X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647

RRID:AB_2535813

Spatial Fold Change of FGF Signaling Encodes Positional Information for Segmental Determination in Zebrafish.

  • Simsek MF
  • Cell Rep
  • 2018 Jul 3

Literature context: or 647 Invitrogen Cat#A-21245; RRID:AB_2535813 Anti-Digoxygenin (DIG)-AP Fab f


Abstract:

Signal gradients encode instructive information for numerous decision-making processes during embryonic development. A striking example of precise, scalable tissue-level patterning is the segmentation of somites-the precursors of the vertebral column-during which the fibroblast growth factor (FGF), Wnt, and retinoic acid (RA) pathways establish spatial gradients. Despite decades of studies proposing roles for all three pathways, the dynamic feature of these gradients that encodes instructive information determining segment sizes remained elusive. We developed a non-elongating tail explant system, integrated quantitative measurements with computational modeling, and tested alternative models to show that positional information is encoded solely by spatial fold change (SFC) in FGF signal output. Neighboring cells measure SFC to accurately position the determination front and thus determine segment size. The SFC model successfully recapitulates results of spatiotemporal perturbation experiments on both explants and intact embryos, and it shows that Wnt signaling acts permissively upstream of FGF signaling and that RA gradient is dispensable.

Funding information:
  • NIGMS NIH HHS - 1R15GM94732-1 A1(United States)

3D Culture Method for Alzheimer's Disease Modeling Reveals Interleukin-4 Rescues Aβ42-Induced Loss of Human Neural Stem Cell Plasticity.

  • Papadimitriou C
  • Dev. Cell
  • 2018 Jul 2

Literature context: Life Technologies Cat# A-21245; RRID:AB_2535813 Goat anti-Rat IgG (H+L), Alexa


Abstract:

Neural stem cells (NSCs) constitute an endogenous reservoir for neurons that could potentially be harnessed for regenerative therapies in disease contexts such as neurodegeneration. However, in Alzheimer's disease (AD), NSCs lose plasticity and thus possible regenerative capacity. We investigate how NSCs lose their plasticity in AD by using starPEG-heparin-based hydrogels to establish a reductionist 3D cell-instructive neuro-microenvironment that promotes the proliferative and neurogenic ability of primary and induced human NSCs. We find that administration of AD-associated Amyloid-β42 causes classical neuropathology and hampers NSC plasticity by inducing kynurenic acid (KYNA) production. Interleukin-4 restores NSC proliferative and neurogenic ability by suppressing the KYNA-producing enzyme Kynurenine aminotransferase (KAT2), which is upregulated in APP/PS1dE9 mouse model of AD and in postmortem human AD brains. Thus, our culture system enables a reductionist investigation of regulation of human NSC plasticity for the identification of potential therapeutic targets for intervention in AD.

Funding information:
  • Howard Hughes Medical Institute - (United States)

Probing nano-organization of astroglia with multi-color super-resolution microscopy.

  • Heller JP
  • J. Neurosci. Res.
  • 2018 May 18

Literature context: # A21245, RRID:AB_2535813), which we


Abstract:

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100β and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc.

Funding information:
  • Medical Research Council - G0600368()
  • Medical Research Council - G0801316()
  • Medical Research Council - G0802216()
  • Medical Research Council - G0900613()

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

  • Haider A
  • Dev. Cell
  • 2018 May 21

Literature context: Fisher Scientific Cat # A21245; RRID:AB_2535813 Mouse monoclonal anti-Rh1 DSHB


Abstract:

Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.

Funding information:
  • Canadian Institutes of Health Research - MOP-312268(Canada)

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

  • Guo Q
  • Cell
  • 2018 Feb 8

Literature context: Fisher Scientific Cat# A-21245; RRID:AB_2535813 Mouse monoclonal GFP NeuroMab C


Abstract:

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

Funding information:
  • Medical Research Council - MR/J007773/1(United Kingdom)
  • NIGMS NIH HHS - P41 GM104601()

Fc gamma receptors are expressed in the developing rat brain and activate downstream signaling molecules upon cross-linking with immune complex.

  • Stamou M
  • J Neuroinflammation
  • 2018 Jan 6

Literature context: ermo Fisher Scientific A-21245, RRID:AB_2535813; and AlexaFluor-488 goat anti-m


Abstract:

BACKGROUND: Exposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function. METHODS: FcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes. RESULTS: FcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG. CONCLUSIONS: Our data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.

Funding information:
  • Eidgenössische Technische Hochschule Zürich - ETH-30 17-1()
  • Medical Research Council - MC_PC_12001(United Kingdom)
  • National Institute of Child Health and Human Development - HD079125()
  • National Institute of Environmental Health Sciences - ES011269()
  • National Institute of Environmental Health Sciences - ES023513()
  • National Institute of Environmental Health Sciences - ES04699()
  • NICHD NIH HHS - U54 HD079125()
  • NIEHS NIH HHS - P01 ES011269()
  • NIEHS NIH HHS - P30 ES023513()
  • NIEHS NIH HHS - P42 ES004699()
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung - SNF_160259()
  • U.S. Environmental Protection Agency - 83543201()

Human-Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for Efficient Early HIV-1 Replication In Vivo.

  • Yamada E
  • Cell Host Microbe
  • 2018 Jan 10

Literature context: Fisher Scientific Cat# A-21245; RRID:AB_2535813 FITC-conjugated anti-HIV-1 p24


Abstract:

The HIV-1-encoded accessory protein Vpu exerts several immunomodulatory functions, including counteraction of the host restriction factor tetherin, downmodulation of CD4, and inhibition of NF-κB activity to facilitate HIV-1 infection. However, the relative contribution of individual Vpu functions to HIV-1 infection in vivo remained unclear. Here, we used a humanized mouse model and HIV-1 strains with selective mutations in vpu to demonstrate that the anti-tetherin activity of Vpu is a prerequisite for efficient viral spread during the early phase of infection. Mathematical modeling and gain-of-function mutations in SIVcpz, the simian precursor of pandemic HIV-1, corroborate this finding. Blockage of interferon signaling combined with transcriptome analyses revealed that basal tetherin levels are sufficient to control viral replication. These results establish tetherin as a key effector of the intrinsic immune defense against HIV-1, and they demonstrate that Vpu-mediated tetherin antagonism is critical for efficient viral spread during the initial phase of HIV-1 replication.

Activating the regenerative potential of Müller glia cells in a regeneration-deficient retina.

  • Lust K
  • Elife
  • 2018 Jan 29

Literature context: 7 (goat) Thermo Fisher A-21245, RRID:AB_2535813 1: 750


Abstract:

Regeneration responses in animals are widespread across phyla. To identify molecular players that confer regenerative capacities to non-regenerative species is of key relevance for basic research and translational approaches. Here, we report a differential response in retinal regeneration between medaka (Oryzias latipes) and zebrafish (Danio rerio). In contrast to zebrafish, medaka Müller glia (olMG) cells behave like progenitors and exhibit a restricted capacity to regenerate the retina. After injury, olMG cells proliferate but fail to self-renew and ultimately only restore photoreceptors. In our injury paradigm, we observed that in contrast to zebrafish, proliferating olMG cells do not maintain sox2 expression. Sustained sox2 expression in olMG cells confers regenerative responses similar to those of zebrafish MG (drMG) cells. We show that a single, cell-autonomous factor reprograms olMG cells and establishes a regeneration-like mode. Our results position medaka as an attractive model to delineate key regeneration factors with translational potential.

Funding information:
  • NIMH NIH HHS - R01 MH073991(United States)

Endosomal Rab cycles regulate Parkin-mediated mitophagy.

  • Yamano K
  • Elife
  • 2018 Jan 23

Literature context: RRID:AB_2535813 1:500 (IF)


Abstract:

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Funding information:
  • Japan Science and Technology Agency - JPMJCR13M7(International)
  • Japan Society for the Promotion of Science - 16K15095(International)
  • Japan Society for the Promotion of Science - JP15H01196(International)
  • Japan Society for the Promotion of Science - JP16K18545(International)
  • Japan Society for the Promotion of Science - JP26000014(International)
  • Japan Society for the Promotion of Science - JP26111729(International)
  • Japan Society for the Promotion of Science - JP26840033(International)
  • NIDCR NIH HHS - R03 DE018415-02(United States)
  • NINDS NIH HHS - Intramural program(United States)

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

  • Clift D
  • Cell
  • 2017 Dec 14

Literature context: or 647 ThermoFisher Cat#A21245; RRID:AB_2535813 Goat anti-Mouse IgG (H+L) Highl


Abstract:

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Funding information:
  • NIDCD NIH HHS - P30 DC04657(United States)

Myosin II Controls Junction Fluctuations to Guide Epithelial Tissue Ordering.

  • Curran S
  • Dev. Cell
  • 2017 Nov 20

Literature context: oFisher Scientific Cat: a21245; RRID:AB_2535813.


Abstract:

Under conditions of homeostasis, dynamic changes in the length of individual adherens junctions (AJs) provide epithelia with the fluidity required to maintain tissue integrity in the face of intrinsic and extrinsic forces. While the contribution of AJ remodeling to developmental morphogenesis has been intensively studied, less is known about AJ dynamics in other circumstances. Here, we study AJ dynamics in an epithelium that undergoes a gradual increase in packing order, without concomitant large-scale changes in tissue size or shape. We find that neighbor exchange events are driven by stochastic fluctuations in junction length, regulated in part by junctional actomyosin. In this context, the developmental increase of isotropic junctional actomyosin reduces the rate of neighbor exchange, contributing to tissue order. We propose a model in which the local variance in tension between junctions determines whether actomyosin-based forces will inhibit or drive the topological transitions that either refine or deform a tissue.

Funding information:
  • NCRR NIH HHS - P51RR165(United States)

Astrocyte-Secreted Glypican 4 Regulates Release of Neuronal Pentraxin 1 from Axons to Induce Functional Synapse Formation.

  • Farhy-Tselnicker I
  • Neuron
  • 2017 Oct 11

Literature context: cular Probes CAT# A21245; RRID:AB_2535813 Goat anti-Rabbit Alexa 680 Mole


Abstract:

The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.

Funding information:
  • NINDS NIH HHS - R01 NS089791()
  • Wellcome Trust - P30 NS072031()

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura.

  • Tischfield MA
  • Dev. Cell
  • 2017 Sep 11

Literature context: (Thermo Fisher)A-11034, A-11037,A-21245Donkey anti-Rabbit IgG (H+L) (1:


Abstract:

Dural cerebral veins (CV) are required for cerebrospinal fluid reabsorption and brain homeostasis, but mechanisms that regulate their growth and remodeling are unknown. We report molecular and cellular processes that regulate dural CV development in mammals and describe venous malformations in humans with craniosynostosis and TWIST1 mutations that are recapitulated in mouse models. Surprisingly, Twist1 is dispensable in endothelial cells but required for specification of osteoprogenitor cells that differentiate into preosteoblasts that produce bone morphogenetic proteins (BMPs). Inactivation of Bmp2 and Bmp4 in preosteoblasts and periosteal dura causes skull and CV malformations, similar to humans harboring TWIST1 mutations. Notably, arterial development appears normal, suggesting that morphogens from the skull and dura establish optimal venous networks independent from arterial influences. Collectively, our work establishes a paradigm whereby CV malformations result from primary or secondary loss of paracrine BMP signaling from preosteoblasts and dura, highlighting unique cellular interactions that influence tissue-specific angiogenesis in mammals.

aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity.

  • Rodriguez J
  • Dev. Cell
  • 2017 Aug 21

Literature context: AB_2576217 / RRID: AB_2534095 / RRID: AB_2535813α-mouse-Alexa488/594/647Molecula


Abstract:

The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.

Neural Circuit-Specialized Astrocytes: Transcriptomic, Proteomic, Morphological, and Functional Evidence.

  • Chai H
  • Neuron
  • 2017 Aug 2

Literature context: t#A21245; RRID:AB_2535813 IRDye 800C


Abstract:

Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested, and the possibility that astrocytes are neural circuit specialized remains largely unexplored. Here, we used multiple integrated approaches, including RNA sequencing (RNA-seq), mass spectrometry, electrophysiology, immunohistochemistry, serial block-face-scanning electron microscopy, morphological reconstructions, pharmacogenetics, and diffusible dye, calcium, and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences in electrophysiological properties, Ca2+ signaling, morphology, and astrocyte-synapse proximity between striatal and hippocampal astrocytes. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits, and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain. VIDEO ABSTRACT.

Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia.

  • Windrem MS
  • Cell Stem Cell
  • 2017 Aug 3

Literature context: #A-21245; RRID:AB_2535813 Goat anti-


Abstract:

In this study, we investigated whether intrinsic glial dysfunction contributes to the pathogenesis of schizophrenia (SCZ). Our approach was to establish humanized glial chimeric mice using glial progenitor cells (GPCs) produced from induced pluripotent stem cells derived from patients with childhood-onset SCZ. After neonatal implantation into myelin-deficient shiverer mice, SCZ GPCs showed premature migration into the cortex, leading to reduced white matter expansion and hypomyelination relative to controls. The SCZ glial chimeras also showed delayed astrocytic differentiation and abnormal astrocytic morphologies. When established in myelin wild-type hosts, SCZ glial mice showed reduced prepulse inhibition and abnormal behavior, including excessive anxiety, antisocial traits, and disturbed sleep. RNA-seq of cultured SCZ human glial progenitor cells (hGPCs) revealed disrupted glial differentiation-associated and synaptic gene expression, indicating that glial pathology was cell autonomous. Our data therefore suggest a causal role for impaired glial maturation in the development of schizophrenia and provide a humanized model for its in vivo assessment.

Funding information:
  • NIMH NIH HHS - R01 MH099578()
  • NIMH NIH HHS - R01 MH104701()

Neuromuscular adaptability of male and female rats to muscle unloading.

  • Deschenes MR
  • J. Neurosci. Res.
  • 2017 Aug 2

Literature context: or 647 (Molecular Probes; RRID; AB_2535813) labeled secondary antibody dil


Abstract:

Previously, it has been shown that following muscle unloading, males and females experience different maladaptations in neuromuscular function. As a follow-up, the present investigation sought to determine if male and female neuromuscular systems demonstrated similar, or disparate morphological adaptations to muscle unloading. Twenty young adult male, and 20 young adult female rats were randomly assigned to one of two treatment protocols: muscle unloading, or control conditions. Following the 2-week intervention period, immunofluorescent procedures were used to quantify pre- and post-synaptic features of neuromuscular junctions (NMJs), and to assess myofiber profiles (size and fiber type composition) of the soleus, plantaris, and EDL muscles. A 2-way ANOVA with main effects for sex and treatment was then used to identify statistically significant (p ≤ .05) differences among structural parameters. Analysis of NMJs showed a consistent lack of differences between males and females. Overall, NMJs were also found to be resistant to the effects of unloading. When examining myofiber profiles, however, male myofibers were revealed to be significantly larger than female ones in each of the muscles examined. Unloading resulted in significant myofiber atrophy only in the primarily weight-bearing soleus muscle. Only the EDL showed unloading-induced differences in myofiber type distribution (Type II → I). These data indicate that different components of the neuromuscular system (NMJs, myofibers) respond uniquely to unloading, and that sex affects myofiber type profiles, but not NMJs. Moreover, it appears that only muscles that have their habitual activity patterns disturbed by unloading (i.e., the soleus, adapt to that intervention).

Selective Erasure of Distinct Forms of Long-Term Synaptic Plasticity Underlying Different Forms of Memory in the Same Postsynaptic Neuron.

  • Hu J
  • Curr. Biol.
  • 2017 Jul 10

Literature context: A-21245; RRID:AB_2535813 Chemicals,


Abstract:

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, we found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant-negative (dn) atypical PKM selectively reversed associative LTF, while a dn classical PKM selectively reversed non-associative LTF. Although both PKMs are formed from calpain-mediated cleavage of protein kinase C (PKC) isoforms, each form of LTF is sensitive to a distinct dn calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dn classical calpain, whereas non-associative LTF is blocked by dn small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs, allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories.

Funding information:
  • NCRR NIH HHS - P40 RR010294()
  • NIMH NIH HHS - R01 MH060387()

Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration.

  • Saini JS
  • Cell Stem Cell
  • 2017 May 4

Literature context: #A-21245; RRID:AB_2535813 Peroxidase


Abstract:

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.

Funding information:
  • NEI NIH HHS - F32 EY025931()
  • NEI NIH HHS - R01 EY022079()
  • NIA NIH HHS - RF1 AG042932()

An inhibitory gate for state transition in cortex.

  • Zucca S
  • Elife
  • 2017 May 16

Literature context: bbit 647 (RRID:AB_2535813, 1:800, In


Abstract:

Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale.

Funding information:
  • NINDS NIH HHS - U01 NS090576()

Dynamic Palmitoylation Targets MAP6 to the Axon to Promote Microtubule Stabilization during Neuronal Polarization.

  • Tortosa E
  • Neuron
  • 2017 May 17

Literature context: , A21245, RRID:AB_2535813), anti-chi


Abstract:

Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediated by the family of α/β Hydrolase domain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6's palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ.

Macrophages Facilitate Electrical Conduction in the Heart.

  • Hulsmans M
  • Cell
  • 2017 Apr 20

Literature context: mo Fisher ScientificCat# A-11036Goat anti-rabbit IgG Alexa Fluor 647Thermo Fisher ScientificCat# A-21245Goat anti-rat IgG Alexa Fluor 56


Abstract:

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Funding information:
  • NHLBI NIH HHS - K24 HL105780()
  • NHLBI NIH HHS - R01 HL092577()
  • NHLBI NIH HHS - R01 HL096576()
  • NHLBI NIH HHS - R01 HL114477()
  • NHLBI NIH HHS - R01 HL117829()
  • NHLBI NIH HHS - R01 HL125428()
  • NHLBI NIH HHS - R01 HL128264()
  • NHLBI NIH HHS - R01 HL131495()
  • NICHD NIH HHS - R01 HD069623()
  • NIDDK NIH HHS - P30 DK043351()
  • NIDDK NIH HHS - P30 DK057521()
  • NINDS NIH HHS - R01 NS084863()

Hallmarks of Alzheimer's Disease in Stem-Cell-Derived Human Neurons Transplanted into Mouse Brain.

  • Espuny-Camacho I
  • Neuron
  • 2017 Mar 8

Literature context: so A21245 RRID:AB_2535813 Anti-rabbi


Abstract:

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.

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

  • Miller AP
  • PLoS ONE
  • 2017 Mar 6

Literature context: A-21245, RRID:AB_2535813; 1:750), a


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.

Leaky Gate Model: Intensity-Dependent Coding of Pain and Itch in the Spinal Cord.

  • Sun S
  • Neuron
  • 2017 Feb 22

Literature context: # A21245; RRID:AB_2535813 goat anti-


Abstract:

Coding of itch versus pain has been heatedly debated for decades. However, the current coding theories (labeled line, intensity, and selectivity theory) cannot accommodate all experimental observations. Here we identified a subset of spinal interneurons, labeled by gastrin-releasing peptide (Grp), that receive direct synaptic input from both pain and itch primary sensory neurons. When activated, these Grp+ neurons generated rarely seen, simultaneous robust pain and itch responses that were intensity dependent. Accordingly, we propose a "leaky gate" model in which Grp+ neurons transmit both itch and weak pain signals; however, upon strong painful stimuli, the recruitment of endogenous opioids works to close this gate, reducing overwhelming pain generated by parallel pathways. Consistent with our model, loss of these Grp+ neurons increased pain responses while itch was decreased. Our new model serves as an example of non-monotonic coding in the spinal cord and better explains observations in human psychophysical studies.

Funding information:
  • NEI NIH HHS - R01 EY024704()
  • NIAID NIH HHS - R01 AI125743()
  • NIDCR NIH HHS - R01 DE022750()
  • NINDS NIH HHS - R01 NS054791()
  • NINDS NIH HHS - R01 NS070814()

Pharmacological Rescue of Long-Term Potentiation in Alzheimer Diseased Synapses.

  • Prieto GA
  • J. Neurosci.
  • 2017 Feb 1

Literature context: #A-21245 RRID:AB_2535813), AlexaFlu


Abstract:

Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.

Funding information:
  • NIA NIH HHS - P01 AG000538()
  • NIA NIH HHS - P50 AG016573()
  • NIA NIH HHS - R01 AG034667()
  • NIA NIH HHS - R21 AG048506()
  • NINDS NIH HHS - P01 NS045260()

Hand2 inhibits kidney specification while promoting vein formation within the posterior mesoderm.

  • Perens EA
  • Elife
  • 2016 Nov 2

Literature context: A21245) (RRID:AB_2535813), all at 1


Abstract:

Proper organogenesis depends upon defining the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that set the boundaries of the IM are poorly understood. Here, we show that the bHLH transcription factor Hand2 limits the size of the embryonic kidney by restricting IM dimensions. The IM is expanded in zebrafish hand2 mutants and is diminished when hand2 is overexpressed. Within the posterior mesoderm, hand2 is expressed laterally adjacent to the IM. Venous progenitors arise between these two territories, and hand2 promotes venous development while inhibiting IM formation at this interface. Furthermore, hand2 and the co-expressed zinc-finger transcription factor osr1 have functionally antagonistic influences on kidney development. Together, our data suggest that hand2 functions in opposition to osr1 to balance the formation of kidney and vein progenitors by regulating cell fate decisions at the lateral boundary of the IM.

Funding information:
  • NEI NIH HHS - R01 EY020533(United States)

In Utero Exposure to Valproic Acid Induces Neocortical Dysgenesis via Dysregulation of Neural Progenitor Cell Proliferation/Differentiation.

  • Fujimura K
  • J. Neurosci.
  • 2016 Oct 19

Literature context: ientific, RRID:AB_2535813; room temp


Abstract:

Valproic acid (VPA), a widely used antiepileptic drug, is an inhibitor of histone deacetylases, which epigenetically modify cell proliferation/differentiation in developing tissues. A series of recent clinical studies in humans reported that VPA exposure in utero impaired histogenesis and the development of the central nervous system, leading to increased risks of congenital malformation and the impairment of higher brain functions in children. In the present study conducted in mice, we report that VPA exposure in utero (1) increases the amount of acetylated histone proteins, (2) alters the expression of G1-phase regulatory proteins, (3) inhibits the cell cycle exit of neural progenitor cells during the early stage of neocortical histogenesis, and (4) increases the production of projection neurons distributed in the superficial neocortical layers in embryonic brains. Together, our findings show that VPA exposure in utero alters proliferation/differentiation characteristics of neural progenitor cells and hence leads to the neocortical dysgenesis. SIGNIFICANCE STATEMENT: This study provides new insight into the mechanisms of how an altered in utero environment, such as drug exposure, affects the generation of neurons prenatally. The antiepileptic drug valproic acid (VPA) is a good target molecule as in utero exposure to VPA has been repeatedly reported to increase the risk of nervous system malformations and to impair higher brain functions in children. We show that VPA decreases the probability of differentiation of the neural progenitor cells (NPCs) in mice, resulting in an abnormally increased number of projection neurons in the superficial layers of the neocortex. Further, we suggest that histone deacetylase inhibition by VPA may be involved in the dysregulation of proliferation/differentiation characteristics of NPCs.

Psychedelics Recruit Multiple Cellular Types and Produce Complex Transcriptional Responses Within the Brain.

  • Martin DA
  • EBioMedicine
  • 2016 Sep 21

Literature context: vitrogen; RRID:AB_2535813). DAPI (1.


Abstract:

There has recently been a resurgence of interest in psychedelics, substances that profoundly alter perception and cognition and have recently demonstrated therapeutic efficacy to treat anxiety, depression, and addiction in the clinic. The receptor mechanisms that drive their molecular and behavioral effects involve activation of cortical serotonin 5-HT2A receptors, but the responses of specific cellular populations remain unknown. Here, we provide evidence that a small subset of 5-HT2A-expressing excitatory neurons is directly activated by psychedelics and subsequently recruits other select cell types including subpopulations of inhibitory somatostatin and parvalbumin GABAergic interneurons, as well as astrocytes, to produce distinct and regional responses. To gather data regarding the response of specific neuronal populations, we developed methodology for fluorescence-activated cell sorting (FACS) to segregate and enrich specific cellular subtypes in the brain. These methods allow for robust neuronal sorting based on cytoplasmic epitopes followed by downstream nucleic acid analysis, expanding the utility of FACS in neuroscience research.

Funding information:
  • NIH HHS - P40 OD010440(United States)
  • NINDS NIH HHS - NS072030(United States)

Filamin A (FLNA) plays an essential role in somatostatin receptor 2 (SST2) signaling and stabilization after agonist stimulation in human and rat somatotroph tumor cells.

  • Peverelli E
  • Endocrinology
  • 2014 Aug 19

Literature context:


Abstract:

Somatostatin receptor type 2 (SST2) is the main pharmacological target of medical therapy for GH-secreting pituitary tumors, but molecular mechanisms regulating its expression and signaling are largely unknown. The aim of this study was to investigate the role of cytoskeleton protein filamin A (FLNA) in SST2 expression and signaling in somatotroph tumor cells. We found a highly variable expression of FLNA in human GH-secreting tumors, without a correlation with SST2 levels. FLNA silencing in human tumoral cells did not affect SST2 expression and localization but abolished the SST2-induced reduction of cyclin D1 (-37% ± 15% in control cells, P < .05 vs basal) and caspase-3/7 activation (+63% ± 31% in control cells, P < .05 vs basal). Overexpression of a FLNA dominant-negative mutant that specifically prevents SST2-FLNA binding reduced SST2 expression after prolonged agonist exposure (-55% ± 5%, P < .01 vs untreated cells) in GH3 cells. Moreover, SST2-induced apoptotic effect (77% ± 54% increase of caspase activity, P < .05 vs basal) and SST2-mediated ERK1/2 inhibition (48% ± 17% reduction of ERK1/2 phosphorylation, P < .01 vs basal) were abrogated in cells overexpressing another FLNA mutant that prevents FLNA interaction with partner proteins but not with SST2, suggesting a scaffold function of FLNA in somatotrophs. In conclusion, these data demonstrate that FLNA is involved in SST2 stabilization and signaling in tumoral somatotrophs, playing both a structural and functional role.

Funding information:
  • NEI NIH HHS - R21 EY023061(United States)