LRP1 mediates internalization and accumulation of Aβ42 in GT1-7 and MEF cells.

<p><i>A,</i> LRP1 knockdown decreases Aβ42 accumulation in GT1-7 cells. GT1-7 cells were transiently transfected with LRP1 siRNA or with control, scrambled siRNA. After 72 h, cells were treated with 500 nM FAM-Aβ42 for 4 h and intracellular Aβ42 was determined by flow cytometric analyses of pronase-treated cells as described in the <i>Experimental Procedures</i>. <i>B,</i> decreased Aβ42 accumulation in mouse embryonic fibroblasts from LRP1 knockout mice. Wild type (MEF1) and LRP1 knockout (MEF2) fibroblasts were treated with 500 nM FAM-Aβ42 for 4 h, and intracellular Aβ42 was determined by flow cytometric analyses. ** p<0.01, <i>t</i>-test. n = 3. <i>C,</i> cell lysates from GT1-7 and MEF cells treated as in <i>A</i> and <i>B</i>, analyzed by 7.5% SDS-PAGE, and Western blotted with anti-LRP1 antibodies. Levels of LRP1 were efficiently decreased in both cell types.</p

Increased accumulation of intracellular Aβ42 within lysosomes in LRP1 minireceptor-expressing cells.

<p><i>A,</i> increased Aβ42 accumulation in LRP1 minireceptor-expressing cells. N2a-mLRP4 and N2a-pcDNA3 cells were treated with 500 nM of FAM-Aβ42 at 37°C for 24, 48 and 72 h, and steady-state levels of intracellular Aβ42 were determined by flow cytometric analyses of pronase-treated cells as described in the <i>Experimental Procedures</i>. N2a-mLRP4 cells showed increased Aβ42 accumulation compared to N2a-pcDNA3 cells starting at 48 h of Aβ42 incubation. ** p<0.01, *** p<0.001, <i>t</i>-test. n = 3. <i>B,</i> increased co-localization of intracellular Aβ42 and lysosomes in N2a-mLRP4 cells. N2a-pcDNA3 and N2a-mLRP4 cells were grown in glass chamber slides and treated with 500 nM of FAM-Aβ42 at 37°C for 24, 48 and 72 h. Lysosomes were labeled with LysoTracker 30 min before the end of each incubation. Cells were then fixed and analyzed by confocal microscopy. Intracellular accumulated Aβ42 was highly co-localized with LysoTracker and increased over time in N2a-mLRP4 cells.</p

LRP1 endocytosis is required for Aβ42 uptake and accumulation in N2a cells.

<p><i>A,</i> clathrin heavy chain (CHC) knockdown increases cell surface levels of mLRP4. N2a-mLRP4 cells were infected with CHC shRNA lentivirus or pLKO, control lentivirus. The levels of cell surface and total pools of mLRP4 were determined by flow cytometric analyses with anti-HA antibody in non-permeabilized and saponin-treated cells, respectively. The surface-to-total ratio were calculated and plotted as fold-change to control-infected cells. <i>Right panel</i>, decreased CHC levels and normal mLRP4 levels in transduced N2a-mLRP4 cells were verified by Western blot from sister cultures. <i>B,</i> CHC knockdown decreases accumulation of Aβ42 in N2a-mLRP4 cells. N2a-mLRP4 cells infected with clathrin heavy chain lentivirus as in <i>A</i>) were treated with 500 nM of FAM-Aβ42 or the corresponding control, scrambled peptide for 48 h. The intracellular level of FAM-Aβ42 was determined by flow cytometric analyses of pronase-treated cells. <i>C,</i> deletion of LRP1 tail increases cell surface levels of mLRP4. The cell surface and total pools of the LRP1 minireceptor were determined by flow cytometric analyses with anti-HA antibody as in <i>A</i> in N2a-mLRP4 cells and in N2a cells stably transfected with a deletion variant lacking the cytoplasmic tail of mLRP4 (mLRP4-Tless). The surface-to-total ratios were then calculated and plotted as fold-change to N2a-mLRP4 cells. <i>Right panel</i>, HA blot showing the expression level of minireceptors in N2a stable cell lines. <i>D,</i> deletion of LRP1 tail decreased the mLRP4 endocytosis rate in N2a cells. N2a-mLRP4 and N2a-mLRP4-Tless cells were incubated with 5 nM ¹²⁵I-RAP at 4°C for 60 min, and then shifted to 37°C for the indicated times. At each time point, the amounts of ligand that is either internalized or that remains at the cell surface were determined and the ratios of internalized to total cell-associated ligand were plotted against time. Values are the average of triple determinations with the S.D. indicated by <i>error bars</i>. <i>E</i>, impaired LRP1 endocytosis rate decreases accumulation of Aβ42 in N2a-mLRP4 cells. N2a-pcDNA3, N2a-mLRP4 and N2a-mLRP4-Tless cells were treated with 500 nM of FAM-Aβ42 or the corresponding control, scrambled peptide for 48 h and the cell-associated (without pronase) and intracellular (with pronase) levels of Aβ42 were determined by flow cytometric analyses.</p

Increased susceptibility to Aβ42-mediated cell death in LRP1 minireceptor-expressing cells.

<p>N2a-pcDNA3 and N2a-mLRP4 cells were incubated with increasing concentrations of Aβ42 for 24, 48 and 72 h and cell viability was assessed by the reduction of the MTS dye. Decreased viability was detected only after 72 h incubation with a high concentration of Aβ42 in LRP1 minireceptor expressing cells. * p<0.05, <i>t</i>-test. n = 3.</p

Increased GABA secretion with GAD-65/GAD-67 co-expression.

MSCs were seeded at 10,000 cells/cm2 in 6 well plates and grew overnight. Cells were then transfected with Fugene6 as described in Methods using 1 μg total plasmid, and cells were recovered for 24 h. Co-expression with GAD-65 and GAD-67 used equivalent amounts of each plasmid. (A) Conditioned media was pre-cleared by centrifugation and secreted GABA levels were enzymatically determined using a fluorescence-coupled assay. Basal levels were 2.48 ± 0.83 μM and results were expressed as fold change to GAD-65 transfected (GAD-65) cells. Maximal secretion of GABA was detected using GADs co-expression. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 5). (B) 15 μg (GAD-65, β-actin) or 30 μg (GAD-67) proteins were separated by 8% SDS-PAGE, proteins transferred to PVDF membranes and GAD-65, GAD-67 or β-actin immunodetected by Western blotting. Note that GAD levels in co-transfected cells were approximately half the levels of GADs expressed individually.</p

Overexpression of Glutamate Decarboxylase in Mesenchymal Stem Cells Enhances Their Immunosuppressive Properties and Increases GABA and Nitric Oxide Levels

<div><p>The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers <i>in vitro</i>, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders.</p></div

GAD-67 gene expression regulation by pro-inflammatory cytokines IL-1β and IFN-γ in MSCs.

MSC were seeded at 4,800 cells/cm2 and grew for 24 h. MSC cultures were then supplemented with 20 ng/mL IL-1β, 25 ng/mL IFN-γ, or the combination of both cytokines, and MSC cells were grew for indicated times. Control cultures were identically treated but cytokines were not added. (A) GAD-67, GAD-65 and ABAT mRNA levels were determined 48 h after cytokine treatment by RT-qPCR. IL-1β selectively increases GAD-67 mRNA levels in MSCs and IFN-γ potentiates IL-1β effects. *, pLower panel, densitometric analysis of Western blot results. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 11).</p

IL-1β pre-treatment increases immunosuppressive properties of MSCs.

<p>MSC were seeded at 4,800 cells/cm² and grew for 24 h. MSC cultures were then supplemented with 20 ng/mL IL-1β, 25 ng/mL IFN-γ, or the combination of both cytokines as described in <i>Methods</i> and MSC cells were grew for additional 48 h. The ending day for the cytokine treatment, splenic TCD4+ lymphocytes were isolated from healthy C57BL/6 mice, labeled with CTV for proliferation assessment and stimulated with anti-CD3/anti-CD28 for 4 h. MSC were then added to CTV-labeled TCD4+ cells (ratio 1:10 MSC:TCD4⁺) and co-cultures were continued for additional 60 h as described in <i>Methods</i>. Proliferation controls included the determination of background and maximal proliferation for the T-cell preparation (basal, No MSC and anti-CD3/anti-CD28, respectively), and basal MSC-mediated immunosuppression was determined using untreated MSC (MSC Ctrl). Priming with IL-1β increases immunosuppressive properties of MSC. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 3). <i>Right Panel</i>, representative histograms showing gating strategy and CTV dilution peaks for each treatment.</p

Increased GABA secretion with GAD-65/GAD-67 co-expression.

<p>MSCs were seeded at 10,000 cells/cm² in 6 well plates and grew overnight. Cells were then transfected with Fugene6 as described in <i>Methods</i> using 1 μg total plasmid, and cells were recovered for 24 h. Co-expression with GAD-65 and GAD-67 used equivalent amounts of each plasmid. (A) Conditioned media was pre-cleared by centrifugation and secreted GABA levels were enzymatically determined using a fluorescence-coupled assay. Basal levels were 2.48 ± 0.83 μM and results were expressed as fold change to GAD-65 transfected (GAD-65) cells. Maximal secretion of GABA was detected using GADs co-expression. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 5). (B) 15 μg (GAD-65, β-actin) or 30 μg (GAD-67) proteins were separated by 8% SDS-PAGE, proteins transferred to PVDF membranes and GAD-65, GAD-67 or β-actin immunodetected by Western blotting. Note that GAD levels in co-transfected cells were approximately half the levels of GADs expressed individually.</p

GADs expression does not increase nitric oxide levels <i>per se</i> in MSC.

<p>MSCs were seeded and transfected as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163735#pone.0163735.g004" target="_blank">Fig 4</a> or treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163735#pone.0163735.s002" target="_blank">S2 Fig</a>. After 48 h, conditioned media was collected and assayed for NO levels as described in <i>Methods</i>. Plated media without cells was also included as control (media). GAD expression does not increase steady state levels of secreted nitric oxide. Results are from 4 independent experiments with determinations performed in triplicate. ***, p<0.001 (Kruskal-Wallis test with Dunn’s correction). NS, not statistically significant.</p