Egr1 binding and histone modification mapping in brain of mouse model of AD

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  • ing generates a

    tracellular sca

    Background:

    In response to variety of environmental factors, expression levels of IEG

    change rapidly and in most of the cases transiently and in a tissue and or-

    gan specific manner. There are two major classes of IEG - with controlling

    and effector functions. IEG with controlling functions are primarily tran-

    may be signifi

    r PremGluR5 accumulation and redistribution to synaptic sites occur in the brain

    of Alzheimers patients. Methods: Human brain samples were obtained

    from the Cognitive Neurology and Alzheimers Disease Center from

    patients who had a neurological and psychometrical evaluation and

    a post-mortem neuropathological analysis of AD pathology permitting

    classification according to CERAD and Braak stages. Frozen middle frontal

    gyrus tissues were obtained from non-demented without AD pathology (n4), probable AD (n 4) and severe AD (n 4). Soluble and membrane pro-teins were extracted by homogenization with and without various detergents

    (Triton, SDS and DOC) followed by differential centrifugation and exam-

    ined by SDS-PAGE. Free floating sections of medial & lateral temporal cor-

    tical areas were used to perform immunohistochemistry of mGluR5 in

    relation to Abeta deposits immunolabeled with NU-1 (anti-ADDL anti-

    body). Results: mGluR5 levels measured by western blot were increased

    in the DOC membrane fractions in the case of probable AD, while they

    were lost in the severe AD. Tests to correlate this pathological redistribution

    with the accumulation of certain forms of oligomers are underway. Immu-

    nohistochemical analysis of mGluR5 and ADDL distribution in the tempo-

    ral cortex showed an increase in the level of immunoreactivity in the

    pyramidal cell layer. This mGluR5 distribution extended into dendritic pro-

    cesses in the proximity of Abeta oligomer deposition in probable AD, but

    was not evident in controls. Conclusions: Data indicate that synaptic reor-

    ganization of mGluR5 at synaptic sites discovered in culture is also evidentin AD brain an

    the early mem

    target.hyperactive calciummobilization and an accumulation of in-

    ffolding molecules. So far, evidence has been lacking thatdeficits in APP transgenic mice. Furthermore, Abca1 knockout mice were

    shown to have a significant decrease of apolipoprotein E (apoE) and apolipo-

    protein A-I (apoA-I) in the brain.Methods: In this study we have examined

    Abeta clearance and transport from the brain in Abca1ko mice. Single apoE

    and apoA-I knockout mice were used as controls.Results:Our data demon-

    strate that among the genotypes that were tested, Abca1ko mice have the

    highest level of amyloid plaques and cognitive deficits. The increased am-

    yloid load and memory deficits in Abca1-ko mice correlated to decreased

    clearance of A-beta as measured by microdialysis experiments. In addition,

    the transport of radioactive labeled A-beta through blood brain barrier was

    decreased in Abca1-ko as compared to Abca1-wt mice and single knockout

    mice. Our in vitro experiments show that apoE-containing lipoproteins iso-

    lated from Abca1wt astrocytes are lipid-rich and decrease A-beta aggrega-

    tion. In contrast, apoE-containing lipoproteins isolated from Abca1ko cells

    are lipid-poor and do not affect A-beta aggregation. Conclusions: In con-

    clusion, our in vivo and in vitro data suggest that lack of Abca1 increases

    A-beta aggregation thus preventing its clearance from the brain ultimately

    leading to cognitive decline.

    P4-014 SYNAPSE DISRUPTION IN AD BRAIN: EVIDENCE

    FOR EARLY PATHOLOGICAL CLUSTERING OF

    THE GROUP I METABOTROPIC GLUTAMATE

    RECEPTOR 5

    Pascale Lacor1, AnilWadhwani2, Nirali Shah2, Jason Pitt2, WilliamKlein2,1Northwestern University, EVANSTON, Illinois, United States;2Northwestern University, Evanston, Illinois, United States.

    Background: Synapse disruption is considered a primary event in the onset

    of memory deficits in Alzheimers disease (AD). Many studies have demon-

    strated that synapse atrophy and elimination can be attributed to the neuro-

    toxic effect of soluble Abeta oligomers (also referred to as "ADDLs").

    ADDLs induce a large-scale loss of surface neurotransmitter receptors in

    cultured hippocampal neurons and deterioration of dendritic morphology,

    consistent with observations in transgenic mouse models. We recently re-

    ported that initiation of this synapse pathology occurs downstream of olig-

    omer-induced clustering of surface mGluR5. These receptors are trapped at

    synapses in loci where ADDLs have aggregated. This extracellular scaffold-

    P4 Posted support the role of these pathological receptors changes in

    ory deficits of AD making mGluR5 an attractive therapeuticbrain samples from APP23 and WT mice and subjected to chromatin im-

    munoprecipitation and second generation massive parallel sequencing(ChIP-seq), h

    plasticity and

    AD. Conclusicant for memory formation. Results: Data generated withscription factors. Plasticity-inducing stimuli in the entorhinal cortex and

    hippocampus of rodents are the strongest and best studied signals that trig-

    ger IEG transcription in brain. Early growth response gene-1 (Egr1) also

    known as Zif268, Krox-24 or NGFI-A, is activity dependent IEG which is

    up-regulated within minutes following just a single trial in a variety of spa-

    cial, object and object-place recognitions memory tests. In the hippocam-

    pal CA3 established ensembles of place cells, an increased mRNA

    synthesis of Egr1 and its targets provides a means for fast encoding of ex-

    periences. Understanding the transcriptional regulatory networks down-

    stream from IEG with controlling functions and Egr1 in particular is

    considered a promising approach to reveal the molecular mechanisms of

    memory consolidation and retrieval. Egr1 target genes and regulatory net-

    works in the context of Alzheimers disease are virtually unexplored.

    Methods: APP23 micea model of AD, and WT littermates were sub-

    jected to a spacial memory test in a MWM for a total time of 10 min

    and sacrificed 30 min after the beginning of the test. Brain samples

    have been processed for second generation highthroughput DNA sequenc-

    ing and gene expression. A series of validation assays have been designed

    afterwards to further confirm Egr1 controlled metabolic pathways thatwere discovered in early 80s during the search for genes and proteins

    up-regulated in response to growth factors in a way similar to viral IEG.Immediate early genes (IEG) in cultured mammalian cellsP4-015 DECREASED RABPHILIN 3A

    IMMUNOREACTIVITY IN ALZHEIMERS

    DISEASE NEOCORTEX

    Mitchell Lai1, Michelle Tan2, Margaret Esiri3, Peter Wong1,

    Christopher Chen1, 1National University of Singapore, Kent Ridge,

    Singapore; 2Singapore General Hospital, Outram, Singapore; 3John

    Radcliffe Hospital, Oxford, United Kingdom.

    Background: Synaptic dysfunction, together with neuritic plaques, neu-

    rofibrillary tangles and degeneration of neurotransmitter source nuclei, is

    a pathological hallmark of Alzheimers disease (AD). The synaptopa-

    thology of AD is known to involve both pre- and postsynaptic compo-

    nents. However, the status of rabphilin3A (RPH), a member of the

    SNARE complex which is essential for synaptic vesicle exocytosis and

    Ca2+-triggered neurotransmitter release, is at present unclear. Methods:

    Using immunoblotting, we measured RPH and several SNARE proteins

    along with scaffold protein PSD-95 in the postmortem parietal cortex of

    patients with AD and in aged controls. Results: Immunoreactivities of

    both RPH and PSD-95 were reduced compared with aged controls.

    RPH reductions also correlated with dementia severity and increased

    -amyloid peptide (A) concentrations. Conclusions: Increased A

    burden may underlie SNARE dysfunction in AD.

    P4-016 EGR1 BINDING AND HISTONE MODIFICATION

    MAPPING IN BRAIN OF MOUSE MODEL

    OFAD

    Iliya Lefterov1, Martina Lefterova2, Andrea Chronican3, Nicholas Fitz3,

    Jonathan Schug2, Radosveta Koldamova1, 1University of Pittsburgh/GSPH,

    Pittsburgh, Pennsylvania, United States; 2University of Pennsylvania,

    Philadelphia, Pennsylvania, United States; 3University of Pittsburgh,

    Pittsburgh, Pennsylvania, United States.

    sentions S703ave revealed a set of Egr1 targets critical for synaptic

    considered important for development and progression of

    ons: ChIP-seq of brain samples from AD model mice can

  • brain metals

    Methods: Subjects of the Brain bank of Brazilian Aging Brain Study

    now study the

    (CG25). Nine

    Tests compris

    r PreGroup were classified according to the clinical and neuropathological

    evaluations in AD (n 14), vascular dementia (n 4) and control (n 20). The clinical diagnosis was established through a postmortem in-terview with an informant including validated scales and questionnaries.

    Neuropathological examinations were carried out based on accepted cri-

    teria, using immunohistochemistry. Neuropathologically, AD was de-

    fined by a CERAD B and a Braak and Braak IV. Small vesselwell-characterized cases and determined whether brain biometals levels

    are associated with A and neurofibrillary tangles burden and dementia.disease, lacun

    of dementia wlevels in AD, vascular dementia and control postmortemhad died. Hospital and autopsy records with systematic neuropathological

    evaluation were available from 10 patients. Results: In the biopsy, A ag-

    gregates were seen in 3 patients being extensive in two. All three out of 10

    cases with A aggregates in their biopsy sample displayed A in post-mor-

    tem samples in an extent fulfilling criteria for phase 4 according to Thal.

    All remaining 7 patients lacking A in biopsy displayed A in the post-

    mortem samples but in a lesser extent (Thal 3 or less). The presence or ab-

    sence of A in the biopsy correlated (Spearmans r 0.815, p 0.004) withA phase defined at autopsy (Thal). Conclusions: High correlation be-

    tween observation of A in a brain biopsy and post-mortem samples vali-

    dates the clinical significance and research use of the surgically obtained

    frontal cortical biopsy.

    P4-018 ABNORMAL BRAIN METALS CONCENTRATION

    IN ALZHEIMERS DISEASE

    Renata Leite1, Mitiko Saiki2, Lea Grinberg3, Renata Ferretti4,

    Claudia Suemoto1, Jose Farfel1, Ana Alho1, Edilaine Tampellini1,

    Mara Andrade1, Livia Polichiso1, Katia Oliveira1, Carlos Pasqualucci1,

    Ricardo Nitrini1, Wilson Jacob-Filho1, 1University of Sao Paulo,

    Sao Paulo, Brazil; 2Nuclear and Energetic Research Institute - IPEN,

    Sao Paulo, Brazil; 3UCSF, San Francisco, Brazil; 4University of S~ao Paulo

    Medical School, Sao Paulo, Brazil.

    Background: Evidence suggests that imbalance of levels of metals in the

    brain may play a role in the development or progression of Alzheimers

    disease (AD) and some of these metals have been implicated in the de-

    position of amyloid peptide (A). To address this question, we measuredpsy immunostained for A and HPt. Altogether 267 patientsbe used to identify transcription factors binding sites and thus the

    complete set of target genes in an unbiased way allowing the design of

    functional studies to further explore the role of certain genes and

    polymorphisms in the development and pathogenesis of Alzheimers

    disease.

    P4-017 POST-MORTEM BETA-AMYLOID PLAQUES

    CORRELATEWITHFRONTALCORTICALBIOPSY

    FINDINGS

    Ville Leinonen1, Anne Maria Koivisto2, Sakari Savolainen1,

    Jaana Rummukainen1, Anna Sutela1, Ritva Vanninen1, Hilkka Soininen2,

    Irina Alafuzoff2, 1Kuopio University Hospital, Kuopio, Finland; 2University

    of Eastern Finland, Kuopio, Finland.

    Background: Amyloid (A) aggregate accumulation in the brain is

    thought to initiate AD pathogenesis and can be detected years before de-

    mentia. We analyzed whether A in frontal cortical biopsies obtained dur-

    ing evaluation of suspected normal pressure hydrocephalus (NPH)

    correlate with later autopsy findings. Methods: Original series included

    468 patients with suspected NPH studied by ICP monitoring and right fron-

    tal cortical bio

    P4 PosteS704ae, or microinfarcts were considered as possible causes

    hen at least three zones or strategic areas were affected.mentia Rating (CDR) and Neuropsychiatric Inventory (NPI). Methods:

    We used semi-quantitative and quantitative immunohistochemical

    methods for the estimation of proteins Abeta, Tau, synaptophysin, PSD-

    95, and NMDA receptor R1, in CG24 and CG25 compared to EC and

    FC9 from 24 control and AD cases. For nine AD cases correlations be-

    tween pathological data including Braak stages and clinical scores were

    performed. Results: Both CG24 and CG25 were significantly affected

    by AD2 stained NFT in AD cases compared to controls, and CG25 ap-

    peared more affected than CG24 and FC9. Abeta deposits were signifi-

    cantly higher in AD in CG25 and almost significantly in CG24. Taking

    into account both controls and AD, age appeared as a significant factor

    for AD2. Neither Abeta nor AD2 or Braak stages were significantly cor-

    related to clinical scores. However, a significant increase in PSD-95 pro-

    tein expression - mainly in positive neurons - was observed in both CG24

    and CG25, and appeared correlated in CG25 to MMSE, CDR and NPI

    scores, while in FC9, it was correlated only to MMSE and CDR.

    MMSE, CDR and NPI scores were nevertheless correlated between

    each other. Data on synaptophysin and NMDAR1 are in progress. Con-

    clusions: Taken together, these data demonstrate that the cingulate cortex

    is damaged in AD, particularly subgenual area 25, but that Amyloid and

    NFT do not seem good correlates of clinical indexes. On the contrary,

    PSD-95 expression appears to correlate with clinical scores, including

    NPI in subgenual area 25. This highlights the role of PSD-95 proteinfor synaptic p

    and behavior.anterior cingulate area 24 (CG24) and subgenual area 25

    AD cases have been clinically tested shortly before death.

    e Mini-Mental State Examination (MMSE), Clinical De-Levels of iron, zinc, rubidium, sodium and potassium were measured

    in the hippocampus using instrumental neutron activation analysis. Pro-

    tocols were approved by the local ethics committee. The statistical anal-

    ysis was performed using the SPSS Statistics Package v.14. Results:

    There was no difference in age and gender between the three groups. Sig-

    nificantly higher concentrations of iron (p < 0.05), zinc (p < 0.01) and

    sodium (p < 0.001) and significantly lower concentrations of rubidium

    (p < 0.01) and potassium (p < 0.01) were found in the hippocampus

    of AD compared to control cases. No differences in the metal concentra-

    tions between vascular dementia and controls individuals between vascu-

    lar dementia and AD individuals were detected. Zinc and sodium were

    found to increase in tandem with levels of A (p < 0.001) and neurofibril-

    lary tangles (p < 0.001). Iron was found to increase in tandem with levels

    of A (p < 0.01). Rubidium and potassium were found to decrease in tan-

    dem with levels of both A (p < 0.001 and p < 0.01 respectively) and

    neurofibrillary tangles (p < 0.001). Conclusions: Abnormal brain metals

    concentration is a characteristic of Alzheimer disease and is associated

    with brain amyloid peptide. However the exactly role of metals alterations

    in brain in the pathoge...

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