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Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) antibody

RRID:AB_2313567

Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice.

  • Bannerman DM
  • Front Mol Neurosci
  • 2018 Jul 11

Literature context:


Abstract:

The GluA1 subunit of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) plays a crucial, but highly selective, role in cognitive function. Here we analyzed AMPAR expression, AMPAR distribution and spatial learning in mice (Gria1R/R ), expressing the "trafficking compromised" GluA1(Q600R) point mutation. Our analysis revealed somatic accumulation and reduction of GluA1(Q600R) and GluA2, but only slightly reduced CA1 synaptic localization in hippocampi of adult Gria1R/R mice. These immunohistological changes were accompanied by a strong reduction of somatic AMPAR currents in CA1, and a reduction of plasticity (short-term and long-term potentiation, STP and LTP, respectively) in the CA1 subfield following tetanic and theta-burst stimulation. Nevertheless, spatial reference memory acquisition in the Morris water-maze and on an appetitive Y-maze task was unaffected in Gria1R/R mice. In contrast, spatial working/short-term memory during both spontaneous and rewarded alternation tasks was dramatically impaired. These findings identify the GluA1(Q600R) mutation as a loss of function mutation that provides independent evidence for the selective role of GluA1 in the expression of short-term memory.

Funding information:
  • NHGRI NIH HHS - R01 HG002899(United States)

Eldecalcitol Causes FGF23 Resistance for Pi Reabsorption and Improves Rachitic Bone Phenotypes in the Male Hyp Mouse.

  • Kaneko I
  • Endocrinology
  • 2018 Jul 1

Literature context:


Abstract:

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.

Funding information:
  • NIEHS NIH HHS - P30 ES003819(United States)

Functional Consequences of Synapse Remodeling Following Astrocyte-Specific Regulation of Ephrin-B1 in the Adult Hippocampus.

  • Koeppen J
  • J. Neurosci.
  • 2018 Jun 20

Literature context:


Abstract:

Astrocyte-derived factors can control synapse formation and functions, making astrocytes an attractive target for regulating neuronal circuits and associated behaviors. Abnormal astrocyte-neuronal interactions are also implicated in neurodevelopmental disorders and neurodegenerative diseases associated with impaired learning and memory. However, little is known about astrocyte-mediated mechanisms that regulate learning and memory. Here, we propose astrocytic ephrin-B1 as a regulator of synaptogenesis in adult hippocampus and mouse learning behaviors. We found that astrocyte-specific ablation of ephrin-B1 in male mice triggers an increase in the density of immature dendritic spines and excitatory synaptic sites in the adult CA1 hippocampus. However, the prevalence of immature dendritic spines is associated with decreased evoked postsynaptic firing responses in CA1 pyramidal neurons, suggesting impaired maturation of these newly formed and potentially silent synapses or increased excitatory drive on the inhibitory neurons resulting in the overall decreased postsynaptic firing. Nevertheless, astrocyte-specific ephrin-B1 knock-out male mice exhibit normal acquisition of fear memory but enhanced contextual fear memory recall. In contrast, overexpression of astrocytic ephrin-B1 in the adult CA1 hippocampus leads to the loss of dendritic spines, reduced excitatory input, and impaired contextual memory retention. Our results suggest that astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and mediate excitatory synapse elimination through its interactions with neuronal EphB receptors. Indeed, a deletion of neuronal EphB receptors impairs the ability of astrocytes expressing functional ephrin-B1 to engulf synaptosomes in vitro Our findings demonstrate that astrocytic ephrin-B1 regulates long-term contextual memory by restricting new synapse formation in the adult hippocampus.SIGNIFICANCE STATEMENT These studies address a gap in our knowledge of astrocyte-mediated regulation of learning and memory by unveiling a new role for ephrin-B1 in astrocytes and elucidating new mechanisms by which astrocytes regulate learning. Our studies explore the mechanisms underlying astrocyte regulation of hippocampal circuit remodeling during learning using new genetic tools that target ephrin-B signaling in astrocytes in vivo On a subcellular level, astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and trigger astrocyte-mediated elimination of EphB receptor-containing synapses. Given the role EphB receptors play in neurodevelopmental disorders and neurodegenerative diseases, these findings establish a foundation for future studies of astrocyte-mediated synaptogenesis in clinically relevant conditions that can help to guide the development of clinical applications for a variety of neurological disorders.

Funding information:
  • NCI NIH HHS - P01CA142106(United States)

Epigenetic Promoter DNA Methylation of miR-124 Promotes HIV-1 Tat-Mediated Microglial Activation via MECP2-STAT3 Axis.

  • Periyasamy P
  • J. Neurosci.
  • 2018 Jun 6

Literature context:


Abstract:

The present study demonstrates HIV-1 Tat-mediated epigenetic downregulation of microglial miR-124 and its association with microglial activation. Exposure of mouse primary microglia isolated from newborn pups of either sex to HIV-1 Tat resulted in decreased expression of primary miR-124-1, primary miR-124-2 as well as the mature miR-124. In parallel, HIV-1 Tat exposure to mouse primary microglial cells resulted in increased expression of DNA methylation enzymes, such as DNMT1, DNMT3A, and DNMT3B, which were also accompanied by increased global DNA methylation. Bisulfite-converted genomic DNA sequencing in the HIV-1 Tat-exposed mouse primary microglial cells further confirmed increased DNA methylation of the primary miR-124-1 and primary miR-124-2 promoters. Bioinformatic analyses identified MECP2 as a novel 3'-UTR target of miR-124. This was further validated in mouse primary microglial cells wherein HIV-1 Tat-mediated downregulation of miR-124 resulted in increased expression of MECP2, leading in turn to further repression of miR-124 via the feedback loop. In addition to MECP2, miR-124 also modulated the levels of STAT3 through its binding to the 3'-UTR, leading to microglial activation. Luciferase assays and Ago2 immunoprecipitation determined the direct binding between miR-124 and 3'-UTR of both MECP2 and STAT3. Gene silencing of MECP2 and DNMT1 and overexpression of miR-124 blocked HIV-1 Tat-mediated downregulation of miR-124 and microglial activation. In vitro findings were also confirmed in the basal ganglia of SIV-infected rhesus macaques (both sexes). In summary, our findings demonstrate a novel mechanism of HIV-1 Tat-mediated activation of microglia via downregulation of miR-124, leading ultimately to increased MECP2 and STAT3 signaling.SIGNIFICANCE STATEMENT Despite the effectiveness of combination antiretroviral therapy in controlling viremia, the CNS continues to harbor viral reservoirs. The persistence of low-level virus replication leads to the accumulation of early viral proteins, including HIV-1 Tat protein. Understanding the epigenetic/molecular mechanism(s) by which viral proteins, such as HIV-1 Tat, can activate microglia is thus of paramount importance. This study demonstrated that HIV-1 Tat-mediated DNA methylation of the miR-124 promoter leads to its downregulation with a concomitant upregulation of the MECP2-STAT3-IL6, resulting in microglial activation. These findings reveal an unexplored epigenetic/molecular mechanism(s) underlying HIV-1 Tat-mediated microglial activation, thereby providing a potential target for the development of therapeutics aimed at ameliorating microglial activation and neuroinflammation in the context of HIV-1 infection.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

  • Sedlyarov V
  • Cell Host Microbe
  • 2018 Jun 13

Literature context:


Abstract:

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

Funding information:
  • NIA NIH HHS - R01 AG012689(United States)

BDNF infusion into the MPN mag is sufficient to restore copulatory behavior in the castrated Syrian hamster.

  • Brague JC
  • Horm Behav
  • 2018 May 12

Literature context:


Abstract:

Testosterone plays a key role in the expression of male sex behavior by influencing cellular activity and synapses within the magnocellular medial preoptic nucleus (MPN mag), a sub-nucleus of the medial preoptic area (MPOA) in the Syrian hamster. Although the mechanisms underlying hormonally-induced synaptic plasticity in this region remain elusive, the data suggests that an increase in synaptic density may mediate testosterone's effects on copulation. As brain derived neurotrophic factor (BDNF) plays an integral role in regulating synaptic plasticity and gonadal steroids regulate the levels of BDNF, we hypothesize that BDNF may mediate the effects of gonadal hormones on copulatory behavior. To test this hypothesis, we infused BDNF or controls into the MPN mag of long-term castrates. Our results indicate that BDNF, but not the controls, restored copulatory behavior in castrated male Syrian hamsters. Furthermore, the rise of BDNF expression in the MPOA preceded the rise of synaptophysin following testosterone replacement in castrated males. These data are consistent with our hypothesis, implicating a role for BDNF in mediating testosterone's action on copulation and suggest that the delay in testosterone's restoration of copulation is, in part, due to the delay in the increase of BDNF and synaptophysin.

Funding information:
  • NIAMS NIH HHS - R01 AR031062-27(United States)

A homozygous loss-of-function CAMK2A mutation causes growth delay, frequent seizures and severe intellectual disability.

  • Chia PH
  • Elife
  • 2018 May 22

Literature context:


Abstract:

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

Funding information:
  • Agency for Science, Technology and Research - GODAFIT Strategic Positioning Fund()
  • NIGMS NIH HHS - GM080399(United States)

Angiogenin/Ribonuclease 5 Is an EGFR Ligand and a Serum Biomarker for Erlotinib Sensitivity in Pancreatic Cancer.

  • Wang YN
  • Cancer Cell
  • 2018 Apr 9

Literature context:


Abstract:

Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment. These results identify a role of ANG as a serum biomarker that may be used to stratify patients for EGFR-targeted therapies, and offer insights into the ligand-receptor relationship between RNase and RTK families.

Funding information:
  • NCI NIH HHS - P30 CA016672()
  • NCI NIH HHS - R01 CA211615()
  • NCI NIH HHS - T32 CA186892()
  • NCI NIH HHS - U01 CA201777()
  • NIGMS NIH HHS - R01 GM098294(United States)

Drosophila Full-Length Amyloid Precursor Protein Is Required for Visual Working Memory and Prevents Age-Related Memory Impairment.

  • Rieche F
  • Curr. Biol.
  • 2018 Mar 5

Literature context:


Abstract:

The β-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). However, its normal physiological functions are still unclear. APP is cleaved by various secretases whereby sequential processing by the β- and γ-secretases produces the β-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPβ fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPα fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPα fragments have been suggested to have a neurotrophic and neuroprotective role [1]. Moreover, expression of sAPPα in APP-deficient mice could rescue their deficits in learning, spatial memory, and long-term potentiation [2]. Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment [3]. We now show that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, our results show that specific memory processes require distinct APPL products.

Funding information:
  • NCI NIH HHS - Z01 BC010313(United States)
  • NIA NIH HHS - R01 AG045830()
  • NIA NIH HHS - R21 AG055943()
  • NIH HHS - P40 OD018537()

Enhanced Expression of the Key Mitosis Regulator Cyclin B1 Is Mediated by PDZ-Binding Kinase in Islets of Pregnant Mice.

  • Uesato T
  • J Endocr Soc
  • 2018 Mar 1

Literature context:


Abstract:

The proliferation of pancreatic β cells is enhanced to enable an increase in β-cell mass and to compensate for insulin resistance during pregnancy. To elucidate the mechanisms involved, we previously investigated islets from pregnant and nonpregnant mice by gene expression profiling and found that the expression of postsynaptic density-95/Discs large/zonula occludens-1 (PDZ)-binding kinase (Pbk), a member of the mitogen-activated protein kinase kinase family, is increased in pregnant mouse islets compared with control mouse islets. Among the pregnancy hormones, treatment with estradiol upregulated Pbk expression. Inhibition of Pbk expression using a small interfering RNA for Pbk reduced bromodeoxyuridine incorporation in mouse insulinoma 6 cells, which was accompanied by a decreased expression of Ccnb1, a regulatory gene involved in mitosis. Ccnb1 expression was augmented in mouse islets during pregnancy. The forced expression of Pbk using an adenovirus system in isolated mouse islets increased Ccnb1 expression, and the Pbk inhibitor HI-TOPK-032 suppressed Ccnb1 expression in islets isolated from pregnant mice. Our results suggest that Pbk contributes to the expansion of islets during pregnancy and that Ccnb1 may assist Pbk in its role in β-cell proliferation.

Funding information:
  • NIDDK NIH HHS - T32 DK063687(United States)

The InR/Akt/TORC1 Growth-Promoting Signaling Negatively Regulates JAK/STAT Activity and Migratory Cell Fate during Morphogenesis.

  • Kang D
  • Dev. Cell
  • 2018 Feb 26

Literature context:


Abstract:

Cell growth and cell differentiation are two distinct yet coupled developmental processes, but how they are coordinated is not well understood. During Drosophila oogenesis, we found that the growth-promoting InR/Akt/TOR pathway was involved in suppressing the fate determination of the migratory border cells. The InR/Akt/TOR pathway signals through TOR and Raptor, components of TORC1, to downregulate the JAK/STAT pathway, which is necessary and sufficient for border cell fate determination. TORC1 promotes the protein stability of SOCS36E, the conserved negative regulator of JAK/STAT signaling, through physical interaction, suggesting that TORC1 acts as a key regulator coordinating both cell growth and cell differentiation.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

SMC1α Substitutes for Many Meiotic Functions of SMC1β but Cannot Protect Telomeres from Damage.

  • Biswas U
  • Curr. Biol.
  • 2018 Jan 22

Literature context:


Abstract:

The cohesin complex is built upon the SMC1/SMC3 heterodimer, and mammalian meiocytes feature two variants of SMC1 named SMC1α and SMC1β. It is unclear why these two SMC1 variants have evolved. To determine unique versus redundant functions of SMC1β, we asked which of the known functions of SMC1β can be fulfilled by SMC1α. Smc1α was expressed under control of the Smc1β promoter in either wild-type or SMC1β-deficient mice. No effect was seen in the former. However, several major phenotypes of SMC1β-deficient spermatocytes were rescued by SMC1α. We observed extended development before apoptosis and restoration of axial element and synaptonemal complex lengths, chromosome synapsis, sex body formation, processing of DNA double-strand breaks, and formation of MLH1 recombination foci. This supports the concept that the quantity rather than the specific quality of cohesin complexes is decisive for meiotic chromosome architecture. It also suggests plasticity in complex composition, because to replace SMC1β in many functions, SMC1α has to more extensively associate with other cohesins. The cells did not complete meiosis but died to the latest at the pachytene-to-diplotene transition. Telomere aberrations known from Smc1β-/- mice persisted, and DNA damage response and repair proteins accumulated there regardless of expression of SMC1α. Thus, whereas SMC1α can substitute for SMC1β in many functions, the protection of telomere integrity requires SMC1β.

Funding information:
  • NIEHS NIH HHS - P42ES004705(United States)

Polo-like Kinase Couples Cytoplasmic Protein Gradients in the C. elegans Zygote.

  • Han B
  • Curr. Biol.
  • 2018 Jan 8

Literature context:


Abstract:

Intracellular protein gradients underlie essential cellular and developmental processes, but the mechanisms by which they are established are incompletely understood. During the asymmetric division of the C. elegans zygote, the RNA-binding protein MEX-5 forms an anterior-rich cytoplasmic gradient that causes the RNA-binding protein POS-1 to form an opposing, posterior-rich gradient. We demonstrate that the polo-like kinase PLK-1 mediates the repulsive coupling between MEX-5 and POS-1 by increasing the mobility of POS-1 in the anterior. PLK-1 is enriched in the anterior cytoplasm and phosphorylates POS-1, which is both necessary and sufficient to increase POS-1 mobility. Regulation of POS-1 mobility depends on both the interaction between PLK-1 and MEX-5 and between MEX-5 and RNA, suggesting that MEX-5 may recruit PLK-1 to RNA in the anterior. The low concentration of MEX-5/PLK-1 in the posterior cytoplasm provides a permissive environment for the retention of POS-1, which depends on POS-1 RNA binding. Our findings describe a novel reaction/diffusion mechanism in which the asymmetric distribution of cytoplasmic PLK-1 couples two RNA-binding protein gradients, thereby partitioning the cytoplasm.

Funding information:
  • European Research Council - 232937(International)
  • NIGMS NIH HHS - R01 GM110194()
  • NIGMS NIH HHS - R01 GM117237()
  • NIH HHS - P40 OD010440()

Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of β-amyloidosis.

  • Huynh TV
  • Neuron
  • 2017 Dec 6

Literature context:


Abstract:

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer disease. Previous studies suggest that reduction of apoE levels through genetic manipulation can reduce Aβ pathology. However, it is not clear how reduction of apoE levels after birth would affect amyloid deposition. We utilize an antisense oligonucleotide (ASO) to reduce apoE expression in the brains of APP/PS1-21 mice homozygous for the APOE-ε4 or APOE-ε3 allele. ASO treatment starting after birth led to a significant decrease in Aβ pathology when assessed at 4 months. Interestingly, ASO treatment starting at the onset of amyloid deposition led to an increase in Aβ plaque size and a reduction in plaque-associated neuritic dystrophy with no change in overall plaque load. These results suggest that lowering apoE levels prior to plaque deposition can strongly affect the initiation of Aβ pathology while lowering apoE after Aβ seeding modulates plaque size and toxicity.

Funding information:
  • NIA NIH HHS - R01 AG047644()
  • NIGMS NIH HHS - R01GM105431(United States)
  • NIGMS NIH HHS - T32 GM007200()
  • NINDS NIH HHS - R01 NS034467()
  • NINDS NIH HHS - R01 NS090934()
  • NINDS NIH HHS - R37 NS034467()

Control of Cell Shape, Neurite Outgrowth, and Migration by a Nogo-A/HSPG Interaction.

  • Kempf A
  • Dev. Cell
  • 2017 Oct 9

Literature context:


Abstract:

Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2.

An Effective Feedback Loop between Cell-Cell Contact Duration and Morphogen Signaling Determines Cell Fate.

  • Barone V
  • Dev. Cell
  • 2017 Oct 23

Literature context:


Abstract:

Cell-cell contact formation constitutes an essential step in evolution, leading to the differentiation of specialized cell types. However, remarkably little is known about whether and how the interplay between contact formation and fate specification affects development. Here, we identify a positive feedback loop between cell-cell contact duration, morphogen signaling, and mesendoderm cell-fate specification during zebrafish gastrulation. We show that long-lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for ppl cell-fate specification. We further show that Nodal signaling promotes ppl cell-cell contact duration, generating a positive feedback loop between ppl cell-cell contact duration and cell-fate specification. Finally, by combining mathematical modeling and experimentation, we show that this feedback determines whether anterior axial mesendoderm cells become ppl or, instead, turn into endoderm. Thus, the interdependent activities of cell-cell signaling and contact formation control fate diversification within the developing embryo.

Nogo-B is the major form of Nogo at the floor plate and likely mediates crossing of commissural axons in the mouse spinal cord.

  • Wang L
  • J. Comp. Neurol.
  • 2017 Sep 1

Literature context:


Abstract:

Using Nogo antibodies with defined binding specificity, Nogo-B, but not Nogo-A, was localized on radial glia in the floor plate of mouse embryos. The presence of Nogo-B was confirmed in Nogo-A knockout mice. In explant cultures of embryonic day (E) 11 and E12 spinal cord, blocking of NgR function with antagonist peptide NEP1-40 reduced the crossing of newly arrived commissural axons, resulting in an accumulation of growth cones in the floor plate. Analysis of growth cone morphology demonstrated an increase in size of growth cones in the floor plate after peptide treatment, which was not detected in axons growing toward the midline. In knockout embryos, midline crossing was not affected by absence of Nogo-A. In co-culture experiments using collagen gel, floor plate showed a strong inhibitory effect on the extension of post-commissural neurites from the spinal cord. This effect was abolished by NEP1-40, and was observed neither in pre-commissural neurites, nor in post-commissural neurites grown with floor plate derived from Nogo-A knockout embryo. Furthermore, western blot analysis of conditioned medium from floor plates showed a truncated form of Nogo with molecular weight of 37 kDa, which could mediate the diffusible effect to axon growth. We conclude that Nogo-B is expressed in the floor plate of mouse embryo, which probably mediates axon crossing in the spinal cord by repelling axons out of the midline when they start upregulate NgR. Nogo acts on axon growth not only through a contact-mediated mechanism, but also through a diffusible mechanism.

Sexual dimorphic expression of TrkB, TrkB-T1, and BDNF in the medial preoptic area of the Syrian hamster.

  • Brague JC
  • Brain Res.
  • 2017 Aug 15

Literature context:


Abstract:

Neurotrophins regulate many aspects of neuronal function and activity. Specifically, the binding of Brain-derived neurotrophic factor (BDNF) to Tyrosine receptor kinase-B (TrkB) or its truncated version, TrkB-T1, can cause growth and differentiation or dominant inhibition of receptor signaling, respectively. There is evidence that these neurotropic effects on nervous tissue, in both the central and peripheral nervous system, behave differently between the sexes. This study used western blots to examine the expression of these neurotrophins in the medial preoptic area (MPOA), a sexually dimorphic region of the hamster brain that controls male sex behavior. We report that TrkB-FL and BDNF show greater expression in male MPOA tissue, when compared to female. On the contrary, TrkB-T1 is expressed in greater abundance in the female MPOA. Our results indicate a clear sexual dimorphism of neurotrophins in the MPOA of the Syrian hamster. Furthermore, the greater expression of TrkB-FL and BDNF in the male MPOA suggests that these neurotrophins could be promoting synaptic growth to facilitate male-typical copulation. In contrast, the greater TrkB-T1 expression in the female MPOA suggests a possible inhibition of synaptic growth, and may contribute to the lack of male-typical copulation. Altogether, our data suggests that neurotrophins may play a larger role sexual differentiation than previously thought.

Protein kinase D exerts neuroprotective functions during oxidative stress via nuclear factor kappa B-independent signaling pathways.

  • Liliom H
  • J. Neurochem.
  • 2017 Jul 19

Literature context:


Abstract:

Members of the protein kinase D (PKD) family of serine/threonine kinases are known to exert diverse roles in neuronal stress responses. Here, we show the transient activation and nuclear translocation of endogenous PKD upon oxidative stress induced by H2 O2 treatment in primary neuronal cultures. Using pharmacological inhibition, we show that PKD activity protects neurons from oxidative stress-induced cell death. Although members of the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF kappaB) pathway were phosphorylated upon H2 O2 treatment, it was found that the neuronal response to oxidative stress is not executed through the nuclear translocation and activity of RelA. On the other hand, we demonstrate for the first time in neuronal cells, the association of green fluorescent protein-tagged kinase inactive PKD1 with mitochondrial membranes in vivo and the presence of PKD activity in the close vicinity of mitochondria in vitro. Our findings thus support the notion that the neuroprotective role of PKD is exerted independently from NF kappaB signaling and suggest a potential mitochondrial function for PKD in cultured neurons.

MLKL, the Protein that Mediates Necroptosis, Also Regulates Endosomal Trafficking and Extracellular Vesicle Generation.

  • Yoon S
  • Immunity
  • 2017 Jul 18

Literature context:


Abstract:

Activation of the pseudokinase mixed lineage kinase domain-like (MLKL) upon its phosphorylation by the protein kinase RIPK3 triggers necroptosis, a form of programmed cell death in which rupture of cellular membranes yields release of intracellular components. We report that MLKL also associated with endosomes and controlled the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of extracellular vesicles. This role was exerted on two quantitative grades: a constitutive one independent of RIPK3, and an enhanced one, triggered by RIPK3, where the association of MLKL with the endosomes was enhanced, and it was found to bind endosomal sorting complexes required for transport (ESCRT) proteins and the flotillins and to be excluded, together with them, from cells within vesicles. We suggest that release of phosphorylated MLKL within extracellular vesicles serves as a mechanism for self-restricting the necroptotic activity of this protein.

Preprotein Conformational Dynamics Drive Bivalent Translocase Docking and Secretion.

  • Sardis MF
  • Structure
  • 2017 Jul 5

Literature context:


Abstract:

Most bacterial secretory proteins destined beyond the plasma membrane are secreted post-translationally by the Sec translocase. In the first step of translocation, preproteins are targeted for binding to their 2-site receptor SecA, the peripheral ATPase subunit of the translocase. We now reveal that secretory preproteins use a dual-key mechanism to bridge the signal peptide and mature domain receptor sites and cooperatively enhance their affinities. Docking of targeting-competent mature domains requires that their extensive disorder is finely tuned. This is achieved through amino-terminal mature domain regions acting as conformational rheostats. By being linked to the rheostats, signal peptides regulate long-range preprotein disorder. Concomitant conformational changes in SecA sterically adapt its two receptor sites to optimally recognize hundreds of dissimilar preproteins. This novel intramolecular conformational crosstalk in the preprotein chains and the dynamic interaction with their receptor are mechanistically coupled to preprotein engagement in the translocase and essential for secretion.

Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata.

  • Courtial L
  • F1000Res
  • 2017 Jul 15

Literature context:


Abstract:

The extracellular signal-regulated protein kinase (ERK) signalling pathway controls key cellular processes, such as cell cycle regulation, cell fate determination and the response to external stressors. Although ERK functions are well studied in a variety of living organisms ranging from yeast to mammals, its functions in corals are still poorly known. The present work aims to give practical tools to study the expression level of ERK protein and the activity of the ERK signalling pathway in corals. The antibody characterisation experiment was performed five times and identical results were obtained. The present study validated the immune-reactivity of commercially available antibodies directed against ERK and its phosphorylated/activated forms on protein extracts of the reef-building coral Stylophora pistillata.

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

  • Jossin Y
  • Dev. Cell
  • 2017 Jun 5

Literature context:


Abstract:

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1fl/fl), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells.

Funding information:
  • NCI NIH HHS - R01 CA131047()
  • NCI NIH HHS - R01 CA179914()
  • NICHD NIH HHS - T32 HD007183()
  • NINDS NIH HHS - R01 NS080194()

RhoD Inhibits RhoC-ROCK-Dependent Cell Contraction via PAK6.

  • Durkin CH
  • Dev. Cell
  • 2017 May 8

Literature context:


Abstract:

RhoA-mediated regulation of myosin-II activity in the actin cortex controls the ability of cells to contract and bleb during a variety of cellular processes, including cell migration and division. Cell contraction and blebbing also frequently occur as part of the cytopathic effect seen during many different viral infections. We now demonstrate that the vaccinia virus protein F11, which localizes to the plasma membrane, is required for ROCK-mediated cell contraction from 2 hr post infection. Curiously, F11-induced cell contraction is dependent on RhoC and not RhoA signaling to ROCK. Moreover, RhoC-driven cell contraction depends on the upstream inhibition of RhoD signaling by F11. This inhibition prevents RhoD from regulating its downstream effector Pak6, alleviating the suppression of RhoC by the kinase. Our observations with vaccinia have now demonstrated that RhoD recruits Pak6 to the plasma membrane to antagonize RhoC signaling during cell contraction and blebbing.

Prion-Like Seeding of Misfolded α-Synuclein in the Brains of Dementia with Lewy Body Patients in RT-QUIC.

  • Sano K
  • Mol. Neurobiol.
  • 2017 May 28

Literature context:


Abstract:

The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD50) values of 107~1010/g brain. Limbic DLB was estimated to have an SD50 value of ~105/g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect seeding activity in LBD, and the culprit that causes prion-like seeding may be oligomeric forms of αSyn.

The Sec61 translocon limits IRE1α signaling during the unfolded protein response.

  • Sundaram A
  • Elife
  • 2017 May 15

Literature context:


Abstract:

IRE1α is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1α forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1α complexes in cells with blue native PAGE immunoblotting. We find that IRE1α forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1α oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1α-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1α to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1α complex defines the extent of IRE1α activity and may determine cell fate decisions during ER stress conditions.

Funding information:
  • NIGMS NIH HHS - T32 GM007223()

Concerted action of neuroepithelial basal shrinkage and active epithelial migration ensures efficient optic cup morphogenesis.

  • Sidhaye J
  • Elife
  • 2017 Apr 4

Literature context:


Abstract:

Organ formation is a multi-scale event that involves changes at the intracellular, cellular and tissue level. Organogenesis often starts with the formation of characteristically shaped organ precursors. However, the cellular mechanisms driving organ precursor formation are often not clear. Here, using zebrafish, we investigate the epithelial rearrangements responsible for the development of the hemispherical retinal neuroepithelium (RNE), a part of the optic cup. We show that in addition to basal shrinkage of RNE cells, active migration of connected epithelial cells into the RNE is a crucial player in its formation. This cellular movement is driven by progressive cell-matrix contacts and actively translocates prospective RNE cells to their correct location before they adopt neuroepithelial fate. Failure of this migration during neuroepithelium formation leads to ectopic determination of RNE cells and consequently impairs optic cup formation. Overall, this study illustrates how spatiotemporal coordination between morphogenic movements and fate determination critically influences organogenesis.

Adult Hippocampal Neurogenesis along the Dorsoventral Axis Contributes Differentially to Environmental Enrichment Combined with Voluntary Exercise in Alleviating Chronic Inflammatory Pain in Mice.

  • Zheng J
  • J. Neurosci.
  • 2017 Apr 12

Literature context:


Abstract:

Cognitive behavioral therapy, such as environmental enrichment combined with voluntary exercise (EE-VEx), is under active investigation as an adjunct to pharmaceutical treatment for chronic pain. However, the effectiveness and underlying mechanisms of EE-VEx remain unclear. In mice with intraplantar injection of complete Freund's adjuvant, our results revealed that EE-VEx alleviated perceptual, affective, and cognitive dimensions of chronic inflammatory pain. These effects of EE-VEx on chronic pain were contingent on the occurrence of adult neurogenesis in the dentate gyrus in a functionally dissociated manner along the dorsoventral axis: neurogenesis in the ventral dentate gyrus participated in alleviating perceptual and affective components of chronic pain by EE-VEx, whereas neurogenesis in the dorsal dentate gyrus was involved in EE-VEx's cognitive-enhancing effects. Chronic inflammatory pain was accompanied by decreased levels of brain-derived neurotrophic factor (BDNF) in the dentate gyrus, which were reversed by EE-VEx. Overexpression of BDNF in the dentate gyrus mimicked the effects of EE-VEx. Our results demonstrate distinct contribution of adult hippocampal neurogenesis along the dorsoventral axis to EE-VEx's beneficial effects on different dimensions of chronic pain.SIGNIFICANCE STATEMENT Environmental enrichment combined with voluntary exercise (EE-VEx) is under active investigation as an adjunct to pharmaceutical treatment for chronic pain, but its effectiveness and underlying mechanisms remain unclear. In a mouse model of inflammatory pain, the present study demonstrates that the beneficial effects of EE-VEx on chronic pain depend on adult neurogenesis with a dorsoventral dissociation along the hippocampal axis. Adult neurogenesis in the ventral dentate gyrus participates in alleviating perceptual and affective components of chronic pain by EE-VEx, whereas that in the dorsal pole is involved in EE-VEx's cognitive-enhancing effects in chronic pain.

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

  • Fu SJ
  • J. Neurosci.
  • 2017 Mar 1

Literature context:


Abstract:

Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and β subunits. We provide a proof of concept that protecting the human CaV2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.

Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro-inflammatory "FADDosome" Complex upon TRAIL Stimulation.

  • Henry CM
  • Mol. Cell
  • 2017 Feb 16

Literature context:


Abstract:

TRAIL is a potent inducer of apoptosis and has been studied almost exclusively in this context. However, TRAIL can also induce NFκB-dependent expression of multiple pro-inflammatory cytokines and chemokines. Surprisingly, whereas inhibition of caspase activity blocked TRAIL-induced apoptosis, but not cytokine production, knock down or deletion of caspase-8 suppressed both outcomes, suggesting that caspase-8 participates in TRAIL-induced inflammatory signaling in a scaffold role. Consistent with this, introduction of a catalytically inactive caspase-8 mutant into CASP-8 null cells restored TRAIL-induced cytokine production, but not cell death. Furthermore, affinity precipitation of the native TRAIL receptor complex revealed that pro-caspase-8 was required for recruitment of RIPK1, via FADD, to promote NFκB activation and pro-inflammatory cytokine production downstream. Thus, caspase-8 can serve in two distinct roles in response to TRAIL receptor engagement, as a scaffold for assembly of a Caspase-8-FADD-RIPK1 "FADDosome" complex, leading to NFκB-dependent inflammation, or as a protease that promotes apoptosis.

Funding information:
  • Worldwide Cancer Research - 14-0323()

Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination.

  • Juszkiewicz S
  • Mol. Cell
  • 2017 Feb 16

Literature context:


Abstract:

Diverse cellular stressors have been observed to trigger site-specific ubiquitination on several ribosomal proteins. However, the ubiquitin ligases, biochemical consequences, and physiologic pathways linked to these modifications are not known. Here, we show in mammalian cells that the ubiquitin ligase ZNF598 is required for ribosomes to terminally stall during translation of poly(A) sequences. ZNF598-mediated stalling initiated the ribosome-associated quality control (RQC) pathway for degradation of nascent truncated proteins. Biochemical ubiquitination reactions identified two sites of mono-ubiquitination on the 40S protein eS10 as the primary ribosomal target of ZNF598. Cells lacking ZNF598 activity or containing ubiquitination-resistant eS10 ribosomes failed to stall efficiently on poly(A) sequences. In the absence of stalling, read-through of poly(A) produces a poly-lysine tag, which might alter the localization and solubility of the associated protein. Thus, ribosome ubiquitination can modulate translation elongation and impacts co-translational quality control to minimize production of aberrant proteins.

Funding information:
  • Medical Research Council - MC_UP_A022_1007()

Isoform-specific localization of Nogo protein in the optic pathway of mouse embryos.

  • Wang L
  • J. Comp. Neurol.
  • 2016 Aug 1

Literature context:


Abstract:

Expression of Nogo protein was investigated in the optic pathway of embryonic mice by using isoform-specific antibodies Bianca and 11C7, which recognize Nogo-A/B and Nogo-A, respectively. Our previous reports from using antibody N18 have shown that Nogo is localized on the radial glia in the retina and at the midline of the ventral diencephalon in mouse embryos during the ingrowth of retinal ganglion cells (RGCs) axons. This glial-specific localization is markedly different from findings in other studies. This study showed Nogo-A/B primarily on radial glia in the retina at E13 and then later on retinal ganglion cells and axons at E14 and E15, whereas Nogo-A was expressed preferentially by RGCs and their axons. In the ventral diencephalon, Nogo-A/B was expressed strongly on radial glia, particularly in those located in the midline region of the chiasm but also on RGC axons. In Nogo-A knockout embryos, the isoform Nogo-B (revealed by Bianca) was observed on radial glia in the ventral diencephalon and on RGCs and their axons. We concluded that Nogo-A is localized on the ganglion cells and retinal axons, whereas Nogo-B is expressed by the radial glia in the optic pathway. Nogo-B may play an important role in guiding axon growth in decisive regions of the visual pathway, which include the optic disc and the optic chiasm. J. Comp. Neurol. 524:2322-2334, 2016. © 2016 Wiley Periodicals, Inc.

Funding information:
  • NHLBI NIH HHS - R01 HL122494(United States)

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

  • Grainger DL
  • F1000Res
  • 2016 Jun 23

Literature context:


Abstract:

REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1 (-/-) mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin.

Using a 3D Culture System to Differentiate Visceral Adipocytes In Vitro.

  • Emont MP
  • Endocrinology
  • 2015 Dec 21

Literature context:


Abstract:

It has long been recognized that body fat distribution and regional adiposity play a major role in the control of metabolic homeostasis. However, the ability to study and compare the cell autonomous regulation and response of adipocytes from different fat depots has been hampered by the difficulty of inducing preadipocytes isolated from the visceral depot to differentiate into mature adipocytes in culture. Here, we present an easily created 3-dimensional (3D) culture system that can be used to differentiate preadipocytes from the visceral depot as robustly as those from the sc depot. The cells differentiated in these 3D collagen gels are mature adipocytes that retain depot-specific characteristics, as determined by imaging, gene expression, and functional assays. This 3D culture system therefore allows for study of the development and function of adipocytes from both depots in vitro and may ultimately lead to a greater understanding of site-specific functional differences of adipose tissues to metabolic dysregulation.

Funding information:
  • NICHD NIH HHS - R01 HD069560(United States)

Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody.

  • van Endert P
  • F1000Res
  • 2015 Jun 23

Literature context:


Abstract:

In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC) whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.

Differential contributions of microglial and neuronal IKKβ to synaptic plasticity and associative learning in alert behaving mice.

  • Kyrargyri V
  • Glia
  • 2015 Apr 16

Literature context:


Abstract:

Microglia are CNS resident immune cells and a rich source of neuroactive mediators, but their contribution to physiological brain processes such as synaptic plasticity, learning, and memory is not fully understood. In this study, we used mice with partial depletion of IκB kinase β, the main activating kinase in the inducible NF-κB pathway, selectively in myeloid lineage cells (mIKKβKO) or excitatory neurons (nIKKβKO) to measure synaptic strength at hippocampal Schaffer collaterals during long-term potentiation (LTP) and instrumental conditioning in alert behaving individuals. Resting microglial cells in mIKKβKO mice showed less Iba1-immunoreactivity, and brain IL-1β mRNA levels were selectively reduced compared with controls. Measurement of field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the CA3-CA1 synapse in mIKKβKO mice showed higher facilitation in response to paired pulses and enhanced LTP following high frequency stimulation. In contrast, nIKKβKO mice showed normal basic synaptic transmission and LTP induction but impairments in late LTP. To understand the consequences of such impairments in synaptic plasticity for learning and memory, we measured CA1 fEPSPs in behaving mice during instrumental conditioning. IKKβ was not necessary in either microglia or neurons for mice to learn lever-pressing (appetitive behavior) to obtain food (consummatory behavior) but was required in both for modification of their hippocampus-dependent appetitive, not consummatory behavior. Our results show that microglia, through IKKβ and therefore NF-κB activity, regulate hippocampal synaptic plasticity and that both microglia and neurons, through IKKβ, are necessary for animals to modify hippocampus-driven behavior during associative learning.

Epigenetic modifications during sex change repress gonadotropin stimulation of cyp19a1a in a teleost ricefield eel (Monopterus albus).

  • Zhang Y
  • Endocrinology
  • 2013 Aug 22

Literature context:


Abstract:

In vertebrates, cytochrome P450 aromatase, encoded by cyp19a1, converts androgens to estrogens and plays important roles in gonadal differentiation and development. The present study examines whether epigenetic mechanisms are involved in cyp19a1a expression and subsequent gonadal development in the hermaphroditic ricefield eel. The expression of the ricefield eel cyp19a1a was stimulated by gonadotropin via the cAMP pathway in the ovary but not the ovotestis or testis. The CpG within the cAMP response element (CRE) of the cyp19a1a promoter was hypermethylated in the ovotestis and testis compared with the ovary. The methylation levels of CpG sites around CRE in the distal region (region II) and around steroidogenic factor 1/adrenal 4 binding protein sites and TATA box in the proximal region (region I) were inversely correlated with cyp19a1a expression during the natural sex change from female to male. In vitro DNA methylation decreased the basal and forskolin-induced activities of cyp19a1a promoter. Chromatin immunoprecipitation assays indicated that histone 3 (Lys9) in both regions I and II of the cyp19a1a promoter were deacetylated and trimethylated in the testis, and in contrast to the ovary, phosphorylated CRE-binding protein failed to bind to these regions. Lastly, the DNA methylation inhibitor 5-aza-2'-deoxycytidine reversed the natural sex change of ricefield eels. These results suggested that epigenetic mechanisms involving DNA methylation and histone deacetylation and methylation may abrogate the stimulation of cyp19a1a by gonadotropins in a male-specific fashion. This may be a mechanism widely used to drive natural sex change in teleosts as well as gonadal differentiation in other vertebrates.

Funding information:
  • NIA NIH HHS - AG039669(United States)
  • NIEHS NIH HHS - U01 ES019480(United States)