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Developmental alcohol exposure leads to a persistent change on astrocyte secretome

ABSTRACT: Glial cells are central players in the pathogenesis of neurodevelopmental disorders like Fetal Alcohol Spectrum Disorder (FASD). Ethanol can modulate glial differentiation and astrocyte function imposing dramatic changes to the developing brain. Since ethanol can affect a vast number of intracellular targets and signaling pathways and many effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to a long-lasting change in astrocyte secretome in vitro. Animals were treated every other day with ethanol (5g/kg) or saline between post-natal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids (SILAC). 24h-conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among 59 proteins found reduced in ET-CM we found components of the extracellular matrix like Laminin subunits α2, α4, β1, β2 and γ1 and the proteoglycans Biglycan, Heparin Sulfate Proteoglycan 2 and Lumican. Proteins with trophic function like Insulin-Like Growth Factor Binding Protein 4, Pigment Epithelium-Derived Factor and Clusterin as well as proteins involved on modulation of proteolysis like TIMP-1 and PAI-1 were also found reduced. On the other hand, pro-synaptogeneic proteins like Thrombospondin-1, Hevin as well as the modulator of extracelular matrix expression, Angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through Ingenuity Pathway Analysis demonstrated that the Amyloid beta A4 protein precursor (APP), which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM, rendering it a suitable target for a more accurate analysis about the effects of ethanol on its biology. Taken together our results strongly suggest that early exposure to teratogens such alcohol may lead to an enduring change in astrocyte secretome, affecting mainly the expression of trophic and matricellular proteins, which in turn may lead to permanent alterations in brain function.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mustela Putorius Furo (european Domestic Ferret) (mustela Furo)

TISSUE(S): Astrocyte Of The Cerebral Cortex

DISEASE(S): Fetal Alcohol Syndrome

SUBMITTER: Pablo Trindade

LAB HEAD: Alexandre E. Medina de Jesus, D.Sci.

PROVIDER: PXD002787 | Pride | 2016-02-15

REPOSITORIES: Pride

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Developmental alcohol exposure leads to a persistent change on astrocyte secretome.

Trindade Pablo P Hampton Brian B Manhães Alex C AC Medina Alexandre E AE

Journal of neurochemistry 20160215 5

Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western world. It has been quite established that acute alcohol exposure can dramatically affect astrocyte function. Because the effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to persistent changes in astrocyte secretome in vitro. Animals were treate ...[more]

PMID: 26801685

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Project description:Research regarding the role of astrocytes as M-NM-2-amyloid (AM-NM-2) degrading cells has broadened our view about the mechanisms how these common glia cells function in AlzheimerM-bM-^@M-^Ys disease (AD). Based on previous studies adult mouse astrocytes are able to degrade AM-NM-2 deposits from AD mouse model and human brain tissue sections ex vivo, contrary to neonatal astrocytes. In this study, we studied the possible altered gene expression profiles of adult and neonatal astrocytes cultured for 22 h on top of the AM-NM-2 burdened tg APdE9 or wild-type mouse brain sections using whole genome microarrays. Quantitative RT-PCR analysis confirmed the significant up-regulation of HtrA serine peptidase 1 (Htra1), metallopeptidase 9 (Mmp9), phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and scavenger receptor class A, member 5 (Scara5) in adult astrocytes, whereas neonatal astrocytes up-regulated Mmp9 and down-regulated genes related to cholesterol transport and synthesis: apolipoprotein E (Apoe), 24-dehydrocholesterol reductase (Dhcr24) and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1). These findings brought out novel genes which expression is altered during AM-NM-2 clearance in adult and neonatal astrocytes ex vivo. Astrocyte cultures: For the adult astrocyte cultures, hippocampi and cortices were isolated from 6-8-week-old C57Bl/6j wild-type and tg eGFP mice and the tissue was suspended in DMEM/F12 (3:1, Gibco BRL, NY, USA) containing 10 % heat-inactivated fetal bovine serum (Gibco BRL) and 100 U / ml penicillin-streptomycin (Gibco BRL). The cells were treated with Percoll (Sigma-Aldrich, St.Louis, USA) and plated onto poly-L-lysine coated flasks in DMEM/F12 (3:1) containing 10 % heat-inactivated fetal bovine serum, 100 U / ml penicillin-streptomycin and G5 supplement (Gibco BRL). Before the experiments the glial cell cultures were shaken to remove microglia at 200 rpm for 2 h. For the neonatal astrocyte cultures, hippocampi and cortices were isolated from 2-day-old wild-type and eGFP mice according to the protocol described above, except that the culture medium was DMEM (+4500mg/l glucose, + L-glutamine, -pyruvate, Gibco BRL) without G5 supplement and the isolation method included no Percoll treatment. Standard anti-GFAP (1:500; Dako Cytomation, Glostrup, Denmark) immunocytochemistry was used to determine the purity of astrocyte cultures. The cells were incubated with biotinylated goat anti-rabbit IgG (1:200 dilution; Vector Laboratories, CA) secondary antibody and Vectastain ABC Elite kit was used (Vector Laboratories) according to manufacturerM-bM-^@M-^Ys protocol. Hydroxen peroxide (H2O2) and nickel enhanced diaminobenzidine (Ni-DAB) was added to detect the immunoreactivity. Three fields per well and at least three wells per cell type were analyzed for the percentage of Ni-DAB stained cells of all cellular profiles in the field. Adult and neonatal astrocyte cultures both contained on average 99.6 % M-BM-1 0.6 % anti-GFAP immunoreactive astrocytes. Anti-Iba (2 M-BM-5g/ml; Wako, Germany) and anti-CD11b (1:500 dilution; Serotec, UK) antibodies were used to detect possible microglial cells. The cultures contained less than 0.4 % of microglial cells in all experiments. Ex vivo treatments: tg APdE9 / wild-type mouse brain section incubated with neonatal (age 3 d) or adult (age 6-8 wks) mouse eGFP expressing astrocytes (4 x 105 cells / well), n=6. The mouse brain sections used were prepared from tg APdE9 mouse line (created by co-injection of chimeric mouse/human APPSwe and PS1-dE9 vectors, both controlled by their own mouse prion protein promoter element [APdE9 mice (Jankowsky et al. 2004) provided by Prof. Heikki Tanila] and their wild type C57Bl/6j littermates. Before the mRNA isolation process, the samples were pooled (n=3). The purity of the samples was checked with FACS (FACSCalibur, Becton Dickinson, USA) using the analyzing program CellQuestTM version 3.3 (Becton Dickinson, USA). After 22 h incubation the culture medium was removed from the wells and the brain sections with cultured eGFP astrocytes were incubated with 1 x trypsin-EDTA (500 M-BM-5l /well) for up to 15 min at 37M-BM-0 C. Partial detachment (at this point, the astrocytes should not lose the grip completely) of the astrocytes from the underlying brain section was monitored with a standard light microscope. After the incubation the brain sections with cultured astrocytes were carefully removed into small petri dishes filled with FBS-containing culture medium to inactivate the trypsin. Subsequently, the brain sections were immediately examined with fluorescence microscope (Olympus IX-71, Japan). The eGFP astrocytes were separated from the underlying brain section using a special suction hose, which was a modified version from suction hoses typically used for the microinjection techniques. In the other end of the tube there is a pipet tip as a mouthpiece, in the middle of the tube a 0.22 M-BM-5m filter and in the other end a stretched glass capillary (prepared with a Bunsen burner). The green fluorescent eGFP astrocytes were picked up gently from the underlying brain section (without breaking the brain tissue) using wavelength 468 nm of the fluorescence microscope and simultaneous light microscopy. Data normalization and analysis: The raw data was preprocessed with the robust multichip algorithm (Irizarry et al. 2003), normalized per chip to the median. 12 samples were grouped into 4 sample types: adult_wt, adult_tg, neonatal_wt, neonatal_tg. The change in the astrocyte gene expression was identified according to the sections the astrocytes were incubated (WT or TG). The differentially expressed probe sets were further processed using statistical analysis with Welch unpaired t-test assuming unequal variances (p<0.05; fold change > 2.0 or < 0.5). Benjamini-Hochberg false-discovery rate (FDR) correction for multiple testing was used to control the number of false-positives results.

Dataset Information

Developmental alcohol exposure leads to a persistent change on astrocyte secretome

Publications

Developmental alcohol exposure leads to a persistent change on astrocyte secretome.

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