and JR Wicking Trust - N/A()
  • NIAMS NIH HHS - AR42309(United States)
  • Royal Hospital Research Foundation - N/A()

The pro-apoptotic JNK scaffold POSH/SH3RF1 mediates CHMP2BIntron5-associated toxicity in animal models of frontotemporal dementia.

  • West RJH
  • Hum. Mol. Genet.
  • 2018 Apr 15

Literature context:


Abstract:

Frontotemporal dementia (FTD) is one of the most prevalent forms of early-onset dementia. However, the pathological mechanisms driving neuronal atrophy in FTD remain poorly understood. Here we identify a conserved role for the novel pro-apoptotic protein plenty of SH3s (POSH)/SH3 domain containing ring finger 1 in mediating neuropathology in Drosophila and mammalian models of charged multivesicular body protein 2B (CHMP2BIntron5) associated FTD. Aberrant, AKT dependent, accumulation of POSH was observed throughout the nervous system of both Drosophila and mice expressing CHMP2BIntron5. Knockdown of POSH was shown to be neuroprotective and sufficient to alleviate aberrant neuronal morphology, behavioral deficits and premature-lethality in Drosophila models, as well as dendritic collapse and cell death in CHMP2BIntron5expressing rat primary neurons. POSH knockdown also ameliorated elevated markers of Jun N-terminal kinase and apoptotic cascades in both Drosophila and mammalian models. This study provides the first characterization of POSH as a potential component of an FTD neuropathology, identifying a novel apoptotic pathway with relevance to the FTD spectrum.

Funding information:
  • NCI NIH HHS - CA096832(United States)

An RNAi screen of Rho signalling networks identifies RhoH as a regulator of Rac1 in prostate cancer cell migration.

  • Tajadura-Ortega V
  • BMC Biol.
  • 2018 Mar 6

Literature context:


Abstract:

BACKGROUND: Cell migration is essential for development and tissue repair, but it also contributes to disease. Rho GTPases regulate cell migration, but a comprehensive analysis of how each Rho signalling component affects migration has not been carried out. RESULTS: Through an RNA interference screen, and using a prostate cancer cell line, we find that approximately 25% of Rho network components alter migration. Some genes enhance migration while others decrease basal and/or hepatocyte growth factor-stimulated migration. Surprisingly, we identify RhoH as a screen hit. RhoH expression is normally restricted to haematopoietic cells, but we find it is expressed in multiple epithelial cancer cell lines. High RhoH expression in samples from prostate cancer patients correlates with earlier relapse. RhoH depletion reduces cell speed and persistence and decreases migratory polarity. Rac1 activity normally localizes to the front of migrating cells at areas of dynamic membrane movement, but in RhoH-depleted cells active Rac1 is localised around the whole cell periphery and associated with membrane regions that are not extending or retracting. RhoH interacts with Rac1 and with several p21-activated kinases (PAKs), which are Rac effectors. Similar to RhoH depletion, PAK2 depletion increases cell spread area and reduces cell migration. In addition, RhoH depletion reduces lamellipodium extension induced by PAK2 overexpression. CONCLUSIONS: We describe a novel role for RhoH in prostate cancer cell migration. We propose that RhoH promotes cell migration by coupling Rac1 activity and PAK2 to membrane protrusion. Our results also suggest that RhoH expression levels correlate with prostate cancer progression.

Funding information:
  • Cancer Research UK - C41786/A132()
  • Cancer Research UK - C6220/A8833()
  • Cancer Research UK - C6620/A15961()
  • National Institutes of Health - PO1-GM103723()
  • National Institutes of Health - R01-GM079271()
  • National Institutes of Health - U01-EB018816()
  • NCI NIH HHS - R01 CA133966-04(United States)

Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury.

  • Dias DO
  • Cell
  • 2018 Mar 22

Literature context:


Abstract:

CNS injury often severs axons. Scar tissue that forms locally at the lesion site is thought to block axonal regeneration, resulting in permanent functional deficits. We report that inhibiting the generation of progeny by a subclass of pericytes led to decreased fibrosis and extracellular matrix deposition after spinal cord injury in mice. Regeneration of raphespinal and corticospinal tract axons was enhanced and sensorimotor function recovery improved following spinal cord injury in animals with attenuated pericyte-derived scarring. Using optogenetic stimulation, we demonstrate that regenerated corticospinal tract axons integrated into the local spinal cord circuitry below the lesion site. The number of regenerated axons correlated with improved sensorimotor function recovery. In conclusion, attenuation of pericyte-derived fibrosis represents a promising therapeutic approach to facilitate recovery following CNS injury.

Funding information:
  • Intramural NIH HHS - Z01 DE000698-10(United States)

Cand1-Mediated Adaptive Exchange Mechanism Enables Variation in F-Box Protein Expression.

  • Liu X
  • Mol. Cell
  • 2018 Mar 1

Literature context:


Abstract:

Skp1⋅Cul1⋅F-box (SCF) ubiquitin ligase assembly is regulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1/2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the koff of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution.

Funding information:
  • NIGMS NIH HHS - F32 GM112308()
  • NIGMS NIH HHS - R01 GM065997()
  • Wellcome Trust - 092076(United Kingdom)

Sense-Antisense lncRNA Pair Encoded by Locus 6p22.3 Determines Neuroblastoma Susceptibility via the USP36-CHD7-SOX9 Regulatory Axis.

  • Mondal T
  • Cancer Cell
  • 2018 Mar 12

Literature context:


Abstract:

Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.

Funding information:
  • NINDS NIH HHS - NS047331(United States)

Histone Deacetylases 1 and 2 Regulate Microglia Function during Development, Homeostasis, and Neurodegeneration in a Context-Dependent Manner.

  • Datta M
  • Immunity
  • 2018 Mar 20

Literature context:


Abstract:

Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo. We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer's disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically.

Funding information:
  • Austrian Science Fund FWF - P 18613(Austria)

Effects of Chemical Chaperones on Thyroid Hormone Transport by MCT8 Mutants in Patient-Derived Fibroblasts.

  • Groeneweg S
  • Endocrinology
  • 2018 Mar 1

Literature context:


Abstract:

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in severe intellectual and motor disability. At present, no effective therapy is available to restore TH signaling in MCT8-dependent tissues. Recent in vitro studies in stable overexpression cell models suggested that the function of certain mutant MCT8 proteins, specifically those that affect protein stability and intracellular trafficking (e.g., p.F501del), could be partially recovered by chemical chaperones. However, the effects of chaperones have not been demonstrated in other commonly used models for MCT8 deficiency, including transient overexpression models and patient-derived fibroblasts. Here, we demonstrate that the chemical chaperone 4-phenylbutyric acid (PBA) similarly potentiates the T3 transport function of wild-type and p.F501del mutant MCT8 in transiently transfected COS-1 cells by increasing MCT8 messenger RNA, total protein, and cell surface expression levels. Although PBA also increased the cell surface expression levels of the p.R445L mutant, no functional improvement was observed, which is in line with the proposed important role of Arg445 in substrate translocation. In contrast, PBA showed only minimal effects in ex vivo studies using control or p.F501del patient-derived fibroblasts. Moreover, the MCT8-specific inhibitor silychristin did not change these minimal effects, suggesting that the underlying mechanism is unrelated to the rescue of functional MCT8. Together, these findings indicate that the potency of chaperones to rescue mutant MCT8 function strongly depends on the cellular model and stress the need for further preclinical studies before clinically available chaperones should be considered as a treatment option in patients with MCT8 deficiency.

Actomyosin-Mediated Tension Orchestrates Uncoupled Respiration in Adipose Tissues.

  • Tharp KM
  • Cell Metab.
  • 2018 Mar 6

Literature context:


Abstract:

The activation of brown/beige adipose tissue (BAT) metabolism and the induction of uncoupling protein 1 (UCP1) expression are essential for BAT-based strategies to improve metabolic homeostasis. Here, we demonstrate that BAT utilizes actomyosin machinery to generate tensional responses following adrenergic stimulation, similar to muscle tissues. The activation of actomyosin mechanics is critical for the acute induction of oxidative metabolism and uncoupled respiration in UCP1+ adipocytes. Moreover, we show that actomyosin-mediated elasticity regulates the thermogenic capacity of adipocytes via the mechanosensitive transcriptional co-activators YAP and TAZ, which are indispensable for normal BAT function. These biomechanical signaling mechanisms may inform future strategies to promote the expansion and activation of brown/beige adipocytes.

Funding information:
  • Austrian Science Fund FWF - P 20522(Austria)
  • NCRR NIH HHS - S10 RR026866()
  • NIBIB NIH HHS - R21 EB025017()
  • NIDDK NIH HHS - R01 DK089202()
  • NIDDK NIH HHS - R01 DK097441()
  • NIDDK NIH HHS - R01 DK101293()
  • NIGMS NIH HHS - R01 GM122375()

The Transcriptionally Permissive Chromatin State of Embryonic Stem Cells Is Acutely Tuned to Translational Output.

  • Bulut-Karslioglu A
  • Cell Stem Cell
  • 2018 Mar 1

Literature context:


Abstract:

A permissive chromatin environment coupled to hypertranscription drives the rapid proliferation of embryonic stem cells (ESCs) and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ESCs. The results revealed that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of ESCs. Acute inhibition of translation rapidly depletes euchromatic marks in mouse ESCs and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Translation inhibition promotes rewiring of chromatin accessibility, which decreases at a subset of active developmental enhancers and increases at histone genes and transposable elements. Proteome-scale analyses revealed that several euchromatin regulators are unstable proteins and continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription, and translation sets the pace of proliferation at peri-implantation and may be employed by other stem/progenitor cells.

Funding information:
  • NICHD NIH HHS - F30 HD093116()
  • NIGMS NIH HHS - R01 GM113014()
  • NIGMS NIH HHS - R01 GM123556()
  • NIGMS NIH HHS - R01 GM55040(United States)

α7-Nicotinic Acetylcholine Receptor Agonist Ameliorates Nicotine Plus High-Fat Diet-Induced Hepatic Steatosis in Male Mice by Inhibiting Oxidative Stress and Stimulating AMPK Signaling.

  • Hasan MK
  • Endocrinology
  • 2018 Feb 1

Literature context:


Abstract:

α7-Nicotinic acetylcholine receptor (α7nAChR) agonists confer protection against a wide variety of cytotoxic insults and suppress oxidative stress and apoptosis in various cell systems, including hepatocytes. We recently demonstrated that nicotine, when combined with a high-fat diet (HFD), triggers oxidative stress, activates hepatocyte apoptosis, and exacerbates HFD-induced hepatic steatosis in male mice. This study evaluates whether PNU-282987 (PNU), a specific α7nAChR agonist, is effective in preventing nicotine plus HFD-induced hepatic steatosis. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice-daily intraperitoneal injections of 0.75 mg/kg body weight (BW) of nicotine, PNU (0.26 mg/kg BW), PNU plus nicotine, or saline for 10 weeks. PNU treatment was effective in attenuating nicotine plus HFD-induced increase in hepatic triglyceride levels, hepatocyte apoptosis, and hepatic steatosis. The preventive effects of PNU on nicotine plus HFD-induced hepatic steatosis were mediated by suppression of oxidative stress and activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) together with inhibition of its downstream target sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), and acetyl-coenzyme A-carboxylase (ACC). We conclude that the α7nAChR agonist PNU protects against nicotine plus HFD-induced hepatic steatosis in obese mice. PNU appears to work at various steps of signaling pathways involving suppression of oxidative stress, activation of AMPK, and inhibition of SREBP1c, FAS, and ACC. α7nAChR agonists may be an effective therapeutic strategy for ameliorating fatty liver disease, especially in obese smokers.

Funding information:
  • NCI NIH HHS - R01CA160514(United States)
  • NIDA NIH HHS - R24 DA017298()
  • NIMHD NIH HHS - U54 MD007598()

GDC-0879, a BRAFV600E Inhibitor, Protects Kidney Podocytes from Death.

  • Sieber J
  • Cell Chem Biol
  • 2018 Feb 15

Literature context:


Abstract:

Progressive kidney diseases affect approximately 500 million people worldwide. Podocytes are terminally differentiated cells of the kidney filter, the loss of which leads to disease progression and kidney failure. To date, there are no therapies to promote podocyte survival. Drug repurposing may therefore help accelerate the development of cures in an area of tremendous unmet need. In a newly developed high-throughput screening assay of podocyte viability, we identified the BRAFV600E inhibitor GDC-0879 and the adenylate cyclase agonist forskolin as podocyte-survival-promoting compounds. GDC-0879 protects podocytes from injury through paradoxical activation of the MEK/ERK pathway. Forskolin promotes podocyte survival by attenuating protein biosynthesis. Importantly, GDC-0879 and forskolin are shown to promote podocyte survival against an array of cellular stressors. This work reveals new therapeutic targets for much needed podocyte-protective therapies and provides insights into the use of GDC-0879-like molecules for the treatment of progressive kidney diseases.

Funding information:
  • NIDDK NIH HHS - R01 DK095045()
  • NIDDK NIH HHS - R01 DK099465()
  • NINDS NIH HHS - 4R00NS057944-03(United States)

Astrocytes from old Alzheimer's disease mice are impaired in Aβ uptake and in neuroprotection.

  • Iram T
  • Neurobiol. Dis.
  • 2018 Feb 9

Literature context:


Abstract:

In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aβ42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aβ clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aβ uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.

Programming of Schwann Cells by Lats1/2-TAZ/YAP Signaling Drives Malignant Peripheral Nerve Sheath Tumorigenesis.

  • Wu LMN
  • Cancer Cell
  • 2018 Feb 12

Literature context:


Abstract:

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann cell (SC)-lineage-derived sarcomas. Molecular events driving SC-to-MPNST transformation are incompletely understood. Here, we show that human MPNSTs exhibit elevated HIPPO-TAZ/YAP expression, and that TAZ/YAP hyperactivity in SCs caused by Lats1/2 loss potently induces high-grade nerve-associated tumors with full penetrance. Lats1/2 deficiency reprograms SCs to a cancerous, progenitor-like phenotype and promotes hyperproliferation. Conversely, disruption of TAZ/YAP activity alleviates tumor burden in Lats1/2-deficient mice and inhibits human MPNST cell proliferation. Moreover, genome-wide profiling reveals that TAZ/YAP-TEAD1 directly activates oncogenic programs, including platelet-derived growth factor receptor (PDGFR) signaling. Co-targeting TAZ/YAP and PDGFR pathways inhibits tumor growth. Thus, our findings establish a previously unrecognized convergence between Lats1/2-TAZ/YAP signaling and MPNST pathogenesis, revealing potential therapeutic targets in these untreatable tumors.

Funding information:
  • NHLBI NIH HHS - R01 HL132211()
  • NIA NIH HHS - R01 AG040990(United States)
  • NINDS NIH HHS - R01 NS072427()
  • NINDS NIH HHS - R01 NS075243()
  • NINDS NIH HHS - R01 NS078092()
  • NINDS NIH HHS - R01 NS086219()
  • NINDS NIH HHS - R37 NS096359()

A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes.

  • Bersuker K
  • Dev. Cell
  • 2018 Jan 8

Literature context:


Abstract:

Lipid droplet (LD) functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to two different cell types identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins. Moreover, quantitative analysis of LD proteome dynamics uncovered a role for endoplasmic reticulum-associated degradation in controlling the composition of the LD proteome. These data provide an important resource for future LD studies and demonstrate the utility of proximity labeling to study the regulation of LD proteomes.

Funding information:
  • NCI NIH HHS - R01 CA172667()
  • NIDDK NIH HHS - R00 DK095921()
  • NIGMS NIH HHS - R01 GM112948()
  • NINDS NIH HHS - NS063953(United States)

Removal of prolyl oligopeptidase reduces alpha-synuclein toxicity in cells and in vivo.

  • Svarcbahs R
  • Sci Rep
  • 2018 Jan 24

Literature context:


Abstract:

Prolyl oligopeptidase (PREP) inhibition by small-molecule inhibitors can reduce alpha-synuclein (aSyn) aggregation, a key player in Parkinson's disease pathology. However, the significance of PREP protein for aSyn aggregation and toxicity is not known. We studied this in vivo by using PREP knock-out mice with viral vector injections of aSyn and PREP. Animal behavior was studied by locomotor activity and cylinder tests, microdialysis and HPLC were used to analyze dopamine levels, and different aSyn forms and loss of dopaminergic neurons were studied by immunostainings. Additionally, PREP knock-out cells were used to characterize the impact of PREP and aSyn on autophagy, proteasomal system and aSyn secretion. PREP knock-out animals were nonresponsive to aSyn-induced unilateral toxicity but combination of PREP and aSyn injections increased aSyn toxicity. Phosphorylated p129, proteinase K resistant aSyn levels and tyrosine hydroxylase positive cells were decreased in aSyn and PREP injected knock-out animals. These changes were accompanied by altered dopamine metabolite levels. PREP knock-out cells showed reduced response to aSyn, while cells were restored to wild-type cell levels after PREP overexpression. Taken together, our data suggests that PREP can enhance aSyn toxicity in vivo.

Funding information:
  • National Cancer Institute - CA163820A1(United States)

Hypochlorite converts cysteinyl-dopamine into a cytotoxic product: A possible factor in Parkinson's Disease.

  • Mehta NJ
  • Free Radic. Biol. Med.
  • 2017 Dec 25

Literature context:


Abstract:

The dopamine oxidation product cysteinyl-dopamine has attracted attention as a contributor to the death of dopaminergic neurons in Parkinson's disease. Treatment of cysteinyl-dopamine with hypochlorite yields an even more cytotoxic product. This product has potent redox-cycling activity and initiates production of superoxide in PC12 cells. Taurine, which scavenges hypochlorite, protects PC12 cells from cysteinyl-dopamine but not from the hypochlorite product, suggesting that the product, not cysteinyl-dopamine itself, is toxic. Furthermore, rotenone, which enhances expression of the hypochlorite-producing enzyme myeloperoxidase, increases the cytotoxicity of cysteinyl-dopamine but not of the hypochlorite product. This suggests that dopamine oxidation to cysteinyl-dopamine followed by hypochlorite-dependent conversion to a cytotoxic redox-cycling product leads to the generation of reactive oxygen species and oxidative stress and may contribute to the death of dopaminergic neurons.

An Alkynyl-Fucose Halts Hepatoma Cell Migration and Invasion by Inhibiting GDP-Fucose-Synthesizing Enzyme FX, TSTA3.

  • Kizuka Y
  • Cell Chem Biol
  • 2017 Dec 21

Literature context:


Abstract:

Fucosylation is a glycan modification critically involved in cancer and inflammation. Although potent fucosylation inhibitors are useful for basic and clinical research, only a few inhibitors have been developed. Here, we focus on a fucose analog with an alkyne group, 6-alkynyl-fucose (6-Alk-Fuc), which is used widely as a detection probe for fucosylated glycans, but is also suggested for use as a fucosylation inhibitor. Our glycan analysis using lectin and mass spectrometry demonstrated that 6-Alk-Fuc is a potent and general inhibitor of cellular fucosylation, with much higher potency than the existing inhibitor, 2-fluoro-fucose (2-F-Fuc). The action mechanism was shown to deplete cellular GDP-Fuc, and the direct target of 6-Alk-Fuc is FX (encoded by TSTA3), the bifunctional GDP-Fuc synthase. We also show that 6-Alk-Fuc halts hepatoma invasion. These results highlight the unappreciated role of 6-Alk-Fuc as a fucosylation inhibitor and its potential use for basic and clinical science.

Funding information:
  • NIGMS NIH HHS - GM068763(United States)

Calreticulin Is Involved in Invasion of Human Extravillous Trophoblasts Through Functional Regulation of Integrin β1.

  • Yamamoto M
  • Endocrinology
  • 2017 Nov 1

Literature context:


Abstract:

Calreticulin (CRT), a molecular chaperone in the endoplasmic reticulum (ER), plays a variety of roles in cell growth, differentiation, apoptosis, immunity, and cancer biology. It has been reported that CRT is expressed in the human placenta, although its function in placental development is poorly understood. Appropriate invasion of extravillous trophoblasts (EVTs) into the maternal decidua is necessary for successful pregnancy. The objective of the present study was to investigate the expression and functional role of CRT in EVTs using the human EVT cell line HTR8/SVneo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in the human placenta in the early stage of pregnancy and localized to the EVTs. CRT knockdown markedly suppressed the invasion ability of HTR8/SVneo cells. Furthermore, the adhesion to fibronectin was suppressed in the CRT-knockdown cells via the dysfunction of integrin α5β1. In the CRT-knockdown cells, terminal sialylation and fucosylation were decreased, and the core galactose-containing structure was increased in the N-glycans of integrin β1. In addition, the expression levels of several critical glycosyltransferases were changed in the CRT-knockdown cells, consistent with the changes in the N-glycans. These results showed that CRT regulates the function of integrin β1 by affecting the synthesis of N-glycans in HTR8/SVneo cells. Collectively, the results of the present study demonstrate that the ER chaperone CRT plays a regulatory role in the invasion of EVTs, suggesting the importance of CRT expression in placental development during early pregnancy.

'Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum.

  • Sargent D
  • J. Neurochem.
  • 2017 Oct 3

Literature context:


Abstract:

The M83 transgenic mouse is a model of human synucleinopathies that develops severe motor impairment correlated with accumulation of the pathological Ser129-phosphorylated α-synuclein (α-synP ) in the brain and spinal cord. M83 disease can be accelerated by intracerebral inoculation of brain extracts from sick M83 mice. This has also recently been described using peripheral routes, injecting recombinant preformed α-syn fibrils into the muscle or the peritoneum. Here, we inoculated homozygous and/or hemizygous M83 neonates via the intraperitoneal and/or intracerebral routes with two different brain extracts: one from sick M83 mice inoculated with brain extract from other sick M83 mice, and the other derived from a human multiple system atrophy source passaged in M83 mice. Detection of α-synP using ELISA and western blot confirmed the disease in mice. The distribution of α-synP in the central nervous system was similar, independently of the inoculum or inoculation route, consistent with previous studies describing M83 disease. ELISA tests revealed higher levels of α-synP in homozygous than in hemizygous sick M83 mice, at least after IC inoculation. Interestingly, the immunoreactivity of α-synP detected by ELISA was significantly lower in M83 mice inoculated with the multiple system atrophy inoculum than in M83 mice inoculated with the M83 inoculum, at the first two passages. 'Prion-like' propagation of the synucleinopathy up to the clinical disease was accelerated by both intracerebral and intraperitoneal inoculations of brain extracts from sick mice. This acceleration, however, depends on the levels of α-syn expression by the mouse and the type of inoculum.

Outward-Open Model of Thyroid Hormone Transporter Monocarboxylate Transporter 8 Provides Novel Structural and Functional Insights.

  • Groeneweg S
  • Endocrinology
  • 2017 Oct 1

Literature context:


Abstract:

Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). Mutations in MCT8 result in severe intellectual and motor disability known as the Allan-Herndon-Dudley syndrome (AHDS). Previous studies have provided valuable insights into the putative mechanism of substrate binding in the inward-open conformation, required for TH efflux. The current study aims to delineate the mechanism of substrate binding in the outward-open conformation, required for TH uptake. Extensive chemical modification and site-directed mutagenesis studies were used to guide protein homology modeling of MCT8 in the outward-open conformation. Arg271 and Arg445 were modified by phenylglyoxal, which was partially prevented in the presence of substrate. Substrate docking in our outward-open model suggested an important role for His192 and Arg445 in substrate binding. Interestingly, mutations affecting these residues have been identified in patients who have AHDS. In addition, our outward-open model predicted the location of Phe189, Met227, Phe279, Gly282, Phe287, and Phe501 at the substrate-binding center, and their Ala substitution differentially affected the apparent Vmax and Km of T3 transport, with F189A, F279A, and F287A showing the highest impact. Thus, here we present an MCT8 homology model in the outward-open conformation, which supports the important role of His192 and Arg445 in substrate docking and identifies critical residues at the putative substrate-binding center. Our findings provide insights into MCT8 structure and function, which add to our understanding of the pathogenic mechanism of mutations found in patients who have AHDS and can be used to screen for novel substrates and inhibitors.

Sonic Hedgehog switches on Wnt/planar cell polarity signaling in commissural axon growth cones by reducing levels of Shisa2.

  • Onishi K
  • Elife
  • 2017 Sep 8

Literature context:


Abstract:

Commissural axons switch on responsiveness to Wnt attraction during midline crossing and turn anteriorly only after exiting the floor plate. We report here that Sonic Hedgehog (Shh)-Smoothened signaling downregulates Shisa2, which inhibits the glycosylation and cell surface presentation of Frizzled3 in rodent commissural axon growth cones. Constitutive Shisa2 expression causes randomized turning of post-crossing commissural axons along the anterior-posterior (A-P) axis. Loss of Shisa2 led to precocious anterior turning of commissural axons before or during midline crossing. Post-crossing commissural axon turning is completely randomized along the A-P axis when Wntless, which is essential for Wnt secretion, is conditionally knocked out in the floor plate. This regulatory link between Shh and planar cell polarity (PCP) signaling may also occur in other developmental processes.

RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2.

  • Zu T
  • Neuron
  • 2017 Sep 13

Literature context:


Abstract:

Several microsatellite-expansion diseases are characterized by the accumulation of RNA foci and RAN proteins, raising the possibility of a mechanistic connection. We explored this question using myotonic dystrophy type 2, a multisystemic disease thought to be primarily caused by RNA gain-of-function effects. We demonstrate that the DM2 CCTG⋅CAGG expansion expresses sense and antisense tetrapeptide poly-(LPAC) and poly-(QAGR) RAN proteins, respectively. In DM2 autopsy brains, LPAC is found in neurons, astrocytes, and glia in gray matter, and antisense QAGR proteins accumulate within white matter. LPAC and QAGR proteins are toxic to cells independent of RNA gain of function. RNA foci and nuclear sequestration of CCUG transcripts by MBNL1 is inversely correlated with LPAC expression. These data suggest a model that involves nuclear retention of expansion RNAs by RNA-binding proteins (RBPs) and an acute phase in which expansion RNAs exceed RBP sequestration capacity, are exported to the cytoplasm, and undergo RAN translation. VIDEO ABSTRACT.

Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages.

  • Escoll P
  • Cell Host Microbe
  • 2017 Sep 13

Literature context:


Abstract:

The intracellular bacteria Legionella pneumophila encodes a type IV secretion system (T4SS) that injects effector proteins into macrophages in order to establish and replicate within the Legionella-containing vacuole (LCV). Once generated, the LCV interacts with mitochondria through unclear mechanisms. We show that Legionella uses both T4SS-independent and T4SS-dependent mechanisms to respectively interact with mitochondria and induce mitochondrial fragmentation that ultimately alters mitochondrial metabolism. The T4SS effector MitF, a Ran GTPase activator, is required for fission of the mitochondrial network. These effects of MitF occur through accumulation of mitochondrial DNM1L, a GTPase critical for fission. Furthermore mitochondrial respiration is abruptly halted in a T4SS-dependent manner, while T4SS-independent upregulation of cellular glycolysis remains elevated. Collectively, these alterations in mitochondrial dynamics promote a Warburg-like phenotype in macrophages that favors bacterial replication. Hence the rewiring of cellular bioenergetics to create a replication permissive niche in host cells is a virulence strategy of L. pneumophila.

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.

  • Vujanovic M
  • Mol. Cell
  • 2017 Sep 7

Literature context:


Abstract:

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

Funding information:
  • NCI NIH HHS - R01 CA197774()
  • NIGMS NIH HHS - R01 GM116616()

Age exacerbates microglial activation, oxidative stress, inflammatory and NOX2 gene expression, and delays functional recovery in a middle-aged rodent model of spinal cord injury.

  • von Leden RE
  • J Neuroinflammation
  • 2017 Aug 18

Literature context:


Abstract:

BACKGROUND: Spinal cord injury (SCI) among people over age 40 has been steadily increasing since the 1980s and is associated with worsened outcome than injuries in young people. Age-related increases in reactive oxygen species (ROS) are suggested to lead to chronic inflammation. The NADPH oxidase 2 (NOX2) enzyme is expressed by microglia and is a primary source of ROS. This study aimed to determine the effect of age on inflammation, oxidative damage, NOX2 gene expression, and functional performance with and without SCI in young adult (3 months) and middle-aged (12 months) male rats. METHODS: Young adult and middle-aged rats were assessed in two groups-naïve and moderate contusion SCI. Functional recovery was determined by weekly assessment with the Basso, Beattie, and Breshnahan general motor score (analyzed two-way ANOVA) and footprint analysis (analyzed by Chi-square analysis). Tissue was analyzed for markers of oxidative damage (8-OHdG, Oxyblot, and 3-NT), microglial-related inflammation (Iba1), NOX2 component (p47PHOX, p22PHOX, and gp91PHOX), and inflammatory (CD86, CD206, TNFα, and NFκB) gene expression (all analyzed by unpaired Student's t test). RESULTS: In both naïve and injured aged rats, compared to young rats, tissue analysis revealed significant increases in 8-OHdG and Iba1, as well as inflammatory and NOX2 component gene expression. Further, injured aged rats showed greater lesion volume rostral and caudal to the injury epicenter. Finally, injured aged rats showed significantly reduced Basso-Beattie-Bresnahan (BBB) scores and stride length after SCI. CONCLUSIONS: These results show that middle-aged rats demonstrate increased microglial activation, oxidative stress, and inflammatory gene expression, which may be related to elevated NOX2 expression, and contribute to worsened functional outcome following injury. These findings are essential to elucidating the mechanisms of age-related differences in response to SCI and developing age-appropriate therapeutics.

Funding information:
  • NINDS NIH HHS - F31 NS090737()
  • NINDS NIH HHS - R01 NS073667()

Ryk controls remapping of motor cortex during functional recovery after spinal cord injury.

  • Hollis ER
  • Nat. Neurosci.
  • 2017 Aug 29

Literature context:


Abstract:

Limited functional recovery can be achieved through rehabilitation after incomplete spinal cord injury. Eliminating the function of a repulsive Wnt receptor, Ryk, in mice and rats by either conditional knockout in the motor cortex or monoclonal antibody infusion resulted in increased corticospinal axon collateral branches with presynaptic puncta in the spinal cord and enhanced recovery of forelimb reaching and grasping function following a cervical dorsal column lesion. Using optical stimulation, we observed that motor cortical output maps underwent massive changes after injury and that hindlimb cortical areas were recruited to control the forelimb over time. Furthermore, a greater cortical area was dedicated to controlling the forelimb in Ryk conditional knockout mice than in controls (wild-type or heterozygotes). In the absence of weekly task-specific training, recruitment of ectopic cortical areas was greatly reduced and there was no significant functional recovery even in Ryk conditional knockout mice. Our study provides evidence that maximal circuit reorganization and functional recovery can be achieved by combining molecular manipulation and targeted rehabilitation.

RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks.

  • Dungrawala H
  • Mol. Cell
  • 2017 Aug 3

Literature context:


Abstract:

RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

Funding information:
  • NCI NIH HHS - P01 CA092584()
  • NIGMS NIH HHS - R01 GM116616()

DGAT1-Dependent Lipid Droplet Biogenesis Protects Mitochondrial Function during Starvation-Induced Autophagy.

  • Nguyen TB
  • Dev. Cell
  • 2017 Jul 10

Literature context:


Abstract:

Lipid droplets (LDs) provide an "on-demand" source of fatty acids (FAs) that can be mobilized in response to fluctuations in nutrient abundance. Surprisingly, the amount of LDs increases during prolonged periods of nutrient deprivation. Why cells store FAs in LDs during an energy crisis is unknown. Our data demonstrate that mTORC1-regulated autophagy is necessary and sufficient for starvation-induced LD biogenesis. The ER-resident diacylglycerol acyltransferase 1 (DGAT1) selectively channels autophagy-liberated FAs into new, clustered LDs that are in close proximity to mitochondria and are lipolytically degraded. However, LDs are not required for FA delivery to mitochondria but instead function to prevent acylcarnitine accumulation and lipotoxic dysregulation of mitochondria. Our data support a model in which LDs provide a lipid buffering system that sequesters FAs released during the autophagic degradation of membranous organelles, reducing lipotoxicity. These findings reveal an unrecognized aspect of the cellular adaptive response to starvation, mediated by LDs.

Funding information:
  • NIDDK NIH HHS - R00 DK095921()
  • NIGMS NIH HHS - R01 GM112948()

PRMT1 regulates astrocytic differentiation of embryonic neural stem/precursor cells.

  • Honda M
  • J. Neurochem.
  • 2017 Jul 11

Literature context:


Abstract:

Arginine methylation is a post-translational modification which is catalyzed by protein arginine methyltransferases (PRMTs). Here, we report that PRMT1 is highly expressed in neural stem/precursor cells (NS/PCs) of mouse embryos, and knockdown of PRMT1 in NS/PCs suppresses the generation of astrocytes. The luciferase assay demonstrated that knockdown of PRMT1 inhibits activation of the promoter of a typical astrocytic marker gene, glial fibrillary acidic protein (Gfap), in NS/PCs. The transcription factor signal transducer and activator of transcription 3 (STAT3) is known to generally be critical for astrocytic differentiation of NS/PCs. We found that PRMT1 methylates arginine residue(s) of STAT3 to regulate its activity positively, resulting in the promotion of astrocytic differentiation of NS/PCs.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat.

  • Sidorova YA
  • Front Pharmacol
  • 2017 Jul 6

Literature context:


Abstract:

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

Serine ADP-ribosylation reversal by the hydrolase ARH3.

  • Fontana P
  • Elife
  • 2017 Jun 26

Literature context:


Abstract:

ADP-ribosylation (ADPr) is a posttranslational modification (PTM) of proteins that controls many cellular processes, including DNA repair, transcription, chromatin regulation and mitosis. A number of proteins catalyse the transfer and hydrolysis of ADPr, and also specify how and when the modification is conjugated to the targets. We recently discovered a new form of ADPr that is attached to serine residues in target proteins (Ser-ADPr) and showed that this PTM is specifically made by PARP1/HPF1 and PARP2/HPF1 complexes. In this work, we found by quantitative proteomics that histone Ser-ADPr is reversible in cells during response to DNA damage. By screening for the hydrolase that is responsible for the reversal of Ser-ADPr, we identified ARH3/ADPRHL2 as capable of efficiently and specifically removing Ser-ADPr of histones and other proteins. We further showed that Ser-ADPr is a major PTM in cells after DNA damage and that this signalling is dependent on ARH3.

Elucidating the Role of the Desmosome Protein p53 Apoptosis Effector Related to PMP-22 in Growth Hormone Tumors.

  • Kiseljak-Vassiliades K
  • Endocrinology
  • 2017 May 1

Literature context:


Abstract:

Densely granulated and sparsely granulated (SG) growth hormone (GH) pituitary adenomas differ in biological behavior, which may be correlated with their known differences in cytoplasmic keratin distribution and E-cadherin expression. We wanted to explore candidate genes that might further explain this behavior. Exon expression microarray was performed on 21 GH tumors (10 SG and 11 densely granulated) and 20 normal control pituitaries from autopsy. Bioinformatic analyses confirmed a differential molecular signature between normal pituitary and GH tumors as well as between the GH tumor subtypes. There was a consistent downregulation of transcripts involved in the structure and function of the desmosome, including desmoplakin (eightfold), desmoglein 2 (sixfold), plakophilin 2 (sevenfold), and p53 apoptosis effector related to PMP-22 (PERP; sixfold) in SG tumors compared with normal pituitary. PERP is lost in more aggressive SG human GH pituitary tumors. PERP re-expression in GH3 rat GH tumor cells resulted in decreased colony formation compared with vector transfectants, confirming the role of PERP as a tumor suppressor with no effects on proliferation. Increased PERP expression was associated with loss of a survival advantage in a hypoxic environment, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (P < 0.05) and cleaved caspase-3 (P < 0.05). Downregulation of desmosomal formation transcripts including PERP may contribute to the aggressive phenotype seen in SG GH pituitary tumors and their behavior in response to surgery and medical therapy.

Funding information:
  • NCI NIH HHS - K12 CA086913()
  • NCI NIH HHS - P30 CA046934()

Follistatin Targets Distinct Pathways To Promote Brown Adipocyte Characteristics in Brown and White Adipose Tissues.

  • Singh R
  • Endocrinology
  • 2017 May 1

Literature context:


Abstract:

We previously demonstrated that Fst expression is highest in brown adipose tissue (BAT) and skeletal muscle, but is also present at substantial levels in epididymal and subcutaneous white adipose tissues (WATs). Fst promotes mouse brown preadipocyte differentiation and promotes browning during differentiation of mouse embryonic fibroblasts. Fst-transgenic (Fst-Tg) mice show substantial increases in circulating Fst levels and increased brown adipose mass. BAT of Fst-Tg mice had increased expression of brown adipose-associated markers including uncoupling protein 1 (UCP1), PRDM16, PGC-1α, and Glut4. WATs from Fst-Tg mice show upregulation of brown/beige adipose markers and significantly increased levels of phosphorylated p38 MAPK/ERK1/2 proteins compared with the wild-type (WT) mice. Pharmacological inhibition of pp38 MAPK/pERK1/2 pathway of recombinant mouse Fst (rFst) treated differentiating 3T3-L1 cells led to significant blockade of Fst-induced UCP1 protein expression. On the other hand, BAT from Fst-Tg mice or differentiating mouse BAT cells treated with rFst show dramatic increase in Myf5 protein levels as well as upregulation of Zic1 and Lhx8 gene expression. Myf5 levels were significantly downregulated in Fst knock-out embryos and small inhibitory RNA-mediated inhibition of Myf5 led to significant inhibition of UCP1, Lhx8, and Zic1 gene expression and significant blockade of Fst-induced induction of UCP1 protein expression in mouse BAT cells. Both interscapular BAT and WAT tissues from Fst-Tg mice display enhanced response to CL316,243 treatment and decreased expression of pSmad3 compared with the WT mice. Therefore, our results indicate that Fst promotes brown adipocyte characteristics in both WAT and BAT depots in vivo through distinct mechanisms.

Funding information:
  • NIA NIH HHS - SC1 AG049682()
  • NIMHD NIH HHS - S21 MD000103()
  • NIMHD NIH HHS - U54 MD007598()

CRISPR/Cas9 Screens Reveal Epstein-Barr Virus-Transformed B Cell Host Dependency Factors.

  • Ma Y
  • Cell Host Microbe
  • 2017 May 10

Literature context:


Abstract:

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify host dependency factors resulting from these EBV+, B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets. Our systematic approach uncovered key mechanisms by which EBV oncoproteins activate the PI3K/AKT pathway and evade tumor suppressor responses. LMP1-induced cFLIP was found to be critical for LCL defense against TNFα-mediated programmed cell death, whereas EBV-induced BATF/IRF4 were critical for BIM suppression and MYC induction in LCLs. Finally, EBV super-enhancer-targeted IRF2 protected LCLs against Blimp1-mediated tumor suppression. Our results identify viral transformation-driven synthetic lethal targets for therapeutic intervention.

Funding information:
  • NIAID NIH HHS - R01 AI123420()

Pharmacogenetic variants in TPMT alter cellular responses to cisplatin in inner ear cell lines.

  • Bhavsar AP
  • PLoS ONE
  • 2017 Apr 13

Literature context:


Abstract:

Cisplatin is a highly-effective and widely-used chemotherapeutic agent that causes ototoxicity in many patients. Pharmacogenomic studies of key genes controlling drug biotransformation identified variants in thiopurine methyltransferase (TPMT) as predictors of cisplatin-induced ototoxicity, although the mechanistic basis of this interaction has not been reported. Expression constructs of TPMT*3A, *3B and *3C variants were generated and monitored in cultured cells. Cellular TPMT*3A levels were detected at >20-fold lower amounts than the wild type confirming the unstable nature of this variant. The expression of wild type TPMT (TPMT*1) in two murine ear cell lines, HEI-OC1 and UB/OC-1, significantly mitigated their susceptibility to cisplatin toxicity. Cisplatin treatment induced Tlr4 gene expression in HEI-OC1 cells and this response was blunted by the expression of wild type TPMT but not TPMT*3A. In line with the significant mitigation of TPMT*1-expressing cells to cisplatin cytotoxicity, these findings demonstrate a drug-gene interaction between increased TPMT activity and decreased susceptibility to cisplatin-induced toxicity of inner ear cells.

Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo.

  • Leanza L
  • Cancer Cell
  • 2017 Apr 10

Literature context:


Abstract:

The potassium channel Kv1.3 is highly expressed in the mitochondria of various cancerous cells. Here we show that direct inhibition of Kv1.3 using two mitochondria-targeted inhibitors alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death of even chemoresistant cells independently of p53 status. These inhibitors killed 98% of ex vivo primary chronic B-lymphocytic leukemia tumor cells while sparing healthy B cells. In orthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma, the compounds reduced tumor size by more than 90% and 60%, respectively, while sparing immune and cardiac functions. Our work provides direct evidence that specific pharmacological targeting of a mitochondrial potassium channel can lead to ROS-mediated selective apoptosis of cancer cells in vivo, without causing significant side effects.

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells.

  • Lobingier BT
  • Cell
  • 2017 Apr 6

Literature context:


Abstract:

Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.

Funding information:
  • NCI NIH HHS - R01 CA186568()
  • NIGMS NIH HHS - P50 GM082250()

Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development.

  • Bulusu V
  • Dev. Cell
  • 2017 Feb 27

Literature context:


Abstract:

How metabolism is rewired during embryonic development is still largely unknown, as it remains a major technical challenge to resolve metabolic activities or metabolite levels with spatiotemporal resolution. Here, we investigated metabolic changes during development of organogenesis-stage mouse embryos, focusing on the presomitic mesoderm (PSM). We measured glycolytic labeling kinetics from 13C-glucose tracing experiments and detected elevated glycolysis in the posterior, more undifferentiated PSM. We found evidence that the spatial metabolic differences are functionally relevant during PSM development. To enable real-time quantification of a glycolytic metabolite with spatiotemporal resolution, we generated a pyruvate FRET-sensor reporter mouse line. We revealed dynamic changes in cytosolic pyruvate levels as cells transit toward a more anterior PSM state. Combined, our approach identifies a gradient of glycolytic activity across the PSM, and we provide evidence that these spatiotemporal metabolic changes are intrinsically linked to PSM development and differentiation.

Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice.

  • Jiang BC
  • J. Neurosci.
  • 2017 Jan 18

Literature context:


Abstract:

DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under neuropathic pain condition remain largely unknown. Here we investigated how chemokine receptor CXCR3 is regulated by DNA methylation and how it contributes to neuropathic pain induced by spinal nerve ligation (SNL) in mice. SNL increased Cxcr3 mRNA and protein expression in the neurons of the spinal cord. Meanwhile, the CpG (5'-cytosine-phosphate-guanine-3') island in the Cxcr3 gene promoter region was demethylated, and the expression of DNA methyltransferase 3b (DNMT3b) was decreased. SNL also increased the binding of CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)/enhancer binding protein α (C/EBPα) with Cxcr3 promoter and decreased the binding of DNMT3b with Cxcr3 promoter in the spinal cord. C/EBPα expression was increased in spinal neurons after SNL, and inhibition of C/EBPα by intrathecal small interfering RNA attenuated SNL-induced pain hypersensitivity and reduced Cxcr3 expression. Furthermore, SNL-induced mechanical allodynia and heat hyperalgesia were markedly reduced in Cxcr3-/- mice. Spinal inhibition of Cxcr3 by shRNA or CXCR3 antagonist also attenuated established neuropathic pain. Moreover, CXCL10, the ligand of CXCR3, was increased in spinal neurons and astrocytes after SNL. Superfusing spinal cord slices with CXCL10 enhanced spontaneous EPSCs and potentiated NMDA-induced and AMPA-induced currents of lamina II neurons. Finally, intrathecal injection of CXCL10 induced CXCR3-dependent pain hypersensitivity in naive mice. Collectively, our results demonstrated that CXCR3, increased by DNA demethylation and the enhanced interaction with C/EBPα, can be activated by CXCL10 to facilitate excitatory synaptic transmission and contribute to the maintenance of neuropathic pain. SIGNIFICANCE STATEMENT: Peripheral nerve injury induces changes of gene expression in the spinal cord that may contribute to the pathogenesis of neuropathic pain. CXCR3 is a chemokine receptor. Whether it is involved in neuropathic pain and how it is regulated after nerve injury remain largely unknown. Our study demonstrates that spinal nerve ligation downregulates the expression of DNMT3b, which may cause demethylation of Cxcr3 gene promoter and facilitate the binding of CCAAT/enhancer binding protein α with Cxcr3 promoter and further increase CXCR3 expression in spinal neurons. The upregulated CXCR3 may contribute to neuropathic pain by facilitating central sensitization. Our study reveals an epigenetic mechanism underlying CXCR3 expression and also suggests that targeting the expression or activation of CXCR3 signaling may offer new therapeutics for neuropathic pain.

Ligand and Target Discovery by Fragment-Based Screening in Human Cells.

  • Parker CG
  • Cell
  • 2017 Jan 26

Literature context:


Abstract:

Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

Funding information:
  • NCI NIH HHS - R01 CA132630()
  • NIDDK NIH HHS - R24 DK099810()
  • NIH HHS - S10 OD016357()

Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

  • Phua SC
  • Cell
  • 2017 Jan 12

Literature context:


Abstract:

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Funding information:
  • NIAMS NIH HHS - R01 AR054396()
  • NIDDK NIH HHS - R01 DK102910()
  • NIGMS NIH HHS - DP2 GM105448()
  • NIGMS NIH HHS - R01 GM095941()
  • NIGMS NIH HHS - R01 GM112988()

Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization.

  • O'Grady GL
  • Am. J. Hum. Genet.
  • 2016 Nov 3

Literature context:


Abstract:

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.

  • Verissimo CS
  • Elife
  • 2016 Nov 15

Literature context:


Abstract:

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.

Funding information:
  • NIMH NIH HHS - MH-095972(United States)

A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins.

  • Sung MK
  • Elife
  • 2016 Aug 23

Literature context:


Abstract:

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.

Funding information:
  • NINDS NIH HHS - R01 NS036715(United States)
  • NINDS NIH HHS - R01 NS078434(United States)

Sodium-dependent vitamin C transporter-2 mediates vitamin C transport at the cortical nerve terminal.

  • Pierce MR
  • J. Neurosci. Res.
  • 2016 Jul 27

Literature context:


Abstract:

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

The Role of Sonic Hedgehog in the Specification of Human Cortical Progenitors In Vitro.

  • Radonjić NV
  • Cereb. Cortex
  • 2016 Jan 15

Literature context:


Abstract:

Impaired sonic hedgehog (Shh) signaling is involved in the pathology of cortical formation found in neuropsychiatric disorders. However, its role in the specification of human cortical progenitors is not known. Here, we report that Shh is expressed in the human developing cortex at mid-gestation by radial glia cells (RGCs) and cortical neurons. We used RGC cultures, established from the dorsal (cortical) telencephalon of human brain at mid-gestation to study the effect of Shh signaling. Cortical RGCs in vitro maintained their regional characteristics, expressed components of Shh signaling, and differentiated into Nkx2.1, Lhx6, and calretinin-positive (CalR(+)) cells, potential cortical interneuron progenitors. Treatment with exogenous Shh increased the pool of Nkx2.1(+) progenitors, decreased Lhx6 expression, and suppressed the generation of CalR(+) cells. The blockade of endogenous Shh signaling increased the number of CalR(+) cells, but did not affect Nkx2.1 expression, implying the existence of parallel Shh-independent pathways for cortical Nkx2.1 regulation. These results support the idea that, during human brain development, Shh plays an important role in the specification of cortical progenitors. Since direct functional studies in humans are limited, the in vitro system that we established here could be of great interest for modeling the development of human cortical progenitors.

Transport of Iodothyronines by Human L-Type Amino Acid Transporters.

  • Zevenbergen C
  • Endocrinology
  • 2015 Nov 17

Literature context:


Abstract:

Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1-5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM (125)I-labeled T4, T3, rT3, 3,3'-T2, and 10 μM [(125)I]3'-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3'-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3'-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3'-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3'-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state.

Funding information:
  • NEI NIH HHS - R01 EY022369(United States)
  • NIDDK NIH HHS - DK52574(United States)

Antinociceptive roles of galanin receptor 1 in nucleus accumbens of rats in a model of neuropathic pain.

  • Duan H
  • J. Neurosci. Res.
  • 2015 Oct 20

Literature context:


Abstract:

It has been reported that galanin and its receptors might be involved in the modulation and transmission of nociception in the central nervous system. Our previous research has also demonstrated that galanin induces antinociception in the nucleus accumbens (NAc) of intact rats. However, the interaction between galanin and its receptors in the NAc and the underlying mechanism of suppressing pain transmission remain unclear. The present study seeks to determine the antinociception induced by galanin receptor (GalR)-1 stimulation in the NAc of rats with neuropathic pain. The left sciatic nerve of rats was ligated to mimic a neuropathic pain model. Western blots showed that the expression of GalR1 was significantly upregulated in the NAc of rats with neuropathic pain. Intra-NAc injection of GalR1 agonist M617 induced a dose-dependent increase in hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulations in rats with neuropathic pain. Also, the effect of M617 was attenuated by M35, a GalR1/2 antagonist; at the same time, M35 reduced the galanin-induced antinociception, suggesting that GalR1 mediates antinociception induced by galanin in the NAc of rats with neuropathic pain. Furthermore, we found that M617-induced antinociception in rats with neuropathic pain was stronger than the antinociception in intact rats. We also found that injections of M617 and galanin each induced significant increases in HWL, but the galanin-induced antinociception was stronger than that of M617. All these results suggest that GalR1 plays an important role in antinociception and that other GalRs also are involved in pain modulation induced by galanin in the NAc of rats with neuropathic pain.

Funding information:
  • NIMH NIH HHS - MH081968(United States)

The Selective Estrogen Receptor Modulator Raloxifene Regulates Arginine-Vasopressin Gene Expression in Human Female Neuroblastoma Cells Through G Protein-Coupled Estrogen Receptor and ERK Signaling.

  • Grassi D
  • Endocrinology
  • 2015 Oct 19

Literature context:


Abstract:

The selective estrogen receptor modulator raloxifene reduces blood pressure in hypertensive postmenopausal women. In the present study we have explored whether raloxifene regulates gene expression of arginine vasopressin (AVP), which is involved in the pathogenesis of hypertension. The effect of raloxifene was assessed in human female SH-SY5Y neuroblastoma cells, which have been recently identified as a suitable cellular model to study the estrogenic regulation of AVP. Raloxifene, within a concentration ranging from 10(-10) M to 10(-6) M, decreased the mRNA levels of AVP in SH-SY5Y cells with maximal effect at 10(-7) M. This effect of raloxifene was imitated by an agonist (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone of G protein-coupled estrogen receptor-1 (GPER) and blocked by an antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline of GPER and by GPER silencing. Raloxifene induced a time-dependent increase in the level of phosphorylated ERK1 and ERK2, by a mechanism blocked by the GPER antagonist. The treatment of SH-SY5Y cells with either a MAPK/ERK kinase 1/2-specific inhibitor (1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadine) or a protein kinase C inhibitor (sotrastaurin) blocked the effects of raloxifene on the phosphorylation of ERK1/2 and the regulation of AVP mRNA levels. These results reveal a mechanism mediating the regulation of AVP expression by raloxifene, involving the activation of GPER, which in turn activates protein kinase C, MAPK/ERK kinase, and ERK. The regulation of AVP by raloxifene and GPER may have implications for the treatment of blood hypertension(.).

Funding information:
  • NIDA NIH HHS - P30 DA035756(United States)
  • NIMH NIH HHS - R01 MH091258(United States)

Fingolimod induces the transition to a nerve regeneration promoting Schwann cell phenotype.

  • Heinen A
  • Exp. Neurol.
  • 2015 Sep 28

Literature context:


Abstract:

Successful regeneration of injured peripheral nerves is mainly attributed to the plastic behavior of Schwann cells. Upon loss of axons, these cells trans-differentiate into regeneration promoting repair cells which provide trophic support to regrowing axons. Among others, activation of cJun was revealed to be involved in this process, initiating the stereotypic pattern of Schwann cell phenotype alterations during Wallerian degeneration. Nevertheless, the ability of Schwann cells to adapt and therefore the nerve's potential to regenerate can be limited in particular after long term denervation or in neuropathies leading to incomplete regeneration only and thus emphasizing the need for novel therapeutic approaches. Here we stimulated primary neonatal and adult rat Schwann cells with Fingolimod/FTY720P and investigated its impact on the regeneration promoting phenotype. FTY720P activated a number of de-differentiation markers including cJun and interfered with maturation marker and myelin expression. Functionally, FTY720P treated Schwann cells upregulated growth factor expression and these cells enhanced dorsal root ganglion neurite outgrowth on inhibitory substrates. Our results therefore provide strong evidence that FTY720P application supports the generation of a repair promoting cellular phenotype and suggest that Fingolimod could be used as treatment for peripheral nerve injuries and diseases.

Erythropoietin produced by genetic-modified NIH/3T3 fibroblasts enhances the survival of degenerating neurons.

  • Li YC
  • Brain Behav
  • 2015 Aug 11

Literature context:


Abstract:

BACKGROUND: Erythropoietin (EPO) has potent neuroprotective effects. The short-term delivery of high-dose EPO seemed to improve patients' neuromuscular functions; however, excessive EPO resulted in systematically high hematocrit and thrombotic risk. In our study, we established a cellular material for future in vivo studies of neurodegenerative diseases based on EPO provided regionally at a nontoxic level. METHODS: A mouse EPO cDNA was subcloned into the pCMS-EGFP vector and transfected into NIH/3T3 fibroblasts to design a biological provider that can regionally release EPO for the treatment of neurological diseases. After G418 selection, a stable EPO-overexpressing cell line, EPO-3T3-EGFP, was established. To further confirm the neuroprotective abilities of secreted EPO from EPO-3T3-EGFP cells, a cell model of neurodegeneration, PC12-INT-EGFP, was applied. RESULTS: The expression level of EPO was highly elevated in EPO-3T3-EGFP cells, and an abundant amount of EPO secreted from EPO-3T3-EGFP cells was detected in the extracellular milieu. After supplementation with conditioned medium prepared from EPO-3T3-EGFP cells, the survival rate of PC12-INT-EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP-tagged α-internexin in PC12-INT-EGFP cells was disaggregated and transported into neurites dynamically. The immunocytochemical distribution of IF proteins, including NF-M, phosphorylated-NF-M, and the α-INT-EGFP fusion protein, were less aggregated in the perikaryal region and transported into neurites after the EPO treatment. CONCLUSION: The established EPO-overexpressing NIH/3T3 cell line, EPO-3T3-EGFP, may provide a material for future studies of cell-based therapies for neurodegenerative diseases via the secretion of EPO on a short-term, high-dose, regional basis.

Classical and membrane-initiated estrogen signaling in an in vitro model of anterior hypothalamic kisspeptin neurons.

  • Mittelman-Smith MA
  • Endocrinology
  • 2015 Jun 18

Literature context:


Abstract:

The neuropeptide kisspeptin is essential for sexual maturation and reproductive function. In particular, kisspeptin-expressing neurons in the anterior rostral periventricular area of the third ventricle are generally recognized as mediators of estrogen positive feedback for the surge release of LH, which stimulates ovulation. Estradiol induces kisspeptin expression in the neurons of the rostral periventricular area of the third ventricle but suppresses kisspeptin expression in neurons of the arcuate nucleus that regulate estrogen-negative feedback. To focus on the intracellular signaling and response to estradiol underlying positive feedback, we used mHypoA51 cells, an immortalized line of kisspeptin neurons derived from adult female mouse hypothalamus. mHypoA51 neurons express estrogen receptor (ER)-α, classical progesterone receptor (PR), and kisspeptin, all key elements of estrogen-positive feedback. As with kisspeptin neurons in vivo, 17β-estradiol (E2) induced kisspeptin and PR in mHypoA51s. The ERα agonist, 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, produced similar increases in expression, indicating that these events were mediated by ERα. However, E2-induced PR up-regulation required an intracellular ER, whereas kisspeptin expression was stimulated through a membrane ER activated by E2 coupled to BSA. These data suggest that anterior hypothalamic kisspeptin neurons integrate both membrane-initiated and classical nuclear estrogen signaling to up-regulate kisspeptin and PR, which are essential for the LH surge.

Funding information:
  • NIDDK NIH HHS - R01 DK103723(United States)

Activation of the dopamine receptor type-2 (DRD2) promoter by 9-cis retinoic acid in a cellular model of Cushing's disease mediates the inhibition of cell proliferation and ACTH secretion without a complete corticotroph-to-melanotroph transdifferentiation.

  • Occhi G
  • Endocrinology
  • 2014 Sep 25

Literature context:


Abstract:

Cushing's disease (CD) is a rare condition in which hypercortisolemia is secondary to excessive ACTH release from a pituitary corticotroph adenoma. CD is associated with significant morbidity and mortality, and a safe therapy that effectively targets the pituitary tumor is still lacking. Retinoic acid (RA) and dopamine agonists (DAs) have recently been considered as monotherapy in CD patients, and satisfactory results have been reported, albeit in a limited number of patients. Given the permissive role of RA on the dopamine receptor type-2 (DRD2), the aim of the present study was to see whether a combination of 9-cis RA and the DA bromocriptine (Br) might represent a possible treatment for CD. Here we show that 9-cis RA induces a functional DRD2 in the pituitary corticotroph cell line AtT20, and increases cell sensitivity to Br via a mechanism only partially related to corticotroph-to-melanotroph transdifferentiation. In addition, 9-cis RA and Br act synergistically to modulate cell viability, with favorable implications for clinical use. In nearly 45% of corticotropinoma-derived primary cultures, the combined administration of 9-cis RA and Br lowered the steady-state level of the ACTH precursor proopiomelanocortin (POMC) more efficiently than either of the drugs alone. In conclusion, the effects of a combination of 9-cis RA and Br on ACTH synthesis/secretion and cell viability in AtT20, and on POMC transcriptional activity in human corticotropinomas might represent a suitable starting point for assessing the potential of this treatment regimen for ACTH-secreting pituitary adenomas. This study thus has potentially important implications for novel therapeutic approaches to CD.

Funding information:
  • NICHD NIH HHS - HD04612(United States)
  • NINDS NIH HHS - R44 NS074540(United States)

A change in liver metabolism but not in brown adipose tissue thermogenesis is an early event in ovariectomy-induced obesity in rats.

  • Nigro M
  • Endocrinology
  • 2014 Aug 19

Literature context:


Abstract:

Menopause is associated with increased visceral adiposity and disrupted glucose homeostasis, but the underlying molecular mechanisms related to these metabolic changes are still elusive. Brown adipose tissue (BAT) plays a key role in energy expenditure that may be regulated by sexual steroids, and alterations in glucose homeostasis could precede increased weight gain after ovariectomy. Thus, the aim of this work was to evaluate the metabolic pathways in both the BAT and the liver that may be disrupted early after ovariectomy. Ovariectomized (OVX) rats had increased food efficiency as early as 12 days after ovariectomy, which could not be explained by differences in feces content. Analysis of isolated BAT mitochondria function revealed no differences in citrate synthase activity, uncoupling protein 1 expression, oxygen consumption, ATP synthesis, or heat production in OVX rats. The addition of GDP and BSA to inhibit uncoupling protein 1 decreased oxygen consumption in BAT mitochondria equally in both groups. Liver analysis revealed increased triglyceride content accompanied by decreased levels of phosphorylated AMP-activated protein kinase and phosphorylated acetyl-CoA carboxylase in OVX animals. The elevated expression of gluconeogenic enzymes in OVX and OVX + estradiol rats was not associated with alterations in glucose tolerance test or in serum insulin but was coincident with higher glucose disposal during the pyruvate tolerance test. Although estradiol treatment prevented the ovariectomy-induced increase in body weight and hepatic triglyceride and cholesterol accumulation, it was not able to prevent increased gluconeogenesis. In conclusion, the disrupted liver glucose homeostasis after ovariectomy is neither caused by estradiol deficiency nor is related to increased body mass.

Funding information:
  • NINDS NIH HHS - NS050465(United States)

An adaptive role of TNFα in the regulation of striatal synapses.

  • Lewitus GM
  • J. Neurosci.
  • 2014 Apr 30

Literature context:


Abstract:

Elevation of inflammatory cytokines in the striatum precedes symptoms in a number of motor dysfunctions, but it is unclear whether this is part of the disease process or an adaptive response to the pathology. In pyramidal cells, TNFα drives the insertion of AMPA-type glutamate receptors into synapses, and contributes to the homeostatic regulation of circuit activity in the developing neocortex. Here we demonstrate that in the mouse dorsolateral striatum, TNFα drives the internalization of AMPARs and reduces corticostriatal synaptic strength, dephosphorylates DARPP-32 and GluA1, and results in a preferential removal of Ca(2+)-permeable AMPARs. Striatal TNFα signaling appears to be adaptive in nature, as TNFα is upregulated in response to the prolonged blockade of D2 dopamine receptors and is necessary to reduce the expression of extrapyramidal symptoms induced by chronic haloperidol treatment. These data indicate that TNFα is a regulator of glutamatergic synaptic strength in the adult striatum in a manner distinct from its regulation of synapses on pyramidal cells and mediates an adaptive response during pathological conditions.

Funding information:
  • NIGMS NIH HHS - R15 GM099054(United States)

The role of Arg445 and Asp498 in the human thyroid hormone transporter MCT8.

  • Groeneweg S
  • Endocrinology
  • 2014 Feb 22

Literature context:


Abstract:

Monocarboxylate transporter 8 (MCT8) facilitates cellular influx and efflux of the thyroid hormones (THs) T(4) and T(3). Mutations in MCT8 lead to severe psychomotor retardation. Here, we studied the importance of 2 highly conserved residues (Arg445 in transmembrane domain 8 and Asp498 in transmembrane domain 10) for substrate recognition and helix interactions. We introduced single and double mutations (R445A, R445C, R445D, R445K, D498A, D498E, D498N, D498R, R445A+D498A, R445D+D498R, and R445K+D498E) in human MCT8 cDNA and studied the effects on MCT8-mediated TH uptake and metabolism in transfected cells. The impact of these mutations on MCT8 protein expression, dimerization capacity, and subcellular localization was studied by Western blotting and confocal microscopy. We found that mutations in Arg445 or Asp498 that alter the local charge resulted in a near-complete loss of TH uptake capacity, whereas the expression, stability, and subcellular localization of these mutant proteins was similar to those for wild-type MCT8. Given the impaired TH uptake, TH efflux could not be adequately studied. The importance of opposite charges at Arg445 and Asp498 was studied by exchanging these residues (R445D+D498R). In particular, T(4) uptake was less severely reduced by the exchange mutation than by the single mutations. Mutations of Arg445 and Asp498 to equally charged residues (R445K and/or D498E) resulted in TH uptake levels similar to wild-type MCT8. The presence of 2 oppositely charged residues at positions Arg445 and Asp498 that are predicted in close structural proximity is crucial for efficient TH uptake, which may indicate the presence of an, at least transient, charge pair between these residues.

Funding information:
  • NIA NIH HHS - 2P01 AG026572-06(United States)
  • NINDS NIH HHS - NS 064135(United States)

EP2 receptor activates dual G protein signaling pathways that mediate contrasting proinflammatory and relaxatory responses in term pregnant human myometrium.

  • Kandola MK
  • Endocrinology
  • 2014 Feb 22

Literature context:


Abstract:

Prostaglandin (PG) E2 (PGE(2)) plays a central role in the regulation of smooth muscle contractions. Classically, PGE(2) stimulates contractions via EP1 and EP3 receptors, whereas EP2 and EP4 maintain quiescence. Labor involves a change from myometrial quiescence to contractions with a shift from anti- to proinflammatory pathways. EP2, a Gαs-coupled receptor, is known to mediate its actions via cAMP signaling. However, we have recently shown that EP2 also activates the proinflammatory PG G/H synthase-2 (PGHS-2). Here, we identify the mechanism underlying the ability of EP2 to maintain uterine quiescence and activate a proinflammatory/prolabor response in term-pregnant human myometrium. Human myometrial biopsies for in vivo and in vitro studies were taken at cesarean section at term, before or after the onset of labor. Activation of EP2 increased intracellular levels of cAMP and reduced contractility. Contrastingly, EP2 stimulation increased levels of PGHS-2, membrane-associated PGE synthase-1, and PGE(2). This was entirely dependent on EP2-mediated activation of calcium signaling. Both calcium signaling and up-regulation of PGHS-2 were insensitive to the Gαi inhibitor pertussis toxin but inhibited by small interfering RNA knockdown of Gαq/11. There were no differences in EP2 mRNA or protein levels between upper or lower segment myometrium or between pre- and postlabor myometrium. However, in myocytes taken after the onset of labor, cAMP signaling was markedly attenuated, whereas activation of calcium and PGHS-2 was preserved. Overall, the dual coupling of EP2 to Gαs-cAMP and Gαq/11-calcium pathways underlies its ability to mediate contrasting functions in term pregnancy and the "switching" to a prolabor receptor.

Funding information:
  • NINDS NIH HHS - R01 NS043330(United States)

Testosterone replacement ameliorates nonalcoholic fatty liver disease in castrated male rats.

  • Nikolaenko L
  • Endocrinology
  • 2014 Feb 22

Literature context:


Abstract:

Nonalcoholic fatty liver disease is common in developed countries and is associated with obesity, metabolic syndrome, and type 2 diabetes. T deficiency is a risk factor for developing these metabolic deficiencies, but its role in hepatic steatosis has not been well studied. We investigated the effects of T on the pathogenesis of hepatic steatosis in rats fed a high-fat diet (HFD). Adult male rats were randomly placed into four groups and treated for 15 weeks: intact rats on regular chow diet (RCD), intact rats on liquid HFD (I+HFD), castrated rats on HFD (C+HFD), and castrated rats with T replacement on HFD (C+HFD+T). Fat contributed 71% energy to the HFD but only 16% of energy to the RCD. Serum T level was undetectable in castrated rats, and T replacement led to 2-fold higher mean serum T levels than in intact rats. C+HFD rats gained less weight but had higher percentage body fat than C+HFD+T. Severe micro- and macrovesicular fat accumulated in hepatocytes with multiple inflammatory foci in the livers of C+HFD. I+HFD and C+HFD+T hepatocytes demonstrated only mild to moderate microvesicular steatosis. T replacement attenuated HFD-induced hepatocyte apoptosis in castrated rats. Serum glucose and insulin levels were not increased with HFD in any group. Immunoblots showed that insulin-regulated proteins were not changed in any group. This study demonstrates that T deficiency may contribute to the severity of hepatic steatosis and T may play a protective role in hepatic steatosis and nonalcoholic fatty liver disease development without insulin resistance.

Funding information:
  • NEI NIH HHS - R01-EY016155(United States)

Growth hormone secretion is correlated with neuromuscular innervation rather than motor neuron number in early-symptomatic male amyotrophic lateral sclerosis mice.

  • Steyn FJ
  • Endocrinology
  • 2013 Dec 25

Literature context:


Abstract:

GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1(G93A) mice throughout the course of ALS disease. Male wild-type and hSOD1(G93A) mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1(G93A) mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1(G93A) mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.

Funding information:
  • NIGMS NIH HHS - 5P20GM103636(United States)

Palmitoylation of estrogen receptors is essential for neuronal membrane signaling.

  • Meitzen J
  • Endocrinology
  • 2013 Nov 21

Literature context:


Abstract:

In addition to activating nuclear estrogen receptor signaling, 17β-estradiol can also regulate neuronal function via surface membrane receptors. In various brain regions, these actions are mediated by the direct association of estrogen receptors (ERs) activating metabotropic glutamate receptors (mGluRs). These ER/mGluR signaling partners are organized into discrete functional microdomains via caveolin proteins. A central question that remains concerns the underlying mechanism by which these subpopulations of ERs are targeted to the surface membrane. One candidate mechanism is S-palmitoylation, a posttranscriptional modification that affects the subcellular distribution and function of the modified protein, including promoting localization to membranes. Here we test for the role of palmitoylation and the necessity of specific palmitoylacyltransferase proteins in neuronal membrane ER action. In hippocampal neurons, pharmacological inhibition of palmitoylation eliminated 17β-estradiol-mediated phosphorylation of cAMP response element-binding protein, a process dependent on surface membrane ERs. In addition, mutation of the palmitoylation site on estrogen receptor (ER) α blocks ERα-mediated cAMP response element-binding protein phosphorylation. Similar results were obtained after mutation of the palmitoylation site on ERβ. Importantly, mutation of either ERα or ERβ did not affect the ability of the reciprocal ER to signal at the membrane. In contrast, membrane ERα and ERβ signaling were both dependent on the expression of the palmitoylacyltransferase proteins DHHC-7 and DHHC-21. Neither mGluR activity nor caveolin or ER expression was affected by knockdown of DHHC-7 and DHHC-21. These data collectively suggest discrete mechanisms that regulate specific isoform or global membrane ER signaling in neurons separate from mGluR activity or nuclear ER function.

Funding information:
  • NINDS NIH HHS - NS-57236(United States)

Importance of His192 in the human thyroid hormone transporter MCT8 for substrate recognition.

  • Groeneweg S
  • Endocrinology
  • 2013 Jul 24

Literature context:


Abstract:

Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). So far, functional domains within MCT8 are not well defined. Mutations in MCT8 result in severe psychomotor retardation due to impaired neuronal differentiation. One such mutation concerns His192 (H192R), located at the border of transmembrane domain (TMD) 1 and extracellular loop (ECL) 1, suggesting that this His residue is important for efficient TH transport. Here, we studied the role of different His residues, predicted within TMDs or ECLs of MCT8, in substrate recognition and translocation. Therefore, we analyzed the effects of the His-modifying reagent diethylpyrocarbonate (DEPC) and of site-directed mutagenesis of several His residues on TH transport by MCT8. Reaction of MCT8 with DEPC inhibited subsequent uptake of T(3) and T(4), whereas T(3) and T(4) efflux were not inhibited. The inhibitory effect of DEPC on TH uptake was prevented in the presence of T(3) or T(4), suggesting that TH blocks access to DEPC-sensitive residues. Three putative DEPC target His residues were replaced by Ala: H192A, H260A, and H450A. The H260A and H450A mutants showed similar TH transport and DEPC sensitivity as wild-type MCT8. However, the H192A mutant showed a significant reduction in TH uptake and was insensitive to DEPC. Taken together, these results indicate that His192 is sensitive to modification by DEPC and may be located close to a putative substrate recognition site within the MCT8 protein, important for efficient TH uptake.

Funding information:
  • NINDS NIH HHS - Z01 NS003119(United States)

Age increase of estrogen receptor-α (ERα) in cortical astrocytes impairs neurotrophic support in male and female rats.

  • Arimoto JM
  • Endocrinology
  • 2013 Jun 20

Literature context:


Abstract:

Rodent models show decreased neuronal responses to estradiol (E2) during aging (E2-desensitization) in association with reduced neuronal estrogen receptor (ER)-α, but little is known about age changes of E2-dependent astrocytic neurotrophic support. Because elevated expression of astrocyte glial fibrillary acidic protein (GFAP) is associated with impaired neurotrophic activity and because the GFAP promoter responds to ERα, we investigated the role of astrocytic ERα and ERβ in impaired astrocyte neurotrophic activity during aging. In vivo and in vitro, ERα was increased greater than 50% with age in astrocytes from the cerebral cortex of male rats (24 vs 3 months), whereas ERβ did not change. In astrocytes from 3-month-old males, experimentally increasing the ERα to ERβ ratio induced the aging phenotype of elevated GFAP and impaired E2-dependent neurite outgrowth. In 24-month-old male astrocytes, lowering ERα reversed the age elevation of GFAP and partially restored E2-dependent neurite outgrowth. Mixed glia (astrocytes to microglia, 3:1) of both sexes also showed these age changes. In a model of perimenopause, mixed glia from 9- to 15-month rats showed E2 desensitization: 9-month regular cyclers retained young-like ERα to ERβ ratios and neurotrophic activity, whereas 9-month noncyclers had elevated ERα and GFAP but low E2-dependent neurotrophic activity. In vivo, ERα levels in cortical astrocytes were also elevated. The persisting effects of ovarian acyclicity in vitro are hypothesized to arise from steroidal perturbations during ovarian senescence. These findings suggest that increased astrocyte ERα expression during aging contributes to the E2 desensitization of the neuronal responses in both sexes.

Funding information:
  • NIDCD NIH HHS - R01 DC014728(United States)

Importance of cysteine residues in the thyroid hormone transporter MCT8.

  • Lima de Souza EC
  • Endocrinology
  • 2013 May 22

Literature context:


Abstract:

The thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) is crucial for brain development as demonstrated by the severe psychomotor retardation in patients with MCT8 mutations. MCT8 contains 10 residues of the reactive amino acid cysteine (Cys) whose functional roles were studied using the Cys-specific reagent p-chloromercurybenzenesulfonate (pCMBS) and by site-directed mutagenesis. Pretreatment of JEG3 cells with pCMBS resulted in a dose- and time-dependent decrease of subsequent T3 uptake. Pretreatment with dithiothreitol did not affect TH transport or its inhibition by pCMBS. However, pCMBS inhibition of MCT8 was reversed by dithiothreitol. Inhibition of MCT8 by pCMBS was prevented in the presence of T3. The single and double mutation of C481A and C497A did not affect T3 transport, but the single mutants were less sensitive and the double mutant was completely insensitive to pCMBS. Similar effects on MCT8 were obtained using HgCl2 instead of pCMBS. In conclusion, we have identified Cys481 and Cys497 in MCT8 as the residues modified by pCMBS or HgCl2. These residues are probably located at or near the substrate-recognition site in MCT8. It remains to be investigated whether MCT8 function is regulated by modification of these Cys residues under pathophysiological conditions.

Funding information:
  • NIDCD NIH HHS - DC007176(United States)

Insulin-like growth factor binding protein 2 (IGFBP-2) promotes growth and survival of breast epithelial cells: novel regulation of the estrogen receptor.

  • Foulstone EJ
  • Endocrinology
  • 2013 May 22

Literature context:


Abstract:

In breast tumors IGF binding protein-2 (IGFBP-2) is elevated, and the presence of IGFBP-2 has been shown to correlate with malignancy. However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity. Exogenous IGFBP-2 acting via integrin receptors to reduce phosphatase and tensin homolog deleted from chromosome 10 (PTEN) levels protected these cells against death induced by various chemotherapeutic agents. This was dependent on a functional estrogen receptor (ER)-α because silencing ER-α blocked the ability of IGFBP-2 to confer cell survival. Loss of IGFBP-2 increased levels of PTEN and improved chemosensitivity of the cells, confirming its role as a survival factor. Silencing IGFBP-2 had no effect on the response to IGF-II, but responses to estrogen and tamoxifen were no longer observed due to loss of ER-α, which could be prevented by the inhibition of PTEN. Conversely, exogenous IGFBP-2 increased ER-α mRNA and protein in both normal and cancer cells via its interaction with integrin receptors. These actions of IGFBP-2 on ER-α involved the IGF-I receptor and activation of phosphatidylinositol 3-kinase in the cancer cells but were independent of this in normal breast cells. The production of IGFBP-2 by breast cancer cells enhances their proliferative potential, increases their survival, and protects them against chemotherapy-induced death. IGFBP-2 not only modulates IGFs and directly regulates PTEN but also has a role in maintaining ER-α expression.

Funding information:
  • NIGMS NIH HHS - R01 GM074830(United States)
  • NINDS NIH HHS - 1F31NS084706-01(United States)

Chronic low-level lead exposure affects the monoaminergic system in the mouse superior olivary complex.

  • Fortune T
  • J. Comp. Neurol.
  • 2009 Apr 10

Literature context:


Abstract:

Low-level lead (Pb) exposure is associated with behavioral and cognitive dysfunction, but it is not clear how Pb produces these behavioral changes. Pb has been shown to alter auditory temporal processing in both humans and animals. Auditory temporal processing occurs in the superior olivary complex (SOC) in the brainstem, where it is an important component in sound detection in noisy environments and in selective auditory attention. The SOC receives a serotonergic innervation from the dorsal raphe, and serotonin has been implicated in auditory temporal processing within the brainstem and inferior colliculus. Because Pb exposure modulates auditory temporal processing, the serotonergic system is a potential target for Pb. The current study was undertaken to determine whether developmental Pb exposure preferentially changes the serotonergic system within the SOC. Pb-treated mice were exposed to no Pb, very low Pb (0.01 mM), or low Pb (0.1 mM) throughout gestation and through 21 days postnatally. Brainstem sections from control and Pb-exposed mice were immunostained for the vesicular monoamine transporter 2 (VMAT2), serotonin (5-HT), and dopamine-beta-hydroxylase (DbetaH; a marker for norepinephrine) in order to elucidate the effect of Pb on monoaminergic input into the SOC. Sections were also immunolabeled with antibodies to vesicular glutamate transporter 1 (VGLUT1), vesicular gamma-aminobutyric acid (GABA) transporter (VGAT), and vesicular acetylcholine transporter (VAChT) to determine whether Pb exposure alters the glutaminergic, GABAergic, or cholinergic systems. Pb exposure caused a significant decrease in VMAT2, 5-HT, and DbetaH expression, whereas VGLUT1, VGAT, and VAChT showed no change. These results provide evidence that Pb exposure during development alters normal monoaminergic expression in the auditory brainstem.

Coexpression of alpha 2A-adrenergic and delta-opioid receptors in substance P-containing terminals in rat dorsal horn.

  • Riedl MS
  • J. Comp. Neurol.
  • 2009 Apr 1

Literature context:


Abstract:

Agonists acting at alpha(2)-adrenergic and opioid receptors (alpha(2)ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of alpha(2)AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between alpha(2)ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between alpha(2A)-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. alpha(2A)AR- and DOP-ir elements were colocalized in subcellular structures of 0.5 mum or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with alpha(2)AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K(+)-stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair alpha(2A)AR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein-protein interactions such as heterooligomerization.

Lead exposure during development results in increased neurofilament phosphorylation, neuritic beading, and temporal processing deficits within the murine auditory brainstem.

  • Jones LG
  • J. Comp. Neurol.
  • 2008 Feb 20

Literature context:


Abstract:

Low-level lead (Pb) exposure is a risk factor for learning disabilities, attention deficit hyperactivity disorder (ADHD), and other neurological dysfunction. It is not known how Pb produces these behavioral deficits, but low-level exposure during development is associated with auditory temporal processing deficits in an avian model, while hearing thresholds remain normal. Similar auditory processing deficits are found in children with learning disabilities and ADHD. To identify cellular changes underlying this functional deficit, Pb-induced alterations of neurons and glia within the mammalian auditory brainstem nuclei were quantified in control and Pb-exposed mice at postnatal day 21 by using immunohistochemistry, Western blotting, and 2D gel electrophoresis. Pb-treated mice were exposed to either 0.1 mM (low) or 2 mM (high) Pb acetate throughout gestation and through 21 days postnatally. Pb exposure results in little change in glial proteins such as glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), or F4/80 as determined by Western blot analysis and immunohistochemistry. In contrast, Pb exposure alters neuronal structural proteins by inducing increased phosphorylation of both the medium (NFM) and high-weight (NFH) forms of neurofilament within auditory brainstem nuclei. Axons immunolabeled for neurofilament protein show neuritic beading following Pb exposure both in vivo and in vitro, suggesting that Pb exposure also impairs axonal transport. Functional assessment shows no significant loss of peripheral function, but does reveal impairments in brainstem conduction time and temporal processing within the brainstem. These results provide evidence that Pb exposure during development alters axonal structure and function within brainstem auditory nuclei.

Funding information:
  • NCI NIH HHS - 5P20CA90578(United States)