Preconditioning of Microglia by α-Synuclein Strongly Affects the Response Induced by Toll-like Receptor (TLR) Stimulation

<div><p>In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson’s disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer’s disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.</p> </div

Impact of Wt αSyn-priming on microglial cytokine release after TLR stimulation.

<p>After treating the microglial cells either with Wt αSyn at 1 µg/mL (‘priming’ or pre-conditioning) or with ‘mock’ solution (no pre-conditioning) for 6 hrs, the TLR agonists were added to their specified final concentrations (see Materials and Methods), and incubated for further 18 hrs at 37 °C. The culture supernatants were harvested and used to measure (<b>A</b>) the levels of the interleukins IL-6, IL-10, IL-13, and IL-17, or (<b>B</b>) of the chemokines IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2, by ELISA. Values are the fold-change calculated as the signal ratio of αSyn-primed, TLR-stimulated cells (‘αSyn+TLR ligand’) relative to non-primed, TLR-stimulated cells (‘TLR ligand’). The results shown (mean ± SEM) are the average of several independent experiments (IL-6: N=5-7; IL-10: N=4-6; IL-13: N=3; IL-17: N=2-3; IP-10, RANTES, MCP-1, and MIP-1α: N=3-5), each containing duplicate samples. Statistically significant differences (* <i>p</i><0.05) were calculated by applying the Student t test between the two sets of results, for all the TLR ligands tested. (#) denotes a result that is significantly different from that obtained after treatment of cells with Wt αSyn alone (p<0.05).</p

Arg1 and iNOS PCR gene expression assays of αSyn-preconditioned microglia stimulated with Pam3 or ssRNA.

<p>Primary microglial cells were either treated with Wt αSyn (Wt), A30P αSyn (A30P), or oligomeric Wt αSyn (oligomers) at 1 µg/mL, or with culture medium alone, for 6 hrs at 37 °C. Subsequently, the TLR agonists or medium alone were added accordingly, and cells were incubated for a further 18 hrs as described before. After treatment, RNA was extracted and reverse transcribed for PCR analysis of arginase-1 (Arg1) and iNOS gene expression. Actin expression was used as a reference and the positive controls for the PCR assays (PCR+) were from bone marrow-derived macrophages stimulated (for Arg1) or not (for iNOS) for 24 hrs with IL-4 (10 ng/mL).</p

Quantitation of activated caspase-3 levels in treated microglial cells, by ELISA and immunofluorescence.

<p>(<b>A</b>) After treatment of the primary microglial cell cultures and incubation for a total of 24 hrs as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079160#pone-0079160-g001" target="_blank">Figure 1</a>, cells were lysed and tested by a specific ELISA assay for cleaved caspase-3 levels quantitation. The results shown (ng/mL cleaved casp-3 per µg of total protein) correspond to the mean of four independent experiments (N=4), each one performed with duplicate samples. Bars correspond to SEM. A discontinuous line represents the mean value obtained for untreated cells. Statistically significant differences were calculated by applying the Student’s t test in relation to the values obtained with the corresponding TLR ligand in the absence of αSyn-preconditioning (* <i>p</i><0.05) and with Wt aSyn alone (# <i>p</i><0.05). Treatment with staurosporine from <i>Streptomyces</i> sp. (5 µM) for 6 hrs was used as a positive control. (<b>B</b>) Cells were cultured in appropriate culture plates and treated as explained above (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079160#pone-0079160-g001" target="_blank">Figure 1</a>) for subsequent labelling of cleaved caspase-3 and nuclear Hoechst 33342 staining for IF analysis, as described in the Methods Section. Samples were analyzed under the fluorescence microscope and three images from random fields containing ca. 80-90 cells each, were recorded, and analyzed for fluorescence quantification. The total specific red fluorescence (RF) and blue fluoresce (BF) were measured and the RF/BF ratio was used as a quantitation method and is represented in this figure. The results shown (RF/BF ratio) correspond to the mean of three images analysed (N=3) within one representative experiment, and bars correspond to SEM. A discontinuous line represents the mean value obtained for images from untreated cells. Statistically significant differences were calculated by applying the Student’s t test in relation to the values obtained with the corresponding TLR ligand in the absence of αSyn-preconditioning (* <i>p</i><0.05) and with Wt αSyn alone (# <i>p</i><0.05). Treatment with staurosporine from <i>Streptomyces</i> sp. (5 µM) for 6 hrs was used as a positive control.</p

Patient disposition.

<p>Low dose = 1x10⁶ AdMSCs/Kg; High dose = 4 x 10⁶ AdMSCs/Kg.</p

Baseline disease characteristics.

<p>Baseline disease characteristics.</p

Adverse events during the trial.

<p>Adverse events during the trial.</p

Changes in treatment effects variables after 12 months of treatment.

<p>Changes in treatment effects variables after 12 months of treatment.</p