Publications by authors named "Anthony J Filiano"

23 Publications

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Mesenchymal stromal cells reprogram monocytes and macrophages with processing bodies.

Stem Cells 2021 Jan 9;39(1):115-128. Epub 2020 Nov 9.

Marcus Center for Cellular Cures, Duke University, Durham, North Carolina, USA.

Mesenchymal stromal cells (MSCs) are widely used in clinical trials because of their ability to modulate inflammation. The success of MSCs has been variable over 25 years, most likely due to an incomplete understanding of their mechanism. After MSCs are injected, they traffic to the lungs and other tissues where they are rapidly cleared. Despite being cleared, MSCs suppress the inflammatory response in the long term. Using human cord tissue-derived MSCs (hCT-MSCs), we demonstrated that hCT-MSCs directly interact and reprogram monocytes and macrophages. After engaging hCT-MSCs, monocytes and macrophages engulfed cytoplasmic components of live hCT-MSCs, then downregulated gene programs for antigen presentation and costimulation, and functionally suppressed the activation of helper T cells. We determined that low-density lipoprotein receptor-related proteins on monocytes and macrophages mediated the engulfment of hCT-MSCs. Since a large amount of cellular information can be packaged in cytoplasmic RNA processing bodies (p-bodies), we generated p-body deficient hCT-MSCs and confirmed that they failed to reprogram monocytes and macrophages in vitro and in vivo. hCT-MSCs suppressed an inflammatory response caused by a nasal lipopolysaccharide challenge. Although both control and p-body deficient hCT-MSCs were engulfed by infiltrating lung monocytes and macrophages, p-body deficient hCT-MSCs failed to suppress inflammation and downregulate MHC-II. Overall, we identified a novel mechanism by which hCT-MSCs indirectly suppressed a T-cell response by directly interacting and reprogramming monocytes and macrophages via p-bodies. The results of this study suggest a novel mechanism for how MSCs can reprogram the inflammatory response and have long-term effects to suppress inflammation.
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http://dx.doi.org/10.1002/stem.3292DOI Listing
January 2021

Risk of epilepsy in rheumatoid arthritis: a meta-analysis of population based studies and bioinformatics analysis.

Ther Adv Chronic Dis 2020 7;11:2040622319899300. Epub 2020 Feb 7.

Laboratory of Rheumatology & Institute of TCM Clinical Basic Medicine, College of Basic Medical Science, Zhejiang Chinese Medical University, No.548 Binwen Road, Hangzhou, 310053, China.

Background: An increasing number of studies support an association between rheumatoid arthritis (RA) and brain disorders. This study aims to determine the association between RA and epilepsy.

Methods: A comprehensive search of databases in both English and Chinese was performed. Data from the selected studies were extracted and analyzed independently by two authors. Genes associated with epilepsy and RA were also collected and analyzed.

Results: We included six nationwide population based studies ( = 7,094,113 cases in total) for the meta-analysis. The risk of epilepsy was increased in RA patients [risk ratio (RR) = 1.601; 95% confidence interval (CI): 1.089-2.354;  = 0.017;  = 3,803,535 cases] and children born to mothers with RA (RR = 1.475; 95% CI: 1.333-1.633;  < 0.001,  = 3,290,578 cases). Subgroup analysis and meta-regression showed the RR of epilepsy in RA was negatively correlated with age. Furthermore, we found that 433 identified genes in a coexpression network from the hippocampi of 129 epileptic patients were enriched in the RA and related Kyoto Encyclopedia of Genes and Genomes pathways, while 13 genes (mainly related to inflammatory cytokines and chemokines) were identified as potential key genes bridging the RA and epilepsy.

Conclusions: Our study, utilizing meta-analysis and bioinformatical data, highlights a close association between epilepsy and RA. Further studies are still warranted to expand these findings, especially for a population that is exposed to RA during fetal and childhood periods.
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http://dx.doi.org/10.1177/2040622319899300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011323PMC
February 2020

Human umbilical cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications.

PLoS One 2019 4;14(9):e0218906. Epub 2019 Sep 4.

Marcus Center for Cellular Cures (MC3), Duke University School of Medicine, Durham, North Carolina, United States of America.

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218906PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726370PMC
March 2020

Cord Blood Connect: The International Congress for Cord Blood and Perinatal Tissue Research.

Stem Cells Transl Med 2019 09;8 Suppl 1:S1-S35

Marcus Center for Cellular Cures at Duke, Duke University School of Medicine, Durham, North Carolina, USA.

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http://dx.doi.org/10.1002/sctm.12550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713533PMC
September 2019

Reduction of microglial progranulin does not exacerbate pathology or behavioral deficits in neuronal progranulin-insufficient mice.

Neurobiol Dis 2019 04 15;124:152-162. Epub 2018 Nov 15.

Departments of Neurology and Neurobiology, Center for Neurodegeneration and Experimental Therapeutics, Alzheimer's Disease Center, Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL, United States. Electronic address:

Loss-of-function mutations in progranulin (GRN), most of which cause progranulin haploinsufficiency, are a major autosomal dominant cause of frontotemporal dementia (FTD). Individuals with loss-of-function mutations on both GRN alleles develop neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. Progranulin is a secreted glycoprotein expressed by a variety of cell types throughout the body, including neurons and microglia in the brain. Understanding the relative importance of neuronal and microglial progranulin insufficiency in FTD pathogenesis may guide development of therapies. In this study, we used mouse models to investigate the role of neuronal and microglial progranulin insufficiency in the development of FTD-like pathology and behavioral deficits. Grn mice model aspects of FTD and NCL, developing lipofuscinosis and gliosis throughout the brain, as well as deficits in social behavior. We have previously shown that selective depletion of neuronal progranulin disrupts social behavior, but does not produce lipofuscinosis or gliosis. We hypothesized that reduction of microglial progranulin would induce lipofuscinosis and gliosis, and exacerbate behavioral deficits, in neuronal progranulin-deficient mice. To test this hypothesis, we crossed Grn mice with mice expressing Cre transgenes targeting neurons (CaMKII-Cre) and myeloid cells/microglia (LysM-Cre). CaMKII-Cre, which is expressed in forebrain excitatory neurons, reduced cortical progranulin protein levels by around 50%. LysM-Cre strongly reduced progranulin immunolabeling in many microglia, but did not reduce total brain progranulin levels, suggesting that, at least under resting conditions, microglia contribute less than neurons to overall brain progranulin levels. Mice with depletion of both neuronal and microglial progranulin failed to develop lipofuscinosis or gliosis, suggesting that progranulin from extracellular sources prevented pathology in cells targeted by the Cre transgenes. Reduction of microglial progranulin also did not exacerbate the social deficits of neuronal progranulin-insufficient mice. These results do not support the hypothesis of synergistic effects between progranulin-deficient neurons and microglia. Nearly complete progranulin deficiency appears to be required to induce lipofuscinosis and gliosis in mice, while partial progranulin insufficiency is sufficient to produce behavioral deficits.
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http://dx.doi.org/10.1016/j.nbd.2018.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363848PMC
April 2019

Meningeal whole mount preparation and characterization of neural cells by flow cytometry.

Curr Protoc Immunol 2018 04 16;121(1):e50. Epub 2018 Apr 16.

Center for Brain Immunology and Glia, School of Medicine, University of Virginia, Charlottesville, Virginia.

Neuroimmunologists aim to understand the interactions between the central nervous system and the immune system under both homeostatic and pathological conditions. The meninges, contrary to the brain parenchyma, are populated by numerous immune cells. Soluble factors produced by these cells are capable to diffuse into the brain parenchyma and influence the brain cells within the parenchyma, including neurons. In this unit, we will describe two protocols: analysis the meningeal compartment of rodents and the use flow cytometry to study the cells of the brain parenchyma (particularly neurons).
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http://dx.doi.org/10.1002/cpim.50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040815PMC
April 2018

Neuronal integrity and complement control synaptic material clearance by microglia after CNS injury.

J Exp Med 2018 07 25;215(7):1789-1801. Epub 2018 Jun 25.

Center for Brain Immunology, and Glia (BIG), University of Virginia, Charlottesville, VA

Phagocytosis of synaptic material by microglia is critical for central nervous system development. Less well understood is this microglial function in the injured adult brain. Assay of microglial phagocytosis is challenging, because peripheral myeloid cells engraft the site of injury, which could obscure interpretation of microglial roles. The model used here, optic nerve crush injury, results in degeneration of synapses in the dorsal lateral geniculate nucleus (dLGN), which stimulates rapid activation and engulfment of synaptic material by resident microglia without myeloid cell engraftment. Pharmacological depletion of microglia causes postinjury accumulation of synaptic debris, suggesting that microglia are the dominant postinjury phagocytes. Genetic or pharmacological manipulations revealed that neuronal activity does not trigger microglia phagocytosis after injury. RNA sequencing reveals C1q and CD11b/CR3 involvement in clearance of debris by dLGN-resident microglia. Indeed, and mice exhibit impaired postinjury debris clearance. Our results show how neurodegenerative debris is cleared by microglia and offers a model for studying its mechanisms and physiological roles.
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http://dx.doi.org/10.1084/jem.20172244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028515PMC
July 2018

Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia.

J Exp Med 2018 06 11;215(6):1627-1647. Epub 2018 Apr 11.

Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA

Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beMφs) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beMφs to populate the niche and that the presence of beMφs does not alter behavior. Furthermore, beMφs maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beMφs as a unique CNS cell type and demonstrates that therapeutic engraftment of beMφs may be possible with irradiation-free conditioning regimens.
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http://dx.doi.org/10.1084/jem.20180247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987928PMC
June 2018

Neuroimmunology in 2017: The central nervous system: privileged by immune connections.

Nat Rev Immunol 2018 02 27;18(2):83-84. Epub 2017 Dec 27.

Robertson Clinical and Translational Cell Therapy Program and the Department of Neurosurgery, Duke University, Durham, North Carolina 27710, USA.

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http://dx.doi.org/10.1038/nri.2017.152DOI Listing
February 2018

Myeloid Cells in the Central Nervous System.

Immunity 2017 06;46(6):943-956

Center for Brain Immunology and Glia, Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA; Gutenberg Research Fellowship Group of Neuroimmunology, Focus Program Translational Neuroscience and Immunotherapy, Rhine Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany. Electronic address:

The central nervous system (CNS) and its meningeal coverings accommodate a diverse myeloid compartment that includes parenchymal microglia and perivascular macrophages, as well as choroid plexus and meningeal macrophages, dendritic cells, and granulocytes. These myeloid populations enjoy an intimate relationship with the CNS, where they play an essential role in both health and disease. Although the importance of these cells is clearly recognized, their exact function in the CNS continues to be explored. Here, we review the subsets of myeloid cells that inhabit the parenchyma, meninges, and choroid plexus and discuss their roles in CNS homeostasis. We also discuss the role of these cells in various neurological pathologies, such as autoimmunity, mechanical injury, neurodegeneration, and infection. We highlight the neuroprotective nature of certain myeloid cells by emphasizing their therapeutic potential for the treatment of neurological conditions.
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http://dx.doi.org/10.1016/j.immuni.2017.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657250PMC
June 2017

How and why do T cells and their derived cytokines affect the injured and healthy brain?

Nat Rev Neurosci 2017 06 27;18(6):375-384. Epub 2017 Apr 27.

Center for Brain Immunology and Glia, Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.

The evolution of adaptive immunity provides enhanced defence against specific pathogens, as well as homeostatic immune surveillance of all tissues. Despite being 'immune privileged', the CNS uses the assistance of the immune system in physiological and pathological states. In this Opinion article, we discuss the influence of adaptive immunity on recovery after CNS injury and on cognitive and social brain function. We further extend a hypothesis that the pro-social effects of interferon-regulated genes were initially exploited by pathogens to increase host-host transmission, and that these genes were later recycled by the host to form part of an immune defence programme. In this way, the evolution of adaptive immunity may reflect a host-pathogen 'arms race'.
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http://dx.doi.org/10.1038/nrn.2017.39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823005PMC
June 2017

Restoring neuronal progranulin reverses deficits in a mouse model of frontotemporal dementia.

Brain 2017 May;140(5):1447-1465

Center for Neurodegeneration and Experimental Therapeutics, Alzheimer's Disease Center, and Evelyn F. McKnight Brain Institute, Departments of Neurology and Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA.

Loss-of-function mutations in progranulin (GRN), a secreted glycoprotein expressed by neurons and microglia, are a common autosomal dominant cause of frontotemporal dementia, a neurodegenerative disease commonly characterized by disrupted social and emotional behaviour. GRN mutations are thought to cause frontotemporal dementia through progranulin haploinsufficiency, therefore, boosting progranulin expression from the intact allele is a rational treatment strategy. However, this approach has not been tested in an animal model of frontotemporal dementia and it is unclear if boosting progranulin could correct pre-existing deficits. Here, we show that adeno-associated virus-driven expression of progranulin in the medial prefrontal cortex reverses social dominance deficits in Grn+/- mice, an animal model of frontotemporal dementia due to GRN mutations. Adeno-associated virus-progranulin also corrected lysosomal abnormalities in Grn+/- mice. The adeno-associated virus-progranulin vector only transduced neurons, suggesting that restoring neuronal progranulin is sufficient to correct deficits in Grn+/- mice. To further test the role of neuronal progranulin in the development of frontotemporal dementia-related deficits, we generated two neuronal progranulin-deficient mouse lines using CaMKII-Cre and Nestin-Cre. Measuring progranulin levels in these lines indicated that most brain progranulin is derived from neurons. Both neuronal progranulin-deficient lines developed social dominance deficits similar to those in global Grn+/- mice, showing that neuronal progranulin deficiency is sufficient to disrupt social behaviour. These data support the concept of progranulin-boosting therapies for frontotemporal dementia and highlight an important role for neuron-derived progranulin in maintaining normal social function.
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http://dx.doi.org/10.1093/brain/awx060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965303PMC
May 2017

Unexpected role of interferon-γ in regulating neuronal connectivity and social behaviour.

Nature 2016 07 13;535(7612):425-9. Epub 2016 Jul 13.

Immune dysfunction is commonly associated with several neurological and mental disorders. Although the mechanisms by which peripheral immunity may influence neuronal function are largely unknown, recent findings implicate meningeal immunity influencing behaviour, such as spatial learning and memory. Here we show that meningeal immunity is also critical for social behaviour; mice deficient in adaptive immunity exhibit social deficits and hyper-connectivity of fronto-cortical brain regions. Associations between rodent transcriptomes from brain and cellular transcriptomes in response to T-cell-derived cytokines suggest a strong interaction between social behaviour and interferon-γ (IFN-γ)-driven responses. Concordantly, we demonstrate that inhibitory neurons respond to IFN-γ and increase GABAergic (γ-aminobutyric-acid) currents in projection neurons, suggesting that IFN-γ is a molecular link between meningeal immunity and neural circuits recruited for social behaviour. Meta-analysis of the transcriptomes of a range of organisms reveals that rodents, fish, and flies elevate IFN-γ/JAK-STAT-dependent gene signatures in a social context, suggesting that the IFN-γ signalling pathway could mediate a co-evolutionary link between social/aggregation behaviour and an efficient anti-pathogen response. This study implicates adaptive immune dysfunction, in particular IFN-γ, in disorders characterized by social dysfunction and suggests a co-evolutionary link between social behaviour and an anti-pathogen immune response driven by IFN-γ signalling.
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http://dx.doi.org/10.1038/nature18626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961620PMC
July 2016

Breaking bad blood: β2-microglobulin as a pro-aging factor in blood.

Nat Med 2015 Aug;21(8):844-5

Center for Brain Immunology and Glia (BIG), Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, Virginia, USA.

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http://dx.doi.org/10.1038/nm.3926DOI Listing
August 2015

Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in progranulin-deficient FTLD.

J Exp Med 2014 Sep 25;211(10):1937-45. Epub 2014 Aug 25.

Gladstone Institute of Neurological Diseases, Department of Neurology, Department of Medicine, Gladstone Institute of Cardiovascular Disease, University of California, San Franciso, San Francisco, CA 94158 Gladstone Institute of Neurological Diseases, Department of Neurology, Department of Medicine, Gladstone Institute of Cardiovascular Disease, University of California, San Franciso, San Francisco, CA 94158

Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3'-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism.
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http://dx.doi.org/10.1084/jem.20140214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172214PMC
September 2014

Interactions of innate and adaptive immunity in brain development and function.

Brain Res 2015 Aug 7;1617:18-27. Epub 2014 Aug 7.

Center for Brain Immunology and Glia (BIG), Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA; Graduate Program in Neuroscience and Medical Scientist Training Program, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA.

It has been known for decades that the immune system has a tremendous impact on behavior. Most work has described the negative role of immune cells on the central nervous system. However, we and others have demonstrated over the last decade that a well-regulated immune system is needed for proper brain function. Here we discuss several neuro-immune interactions, using examples from brain homeostasis and disease states. We will highlight our understanding of the consequences of malfunctioning immunity on neurodevelopment and will discuss the roles of the innate and adaptive immune system in neurodevelopment and how T cells maintain a proper innate immune balance in the brain surroundings and within its parenchyma. Also, we describe how immune imbalance impairs higher order brain functioning, possibly leading to behavioral and cognitive impairment. Lastly, we propose our hypothesis that some behavioral deficits in neurodevelopmental disorders, such as in autism spectrum disorder, are the consequence of malfunctioning immunity. This article is part of a Special Issue entitled SI: Neuroimmunology in Health And Disease.
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http://dx.doi.org/10.1016/j.brainres.2014.07.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320678PMC
August 2015

ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects.

J Clin Invest 2014 Mar 10;124(3):981-99. Epub 2014 Feb 10.

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.
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http://dx.doi.org/10.1172/JCI72723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938263PMC
March 2014

Dissociation of frontotemporal dementia-related deficits and neuroinflammation in progranulin haploinsufficient mice.

J Neurosci 2013 Mar;33(12):5352-61

Center for Neurodegeneration and Experimental Therapeutics, Departments of Neurology and Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Frontotemporal dementia (FTD) is a neurodegenerative disease with hallmark deficits in social and emotional function. Heterozygous loss-of-function mutations in GRN, the progranulin gene, are a common genetic cause of the disorder, but the mechanisms by which progranulin haploinsufficiency causes neuronal dysfunction in FTD are unclear. Homozygous progranulin knock-out (Grn(-/-)) mice have been studied as a model of this disorder and show behavioral deficits and a neuroinflammatory phenotype with robust microglial activation. However, homozygous GRN mutations causing complete progranulin deficiency were recently shown to cause a different neurological disorder, neuronal ceroid lipofuscinosis, suggesting that the total absence of progranulin may have effects distinct from those of haploinsufficiency. Here, we studied progranulin heterozygous (Grn(+/-)) mice, which model progranulin haploinsufficiency. We found that Grn(+/-) mice developed age-dependent social and emotional deficits potentially relevant to FTD. However, unlike Grn(-/-) mice, behavioral deficits in Grn(+/-) mice occurred in the absence of gliosis or increased expression of tumor necrosis factor-α. Instead, we found neuronal abnormalities in the amygdala, an area of selective vulnerability in FTD, in Grn(+/-) mice. Our findings indicate that FTD-related deficits resulting from progranulin haploinsufficiency can develop in the absence of detectable gliosis and neuroinflammation, thereby dissociating microglial activation from functional deficits and suggesting an important effect of progranulin deficiency on neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.6103-11.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740510PMC
March 2013

The application of permanent middle cerebral artery ligation in the mouse.

J Vis Exp 2011 Jul 25(53). Epub 2011 Jul 25.

Department of Pharmacology and Physiology, University of Rochester, USA.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents. The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents. This method was originally described for rats by Tamura et al. in 1981. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories. Early use of this technique led to the definition and description of "infarct core" and "penumbra", and it is often used to evaluate potential neuroprotective compounds. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications. This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.
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http://dx.doi.org/10.3791/3039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196186PMC
July 2011

Transglutaminase 2 protects against ischemic insult, interacts with HIF1beta, and attenuates HIF1 signaling.

FASEB J 2008 Aug 28;22(8):2662-75. Epub 2008 Mar 28.

Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Transglutaminase 2 (TG2) is a multifunctional enzyme that has been implicated in the pathogenesis of neurodegenerative diseases, ischemia, and stroke. The mechanism by which TG2 modulates disease progression have not been elucidated. In this study we investigate the role of TG2 in the cellular response to ischemia and hypoxia. TG2 is up-regulated in neurons exposed to oxygen and glucose deprivation (OGD), and increased TG2 expression protects neurons against OGD-induced cell death independent of its transamidating activity. We identified hypoxia inducible factor 1beta (HIF1beta) as a TG2 binding partner. HIF1beta and HIF1alpha together form the heterodimeric transcription factor hypoxia inducible factor 1 (HIF1). TG2 and the transaminase-inactive mutant C277S-TG2 inhibited a HIF-dependent transcription reporter assay under hypoxic conditions without affecting nuclear protein levels for HIF1alpha or HIF1beta, their ability to form the HIF1 heterodimeric transcription factor, or HIF1 binding to its DNA response element. Interestingly, TG2 attenuates the up-regulation of the HIF-dependent proapoptotic gene Bnip3 in response to OGD but had no effect on the expression of VEGF, which has been linked to prosurvival processes. This study demonstrates for the first time that TG2 protects against OGD, interacts with HIF1beta, and attenuates the HIF1 hypoxic response pathway. These results indicate that TG2 may play an important role in protecting against the delayed neuronal cell death in ischemia and stroke.
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http://dx.doi.org/10.1096/fj.07-097709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493449PMC
August 2008

Glutamate decreases mitochondrial size and movement in primary forebrain neurons.

J Neurosci 2003 Aug;23(21):7881-8

Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

Mitochondria are essential to maintain neuronal viability. In addition to the generation of ATP and maintenance of calcium homeostasis, the effective delivery of mitochondria to the appropriate location within neurons is also likely to influence their function. In this study we examined mitochondrial movement and morphology in primary cultures of rat forebrain using a mitochondrially targeted enhanced yellow fluorescent protein (mt-eYFP). Mt-eYFP-labeled mitochondria display a characteristic elongated phenotype and also move extensively. Application of glutamate to cultures results in a rapid diminution of movement and also an alteration from elongated to rounded morphology. This effect required the entry of calcium and was mediated by activation of the NMDA subtype of glutamate receptor. Treatment of cultures with an uncoupler or blocking ATP synthesis with oligomycin also stopped movement but did not alter morphology. Interestingly, application of glutamate together with the uncoupler did not prevent the changes in movement or shape but facilitated recovery after washout of the stimuli. This suggests that the critical target for calcium in this paradigm is cytosolic. These studies demonstrate that in addition to altering the bioenergetic properties of mitochondria, neurotoxins can also alter mitochondrial movement and morphology. We speculate that neurotoxin-mediated impairment of mitochondrial delivery may contribute to the injurious effects of neurotoxins.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6740596PMC
August 2003

Hypothermic reperfusion after cardiac arrest augments brain-derived neurotrophic factor activation.

J Cereb Blood Flow Metab 2002 Jul;22(7):843-51

Department of Emergency Medicine, University of Pittsburgh, Pennsylvania 15213, USA.

Induction of mild hypothermia improves neurologic outcome after global cerebral ischemia. This study measured levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in hippocampal tissue of rats after resuscitation from 8 minutes of normothermic, asphyxial cardiac arrest. After resuscitation, rats were maintained either at normal temperature (37 degrees C) or cooled to mild hypothermia (33 degrees C, beginning 60 minutes after resuscitation). After 12 or 24 hours, neurotrophin levels in hippocampus were measured by immunoblotting. Ischemia and reperfusion increased hippocampal levels of BDNF. Induction of hypothermia during reperfusion potentiated the increase in BDNF after 24 hours, but not after 12 hours. Levels of NGF were not increased by postresuscitation hypothermia. Hypothermia also increased tissue levels and tyrosine phosphorylation of TrkB, the receptor for BDNF. Increased BDNF levels were correlated with activation of the extracellularly regulated kinase (ERK), a downstream element in the signal transduction cascade induced by BDNF. In contrast to the many deleterious processes during ischemia and reperfusion that are inhibited by induced hypothermia, increasing BDNF levels is a potentially restorative process that is augmented. Increased activation of BDNF signaling is a possible mechanism by which mild hypothermia is able to reduce the neuronal damage typically occurring after cardiac arrest.
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http://dx.doi.org/10.1097/00004647-200207000-00009DOI Listing
July 2002