Isolation, Culture and Functional Characterization of Glia and Endothelial Cells From Adult Pig Brain

Primary cultures of glial and endothelial cells are important tools for basic and translational neuroscience research. Primary cell cultures are usually generated from rodent brain although considerable differences exist between human and rodent glia and endothelial cells. Because many translational research projects aim to identify mechanisms that eventually lead to diagnostic and therapeutic approaches to target human diseases, glia, and endothelial cultures are needed that better reflect the human central nervous system (CNS). Pig brain is easily accessible and, in many aspects, close to the human brain. We established an easy and cost-effective method to isolate and culture different primary glial and endothelial cells from adult pig brain. Oligodendrocyte, microglia, astrocyte, and endothelial primary cell cultures were generated from the same brain tissue and grown for up to 8 weeks. Primary cells showed lineage-specific morphology and expressed specific markers with a purity ranging from 60 to 95%. Cultured oligodendrocytes myelinated neurons and microglia secreted tumor necrosis factor alpha when induced with lipopolysaccharide. Endothelial cells showed typical tube formation when grown on Matrigel. Astrocytes enhanced survival of co-cultured neurons and were killed by Aquaporin-4 antibody positive sera from patients with Neuromyelitis optica. In summary, we established a new method for primary oligodendrocyte, microglia, endothelial and astrocyte cell cultures from pig brain that provide a tool for translational research on human CNS diseases

Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize Neomycin-Resistant Mammalian Cells

<div><h3>Background</h3><p>Previously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.</p> <h3>Methodology</h3><p>Using fluorescence spectroscopy we have shown that the inhibitor, known as <u>A</u>ctive <u>D</u>NA-<u>d</u>ependent <u>A</u>TPase <u>A</u><u>D</u>omain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.</p> <h3>Significance</h3><p>The use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.</p> </div

Delineating the motifs required for the interaction of ADAADiN with ADAAD.

<p>ND  =  not determined.</p><p>ADAAD as well as the deletion constructs were expressed as GST fusion protein and purified using glutathione agarose beads. In case of ADAAD and AMD47, GST was cleaved using PreScission protease and the purified protein was used in these studies.</p

Expression of endogenous SG2NA is influenced by ADAADi production.

<p>The transcript as well as protein expression was monitored in the untransfected cells as well as in cells stably transfected with pcDNA 3.1 myc/his (−) vector at passage 13. (A). Endogenous SG2NA transcript was analyzed by quantitative RT-PCR in untransfected and transfected Neuro 2Acells. (B). Endogenous SG2NA protein analyzed by western blot using antibody against SG2NA. (C). <i>sg2na</i> promoter occupancy by RNAPII, Brg1, H3K9Ac, and H3K9Me2 was analysed in untransfected and transfected Neuro 2A cells using ChIP. Fold enrichment was calculated with respect to the mock ChIP done using IgG antibodies. (D). SG2NA transcript level in transfected cells at passage 4. (E). <b>Expression of exogenous SG2NA expressed using pcDNA 3.1 myc/his (−) vector </b><b>is also influenced by ADAADi production.</b> Overexpression of three variants of SG2NA in Neuro2A cells were monitored using anti-myc antibody. Transfected cells (passage 4) were grown as indicated for 12 hours before analysis. Two clones of 87 kDa (87.1 and 87.2), one clone each of 78 kDa (78.1) and of 52 kDa (52.2) were analyzed for protein expression. The cells transfected with vector alone were used as control. Protein expression was observed only when cells were grown in the absence of both antibiotics. (F). Western blot analysis of expression of 87- and 78-kDa proteins in clones 87.1 and 78.1 in stably transfected cells grown in the presence of antibiotics using anti-myc antibody. Lane 1: vector alone transfected cells; Lane 2: 78.1 clone; Lane 3: 87.1 clone. N.S., indicating non-specific band, was used as loading control. (G). The expression of 78- and 87- kDa protein in 78.1, 78.2, 87.1 and 87.2 clones was monitored in stably transfected cells grown in the absence of antibiotics. Lane 1: vector transfected cells; Lane 2: 78.1 clone; Lane 3: 78.2 clone; M: marker; Lane 4: 87.1 clone; Lane 5: 87.2 clone. N.S., indicating non-specific band, was used as loading control. (H). Semi-quantitative RT-PCR analysis done using insert-specific forward primer and vector-specific reverse primer confirms that 87 kDa transcript expression is observed only when the cells are grown the absence of antibiotics. (I). The expression of 87 kDa, 78 kDa, and 52 kDa was not observed in stably transfected cells even after removal of antibiotics when the cells were freeze-thawed at passage 9. (J). Semi-quantitative RT-PCR analysis of APH transcript. Lane1: Untransfected Neuro2A cells; Lane 2: Stably transfected Neuro2A cells; Lane 3: control reaction using purified pcDNA 3.1 myc/his (−) vector.</p

Effect of ADAADi on untransfected and transfected mammalian cells.

<p>(A). Untransfected Neuro2A cells treated with neomycin (•) or ADAADiN (○). (B). Untransfected Neuro2A cells treated with G418 (•) or ADAADiG418 (○). (C). Untransfected Neuro2A cells treated with kanamycin (•) or ADAADiK (○). (D). Comparing the effect of 50 μM ADAADiN on untransfected and stably transfected Neuro2A cells grown as indicated post-selection. (E). Parental C2C12 cells grown in the absence (•) and presence (○) of 200 μM ADAADiK. (F). Puromycin-resistant C2C12 cells carrying the pCPLX retroviral vector and grown in the absence (▾) and presence (▿) of 200 μM ADAADiK. (G). G418-resistant C2C12 cells carrying the pLNCX retroviral vector and grown in the absence (▪) and presence (□) of 200 μM ADAADiK.</p

Interaction of ADAADiN and ADAADiK with ADAAD in absence and presence of ATP and slDNA.

*<p>The values were reported in Nongkhlaw <i>et al</i>. (Nongkhlaw, 2009).</p><p>His-ADAAD was used for these studies.</p

ADAADi formation is catalyzed by different isoforms of APH using different aminoglycoside substrates. (A). APH (3′)-I, APH (3′)-IIa, and APH (3′)-IIIa catalyze ADAADi formation.

<p>ADAADi, synthesized by the three isozymes of APH, was purified and ATPase assays with 0.22 μM His-ADAAD and 68 μM kanamycin (Kan), 44 μM neomycin (Neo), 1.6 μM ADAADiK from APH(3′)-I (I) and APH(3′)-IIa (IIa), 1.2 μM ADAADiK from APH(3′)-IIIa (IIIa), 1.6 μM ADAADiN from APH (3′)-I (I), 2 μM ADAADiN from APH(3′)-IIa (IIa) , and 1.4 μM ADAADiN from APH(3′)-IIIa (IIIa) were done as described. (B). <b>ADAADi is produced from G418 as well as streptomycin</b> by APH (3′)-IIIa. ATPase assays were done either in the absence or presence of 200 μM streptomycin, 2 μM G418, 4 μM ADAADiS, 4 μM ADAADiG418. (C). ADAADi produced using APH (3′)-IIIa from commercially available aminoglycosides.</p

ADAADi is produced by APH.

<p>(A). Analysis of ADAADiK on silica 60A plate by TLC after purification using Bio-Rex 70 followed by G-15 desalting column. Kanamycin (lanes 1-6), ADAADiK (lanes 7–9), and phosphokanamycin (lanes 18–19) migrate with different mobilities and thus can be separated on these plates. (B). Inhibition profile of fractions eluted from G-15 column. (C). Purification of ADAAD using TSK gel SP-5PW column. The ninhydrin sensitive spots (fractions 15–20) correspond to phosphokanamycin and kanamycin, while the ADAADiK concentration (fractions 9–14) is too low to be detected by ninhydrin. (D). Inhibition profile of fractions eluted from SP column.</p

Production of ADAADi in Neuro2A cells leads to alterations in the epigenome.

<p>(A). Comparing the <i>in vivo</i> ATPase activity of SMARCAL1 present in untransfected Neuro2A cells and stably transfected Neuro2A cells grown as indicated post-selection. (B). APH transcript expression in untransfected and transfected Neuro2A cells. (C). SWI2/SNF2 expression in untransfected and stably transfected Neuro2A cells grown as indicated was analysed using polyclonal anti-SMARCAL1 antibody, anti-Brg1 antibody, and anti- Rad54B antibody. (D). Western blot analysis of H3K9Ac levels in untransfected and stably transfected Neuro2A cells. (E). Western blot analysis of H3K9Me2 levels in untransfected and stably transfected Neuro2A cells grown as indicated by western blot. β-actin was used as loading control in these experiments. (F). The levels of ADH4, Nanog, Runx2, EP300, and Dicer1 was estimated by quantitative RT-PCR. The transcript levels in stably transfected cells were calculated with respect to the levels present in untransfected cells. The data are an average of two independent experiments, each experiment done in duplicate. Error bars indicate standard deviation and stars indicate statistical significance at P<0.05. The P-values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049822#pone.0049822.s011" target="_blank">Table S3</a>.</p

Validating a minipig model of reversible cerebral demyelination using human diagnostic modalities and electron microscopy

Background Inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, are significant sources of morbidity in young adults despite therapeutic advances. Current murine models of remyelination have limited applicability due to the low white matter content of their brains, which restricts the spatial resolution of diagnostic imaging. Large animal models might be more suitable but pose significant technological, ethical and logistical challenges. Methods We induced targeted cerebral demyelinating lesions by serially repeated injections of lysophosphatidylcholine in the minipig brain. Lesions were amenable to follow-up using the same clinical imaging modalities (3T magnetic resonance imaging, C-11-PIB positron emission tomography) and standard histopathology protocols as for human diagnostics (myelin, glia and neuronal cell markers), as well as electron microscopy (EM), to compare against biopsy data from two patients. Findings We demonstrate controlled, clinically unapparent, reversible and multimodally trackable brain white matter demyelination in a large animal model. De-/remyelination dynamics were slower than reported for rodent models and paralleled by a degree of secondary axonal pathology. Regression modelling of ultrastructural parameters (g -ratio, axon thickness) predicted EM features of cerebral de- and remyelination in human data. Interpretation We validated our minipig model of demyelinating brain diseases by employing human diagnostic tools and comparing it with biopsy data from patients with cerebral demyelination