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Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP

RRID:AB_228307

Antibody ID

AB_228307

Target Antigen

Mouse IgG (H+L) mouse

Proper Citation

(Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)

Clonality

polyclonal antibody

Comments

Applications: IHC (1:5,000-1:100,000), IP (1:500-1:5,000), WB (1:5,000-1:200,000), ELISA (1:10,000-1:25,000)

Host Organism

goat

Vendor

Thermo Fisher Scientific Go To Vendor

MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts.

  • Lee SW
  • Dev. Cell
  • 2018 Jul 2

Literature context:


Abstract:

The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9∗-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9∗-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.

Funding information:
  • NHLBI NIH HHS - P50 HL077107(United States)

Naive B Cells with High-Avidity Germline-Encoded Antigen Receptors Produce Persistent IgM+ and Transient IgG+ Memory B Cells.

  • Pape KA
  • Immunity
  • 2018 Jun 19

Literature context:


Abstract:

Although immune memory often lasts for life, this is not the case for certain vaccines in some individuals. We sought a mechanism for this phenomenon by studying B cell responses to phycoerythrin (PE). PE immunization of mouse strains with Ighb immunoglobulin (Ig) variable heavy chain (VH) genes elicited affinity-matured switched Ig memory B cells that declined with time, while the comparable population from an Igha strain was numerically stable. Ighb strains had larger numbers of PE-specific naive B cells and generated smaller germinal center responses and larger numbers of IgM memory cells than the Igha strain. The properties of PE-specific B cells in Ighb mice correlated with usage of a single VH that afforded high-affinity PE binding in its germline form. These results suggest that some individuals may be genetically predisposed to generate non-canonical memory B cell responses to certain antigens because of avid antigen binding via germline-encoded VH elements.

Funding information:
  • NIDCR NIH HHS - R01 DE017413-01A1(United States)

lncRNA Epigenetic Landscape Analysis Identifies EPIC1 as an Oncogenic lncRNA that Interacts with MYC and Promotes Cell-Cycle Progression in Cancer.

  • Wang Z
  • Cancer Cell
  • 2018 Apr 9

Literature context:


Abstract:

We characterized the epigenetic landscape of genes encoding long noncoding RNAs (lncRNAs) across 6,475 tumors and 455 cancer cell lines. In stark contrast to the CpG island hypermethylation phenotype in cancer, we observed a recurrent hypomethylation of 1,006 lncRNA genes in cancer, including EPIC1 (epigenetically-induced lncRNA1). Overexpression of EPIC1 is associated with poor prognosis in luminal B breast cancer patients and enhances tumor growth in vitro and in vivo. Mechanistically, EPIC1 promotes cell-cycle progression by interacting with MYC through EPIC1's 129-283 nt region. EPIC1 knockdown reduces the occupancy of MYC to its target genes (e.g., CDKN1A, CCNA2, CDC20, and CDC45). MYC depletion abolishes EPIC1's regulation of MYC target and luminal breast cancer tumorigenesis in vitro and in vivo.

Funding information:
  • Intramural NIH HHS - Z01 ES100485(United States)

Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus.

  • Gentsch GE
  • Dev. Cell
  • 2018 Mar 12

Literature context:


Abstract:

Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.

Funding information:
  • NICHD NIH HHS - R01HD046236(United States)

NMDA receptor blockade ameliorates abnormalities of spike firing of subthalamic nucleus neurons in a parkinsonian nonhuman primate.

  • Bhattacharya S
  • J. Neurosci. Res.
  • 2018 Mar 27

Literature context:


Abstract:

N-methyl-D-aspartate receptors (NMDARs) are ion channels comprising tetrameric assemblies of GluN1 and GluN2 receptor subunits that mediate excitatory neurotransmission in the central nervous system. Of the four different GluN2 subunits, the GluN2D subunit-containing NMDARs have been suggested as a target for antiparkinsonian therapy because of their expression pattern in some of the basal ganglia nuclei that show abnormal firing patterns in the parkinsonian state, specifically the subthalamic nucleus (STN). In this study, we demonstrate that blockade of NMDARs altered spike firing in the STN in a male nonhuman primate that had been rendered parkinsonian by treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In accompanying experiments in male rodents, we found that GluN2D-NMDAR expression in the STN was reduced in acutely or chronically dopamine-depleted animals. Taken together, our data suggest that blockade of NMDARs in the STN may be a viable antiparkinsonian strategy, but that the ultimate success of this approach may be complicated by parkinsonism-associated changes in NMDAR expression in the STN.

Funding information:
  • NIDCR NIH HHS - T32 DE014320(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)

Role of the GM1 ganglioside oligosaccharide portion in the TrkA-dependent neurite sprouting in neuroblastoma cells.

  • Chiricozzi E
  • J. Neurochem.
  • 2017 Dec 20

Literature context:


Abstract:

GM1 ganglioside (II3 NeuAc-Gg4 Cer) is known to promote neurite formation in neuroblastoma cells by activating TrkA-MAPK pathway. The molecular mechanism by which GM1 is involved in the neurodifferentiation process is still unknown, however, in vitro and in vivo evidences have suggested that the oligosaccharide portion of this ganglioside could be involved. Here, we report that, similarly to the entire GM1 molecule, its oligosaccharide II3 NeuAc-Gg4, rather than its ceramide (Cer) portion is responsible for the neurodifferentiation process by augmenting neurite elongation and increasing the neurofilament protein expression in murine neuroblastoma cells, Neuro2a. Conversely, asialo-GM1, GM2 and GM3 oligosaccharides are not effective in neurite elongation on Neuro2a cells, whereas the effect exerted by the Fuc-GM1 oligosaccharide (IV2 αFucII3 Neu5Ac-Gg4 ) is similar to that exerted by GM1 oligosaccharide. The neurotrophic properties of GM1 oligosaccharide are exerted by activating the TrkA receptor and the following phosphorylation cascade. By photolabeling experiments performed with a nitrophenylazide containing GM1 oligosaccharide, labeled with tritium, we showed a direct interaction between the GM1 oligosaccharide and the extracellular domain of TrkA receptor. Moreover, molecular docking analyses confirmed that GM1 oligosaccharide binds the TrkA-nerve growth factor complex leading to a binding free energy of approx. -11.5 kcal/mol, acting as a bridge able to increase and stabilize the TrkA-nerve growth factor molecular interactions.

Enhanced AMPA Receptor Trafficking Mediates the Anorexigenic Effect of Endogenous Glucagon-like Peptide-1 in the Paraventricular Hypothalamus.

  • Liu J
  • Neuron
  • 2017 Nov 15

Literature context:


Abstract:

Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA)-dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1. VIDEO ABSTRACT.

Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins.

  • Smith KR
  • J. Neurosci.
  • 2017 Nov 15

Literature context:


Abstract:

Appropriate excitatory/inhibitory (E/I) balance is essential for normal cortical function and is altered in some psychiatric disorders, including autism spectrum disorders (ASDs). Cell-autonomous molecular mechanisms that control the balance of excitatory and inhibitory synapse function remain poorly understood; no proteins that regulate excitatory and inhibitory synapse strength in a coordinated reciprocal manner have been identified. Using super-resolution imaging, electrophysiology, and molecular manipulations, we show that cadherin-10, encoded by CDH10 within the ASD risk locus 5p14.1, maintains both excitatory and inhibitory synaptic scaffold structure in cultured cortical neurons from rats of both sexes. Cadherin-10 localizes to both excitatory and inhibitory synapses in neocortex, where it is organized into nanoscale puncta that influence the size of their associated PSDs. Knockdown of cadherin-10 reduces excitatory but increases inhibitory synapse size and strength, altering the E/I ratio in cortical neurons. Furthermore, cadherin-10 exhibits differential participation in complexes with PSD-95 and gephyrin, which may underlie its role in maintaining the E/I ratio. Our data provide a new mechanism whereby a protein encoded by a common ASD risk factor controls E/I ratios by regulating excitatory and inhibitory synapses in opposing directions.SIGNIFICANCE STATEMENT The correct balance between excitatory/inhibitory (E/I) is crucial for normal brain function and is altered in psychiatric disorders such as autism. However, the molecular mechanisms that underlie this balance remain elusive. To address this, we studied cadherin-10, an adhesion protein that is genetically linked to autism and understudied at the cellular level. Using a combination of advanced microscopy techniques and electrophysiology, we show that cadherin-10 forms nanoscale puncta at excitatory and inhibitory synapses, maintains excitatory and inhibitory synaptic structure, and is essential for maintaining the correct balance between excitation and inhibition in neuronal dendrites. These findings reveal a new mechanism by which E/I balance is controlled in neurons and may bear relevance to synaptic dysfunction in autism.

Diadenosine tetraphosphate (Ap4A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

  • Reigada D
  • Purinergic Signal.
  • 2017 Nov 17

Literature context:


Abstract:

Reducing cell death during the secondary injury is a major priority in the development of a cure for traumatic spinal cord injury (SCI). One of the earliest processes that follow SCI is the excitotoxicity resulting from the massive release of excitotoxicity mediators, including ATP, which induce an excessive and/or prolonged activation of their receptors and a deregulation of the calcium homeostasis. Diadenosine tetraphosphate (Ap4A) is an endogenous purinergic agonist, present in both extracellular and intracellular fluids, with promising cytoprotective effects in different diseases including neurodegenerative processes. In a search for efficient neuroprotective strategies for SCI, we have tested the capability of Ap4A to reduce the excitotoxic death mediated by the ATP-induced deregulation of calcium homeostasis and its consequences on tissue preservation and functional recovery in a mouse model of moderate contusive SCI. Our analyses with the murine neural cell line Neuro2a demonstrate that treatment with Ap4A reduces ATP-dependent excitotoxic death by both lowering the intracellular calcium response and decreasing the expression of specific purinergic receptors. Follow-up analyses in a mouse model of contusive SCI showed that acute administration of Ap4A following SCI reduces tissue damage and improves motor function recovery. These results suggest that Ap4A cytoprotection results from a decrease of the purinergic tone preventing the effects of a massive release of ATP after SCI, probably together with a direct induction of anti-apoptotic and pro-survival pathways via activation of P2Y2 proposed in previous studies. In conclusion, Ap4A may be a good candidate for an SCI therapy, particularly to reduce excitotoxicity in combination with other modulators and/or inhibitors of the excitotoxic process that are being tested.

A Split-Abl Kinase for Direct Activation in Cells.

  • Diaz JE
  • Cell Chem Biol
  • 2017 Oct 19

Literature context:


Abstract:

To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells.

Funding information:
  • NCI NIH HHS - R01 CA191018()
  • NIGMS NIH HHS - F32 GM089082()
  • NIGMS NIH HHS - P41 GM108538()
  • NIGMS NIH HHS - R01 GM097316()
  • NLM NIH HHS - T15 LM007359()

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

  • Cooke TF
  • Cell
  • 2017 Oct 5

Literature context:


Abstract:

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Funding information:
  • NIGMS NIH HHS - T32 GM007276()

Ribosome Collision Is Critical for Quality Control during No-Go Decay.

  • Simms CL
  • Mol. Cell
  • 2017 Oct 19

Literature context:


Abstract:

No-go decay (NGD) is a eukaryotic quality control mechanism that evolved to cope with translational arrests. The process is characterized by an endonucleolytic cleavage near the stall sequence, but the mechanistic details are unclear. Our analysis of cleavage sites indicates that cleavage requires multiple ribosomes on the mRNA. We also show that reporters harboring stall sequences near the initiation codon, which cannot accommodate multiple ribosomes, are not subject to NGD. Consistent with our model, we uncover an inverse correlation between ribosome density per mRNA and cleavage efficiency. Furthermore, promoting global ribosome collision in vivo resulted in ubiquitination of ribosomal proteins, suggesting that collision is sensed by the cell to initiate downstream quality control processes. Collectively, our data suggest that NGD and subsequent quality control are triggered by ribosome collision. This model provides insight into the regulation of quality control processes and the manner by which they reduce off-target effects.

A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice.

  • Roberts MN
  • Cell Metab.
  • 2017 Sep 5

Literature context:


Abstract:

Calorie restriction, without malnutrition, has been shown to increase lifespan and is associated with a shift away from glycolysis toward beta-oxidation. The objective of this study was to mimic this metabolic shift using low-carbohydrate diets and to determine the influence of these diets on longevity and healthspan in mice. C57BL/6 mice were assigned to a ketogenic, low-carbohydrate, or control diet at 12 months of age and were either allowed to live their natural lifespan or tested for physiological function after 1 or 14 months of dietary intervention. The ketogenic diet (KD) significantly increased median lifespan and survival compared to controls. In aged mice, only those consuming a KD displayed preservation of physiological function. The KD increased protein acetylation levels and regulated mTORC1 signaling in a tissue-dependent manner. This study demonstrates that a KD extends longevity and healthspan in mice.

Funding information:
  • NIA NIH HHS - P01 AG025532()
  • NIDDK NIH HHS - U24 DK092993()

Post-meiotic DNA double-strand breaks occur in Tetrahymena, and require Topoisomerase II and Spo11.

  • Akematsu T
  • Elife
  • 2017 Jun 16

Literature context:


Abstract:

Based on observations of markers for DNA lesions, such as phosphorylated histone H2AX (γH2AX) and open DNA ends, it has been suggested that post-meiotic DNA double-strand breaks (PM-DSBs) enable chromatin remodeling during animal spermiogenesis. However, the existence of PM-DSBs is unconfirmed, and the mechanism responsible for their formation is unclear. Here, we report the first direct observation of programmed PM-DSBs via the electrophoretic separation of DSB-generated DNA fragments in the ciliate Tetrahymena thermophila. These PM-DSBs are accompanied by switching from a heterochromatic to euchromatic chromatin structure in the haploid pronucleus. Both a topoisomerase II paralog with exclusive pronuclear expression and Spo11 are prerequisites for PM-DSB induction. Reduced PM-DSB induction blocks euchromatin formation, characterized by histone H3K56 acetylation, leading to a failure in gametic nuclei production. We propose that PM-DSBs are responsible for histone replacement during the reprogramming of generative to undifferentiated progeny nuclei.

Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.

  • Yu J
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

Human endometrial stromal decidualization is required for embryo receptivity, angiogenesis, and placentation. Previous studies from our laboratories established that connexin (Cx)-43 critically regulates endometrial stromal cell (ESC) differentiation, whereas gap junction blockade prevents it. The current study evaluated the plasticity of ESC morphology and Cx43 expression, as well as other biochemical markers of cell differentiation, in response to decidualizing hormones. Primary human ESC cultures were exposed to 10 nM estradiol, 100 nM progesterone, and 0.5 mM cAMP for up to 14 days, followed by hormone withdrawal for 14 days, mimicking a biphasic ovulatory cycle. Reversible differentiation was documented by characteristic changes in cell shape. Cx43 was reversibly up- and down-regulated after the estradiol, progesterone, and cAMP treatment and withdrawal, respectively, paralleled by fluctuations in prolactin, vascular endothelial growth factor, IL-11, and glycodelin secretion. Markers of mesenchymal-epithelial transition (MET), and its counterpart epithelial-mesenchymal transition, followed reciprocal patterns corresponding to the morphological changes. Incubation in the presence of 18α-glycyrrhetinic acid, an inhibitor of gap junctions, partially reversed the expression of decidualization and MET markers. In the absence of hormones, Cx43 overexpression promoted increases in vascular endothelial growth factor and IL-11 secretion, up-regulated MET markers, and reduced N-cadherin, an epithelial-mesenchymal transition marker. The combined results support the hypothesis that Cx43-containing gap junctions and endocrine factors cooperate to regulate selected biomarkers of stromal decidualization and MET and suggest roles for both phenomena in endometrial preparation for embryonic receptivity.

Funding information:
  • NIBIB NIH HHS - R37 EB003320(United States)

JMJD3 Is Crucial for the Female AVPV RIP-Cre Neuron-Controlled Kisspeptin-Estrogen Feedback Loop and Reproductive Function.

  • Song A
  • Endocrinology
  • 2017 Jun 1

Literature context:


Abstract:

The hypothalamic-pituitary-gonadal axis controls development, reproduction, and metabolism. Although most studies have focused on the hierarchy from the brain to the gonad, many questions remain unresolved concerning the feedback from the gonad to the central nervous system, especially regarding the potential epigenetic modifications in hypothalamic neurons. In the present report, we generated genetically modified mice lacking histone H3 lysine 27 (H3K27) demethylase Jumonji domain-containing 3 (JMJD3) in hypothalamic rat-insulin-promoter-expressing neurons (RIP-Cre neurons). The female mutant mice displayed late-onset obesity owing to reduced locomotor activity and decreased energy expenditure. JMJD3 deficiency in RIP-Cre neurons also results in delayed pubertal onset, an irregular estrous cycle, impaired fertility, and accelerated ovarian failure in female mice owing to the dysregulation of the hypothalamic-ovarian axis. We found that JMJD3 directly regulates Kiss1 gene expression by binding to the Kiss1 promoter and triggering H3K27me3 demethylation in the anteroventral periventricular (AVPV) nucleus. Further study confirmed that the aberrations arose from impaired kisspeptin signaling in the hypothalamic AVPV nucleus and subsequent estrogen deficiency. Estrogen replacement therapy can reverse obesity in mutant mice. Moreover, we demonstrated that Jmjd3 is an estrogen target gene in the hypothalamus. These results provide direct genetic and molecular evidence that JMJD3 is a key mediator for the kisspeptin-estrogen feedback loop.

Covalent Protein Labeling at Glutamic Acids.

  • Martín-Gago P
  • Cell Chem Biol
  • 2017 May 18

Literature context:


Abstract:

Covalent labeling of amino acids in proteins by reactive small molecules, in particular at cysteine SH and lysine NH groups, is a powerful approach to identify and characterize proteins and their functions. However, for the less-reactive carboxylic acids present in Asp and Glu, hardly any methodology is available. Employing the lipoprotein binding chaperone PDE6δ as an example, we demonstrate that incorporation of isoxazolium salts that resemble the structure and reactivity of Woodward's reagent K into protein ligands provides a novel method for selective covalent targeting of binding site carboxylic acids in whole proteomes. Covalent adduct formation occurs via rapid formation of enol esters and the covalent bond is stable even in the presence of strong nucleophiles. This new method promises to open up hitherto unexplored opportunities for chemical biology research.

Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence.

  • Neubauer HA
  • F1000Res
  • 2017 Apr 14

Literature context:


Abstract:

Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( Sphk2-/-) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

A Drug Screen using Human iPSC-Derived Hepatocyte-like Cells Reveals Cardiac Glycosides as a Potential Treatment for Hypercholesterolemia.

  • Cayo MA
  • Cell Stem Cell
  • 2017 Apr 6

Literature context:


Abstract:

Efforts to identify pharmaceuticals to treat heritable metabolic liver diseases have been hampered by the lack of models. However, cells with hepatocyte characteristics can be produced from induced pluripotent stem cells (iPSCs). Here, we have used hepatocyte-like cells generated from homozygous familial hypercholesterolemia (hoFH) iPSCs to identify drugs that can potentially be repurposed to lower serum LDL-C. We found that cardiac glycosides reduce the production of apolipoprotein B (apoB) from human hepatocytes in culture and the serum of avatar mice harboring humanized livers. The drugs act by increasing the turnover of apoB protein. Analyses of patient medical records revealed that the treatment of patients with cardiac glycosides reduced serum LDL-C levels. These studies highlight the effectiveness of using iPSCs to screen for potential treatments for inborn errors of hepatic metabolism and suggest that cardiac glycosides could provide an approach for reducing hepatocyte production of apoB and treating hypercholesterolemia.

Funding information:
  • NCATS NIH HHS - UL1 TR000055()
  • NHGRI NIH HHS - U01 HG006398()
  • NICHD NIH HHS - R21 HD082570()
  • NIDDK NIH HHS - F30 DK091994()
  • NIDDK NIH HHS - R01 DK055743()
  • NIDDK NIH HHS - R01 DK102716()
  • NIDDK NIH HHS - RC1 DK087377()

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures.

  • Miller AP
  • PLoS ONE
  • 2017 Mar 6

Literature context:


Abstract:

Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

  • Vernon CG
  • J. Neurosci.
  • 2017 Mar 22

Literature context:


Abstract:

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth.SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits.

Funding information:
  • NCI NIH HHS - P30 CA060553()
  • NINDS NIH HHS - R01 NS071952()

Inhibition of Prolyl Oligopeptidase Restores Spontaneous Motor Behavior in the α-Synuclein Virus Vector-Based Parkinson's Disease Mouse Model by Decreasing α-Synuclein Oligomeric Species in Mouse Brain.

  • Svarcbahs R
  • J. Neurosci.
  • 2016 Dec 7

Literature context:


Abstract:

Decreased clearance of α-synuclein (aSyn) and aSyn protein misfolding and aggregation are seen as major factors in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies that leads to disruption in neuronal function and eventually to cell death. Prolyl oligopeptidase (PREP) can accelerate the aSyn aggregation process, while inhibition of PREP by a small molecule inhibitor decreases aSyn oligomer formation and enhances its clearance via autophagy in different aSyn overexpressing cell types and in transgenic PD animal models. In this study, we investigated the impact of chronic PREP inhibition by a small molecule inhibitor, 4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrrolidine (KYP-2047), on aSyn oligomerization, clearance, and underlying spontaneous motor behavior in a virus vector-based aSyn overexpression mouse model 4 weeks after aSyn microinjections and after the onset of symptomatic forepaw bias. Following 4 weeks of PREP inhibition, we saw an improved spontaneous forelimb use in mice that correlated with a decreased immunoreactivity against oligomer-specific forms of aSyn. Additionally, KYP-2047 had a trend to enhance dopaminergic systems activity. Our results suggest that PREP inhibition exhibits a beneficial effect on the aSyn clearance and aggregation in a virus mediated aSyn overexpression PD mouse model and that PREP inhibitors could be a novel therapeutic strategy for synucleinopathies. SIGNIFICANCE STATEMENT: Alpha-synuclein (aSyn) has been implicated in Parkinson's disease, with aSyn aggregates believed to exert toxic effects on neurons, while prolyl oligopeptidase (PREP) has been shown to interact with aSyn both in cells and cell free conditions, thus enhancing its aggregation. We demonstrate the possibility to abolish motor imbalance caused by aSyn viral vector injection with chronic 4 week PREP inhibition by a potent small-molecule PREP inhibitor, 4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrrolidine (KYP-2047). Treatment was initiated postsymptomatically, 4 weeks after aSyn injection. KYP-2047-treated animals had a significantly decreased amount of oligomeric aSyn particles and improved dopamine system activity compared to control animals. To our knowledge, this is the first time viral overexpression of aSyn has been countered and movement impairments abolished after their onset.

TRIB3 increases cell resistance to arsenite toxicity by limiting the expression of the glutathione-degrading enzyme CHAC1.

  • Örd D
  • Biochim. Biophys. Acta
  • 2016 Nov 27

Literature context:


Abstract:

Arsenic, a metalloid with cytotoxic and carcinogenic effects related to the disruption of glutathione homeostasis, induces the expression of ATF4, a central transcription factor in the cellular stress response. However, the interplay between factors downstream of ATF4 is incompletely understood. In this article, we investigate the role of Tribbles homolog 3 (TRIB3), a regulatory member of the ATF4 pathway, in determining cell sensitivity to arsenite. Our results show that arsenite potently upregulates Trib3 mRNA and protein in an ATF4-dependent manner in mouse embryonic fibroblasts. Trib3-deficient cells display increased susceptibility to arsenite-induced cell death, which is rescued by re-expressing TRIB3. In cells lacking TRIB3, arsenite stress leads to markedly elevated mRNA and protein levels of Chac1, a gene that encodes a glutathione-degrading enzyme and is not previously known to be repressed by TRIB3. Analysis of the Chac1 promoter identified two regulatory elements that additively mediate the induction of Chac1 by arsenite and ATF4, as well as the robust suppression of Chac1 by TRIB3. Crucially, Chac1 silencing enhances glutathione levels and eliminates the increased susceptibility of Trib3-deficient cells to arsenite stress. Moreover, Trib3-deficient cells demonstrate an increased rate of glutathione consumption, which is abolished by Chac1 knockdown. Taken together, these data indicate that excessive Chac1 expression is detrimental to arsenite-treated cell survival and that TRIB3 is critical for restraining the pro-death potential of Chac1 during arsenite stress, representing a novel mechanism of cell viability regulation that occurs within the ATF4 pathway.

Funding information:
  • NCATS NIH HHS - UL1 TR001082(United States)

Association of N-cadherin levels and downstream effectors of Rho GTPases with dendritic spine loss induced by chronic stress in rat hippocampal neurons.

  • Castañeda P
  • J. Neurosci. Res.
  • 2015 Oct 20

Literature context:


Abstract:

Chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons. In this animal model of depression, spine loss probably involves a weakening of the interaction between pre- and postsynaptic cell adhesion molecules, such as N-cadherin, followed by disruption of the cytoskeleton. N-cadherin, in concert with catenin, stabilizes the cytoskeleton through Rho-family GTPases. Via their effector LIM kinase (LIMK), RhoA and ras-related C3 botulinum toxin substrate 1 (RAC) GTPases phosphorylate and inhibit cofilin, an actin-depolymerizing molecule, favoring spine growth. Additionally, RhoA, through Rho kinase (ROCK), inactivates myosin phosphatase through phosphorylation of the myosin-binding subunit (MYPT1), producing actomyosin contraction and probable spine loss. Some micro-RNAs negatively control the translation of specific mRNAs involved in Rho GTPase signaling. For example, miR-138 indirectly activates RhoA, and miR-134 reduces LIMK1 levels, resulting in spine shrinkage; in contrast, miR-132 activates RAC1, promoting spine formation. We evaluated whether N-cadherin/β-catenin and Rho signaling is sensitive to chronic restraint stress. Stressed rats exhibit anhedonia, impaired associative learning, and immobility in the forced swim test and reduction in N-cadherin levels but not β-catenin in the hippocampus. We observed a reduction in spine number in the apical dendrites of CA1 pyramidal neurons, with no effect on the levels of miR-132 or miR-134. Although the stress did not modify the RAC-LIMK-cofilin signaling pathway, we observed increased phospho-MYPT1 levels, probably mediated by RhoA-ROCK activation. Furthermore, chronic stress raises the levels of miR-138 in accordance with the observed activation of the RhoA-ROCK pathway. Our findings suggest that a dysregulation of RhoA-ROCK activity by chronic stress could potentially underlie spine loss in hippocampal neurons.

Acute administration of ucf-101 ameliorates the locomotor impairments induced by a traumatic spinal cord injury.

  • Reigada D
  • Neuroscience
  • 2015 Aug 6

Literature context:


Abstract:

Secondary death of neural cells plays a key role in the physiopathology and the functional consequences of traumatic spinal cord injury (SCI). Pharmacological manipulation of cell death pathways leading to the preservation of neural cells is acknowledged as a main therapeutic goal in SCI. In the present work, we hypothesize that administration of the neuroprotective cell-permeable compound ucf-101 will reduce neural cell death during the secondary damage of SCI, increasing tissue preservation and reducing the functional deficits. To test this hypothesis, we treated mice with ucf-101 during the first week after a moderate contusive SCI. Our results reveal that ucf-101 administration protects neural cells from the deleterious secondary mechanisms triggered by the trauma, reducing the extension of tissue damage and improving motor function recovery. Our studies also suggest that the effects of ucf-101 may be mediated through the inhibition of HtrA2/OMI and the concomitant increase of inhibitor of apoptosis protein XIAP, as well as the induction of ERK1/2 activation and/or expression. In vitro assays confirm the effects of ucf-101 on both pathways as well as on the reduction of caspase cascade activation and apoptotic cell death in a neuroblastoma cell line. These results suggest that ucf-101 can be a promising therapeutic tool for SCI that deserves more detailed analyses.