Elevated Alpha-Synuclein Impairs Innate Immune Cell Function and Provides a Potential Peripheral Biomarker for Parkinson's Disease

<div><p>Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson's disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist <i>in vivo</i>. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected <i>in vivo</i> as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson's disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure α-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinson's disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.</p></div

Alteration in cytokine secretion in BAC transgenic mice.

<p>In all graphs, each dot represents measurements from cells isolated from an individual pup or animal (<b>A</b>) Microglia from line 26 and non TG littermates were stimulated with LPS and TNFα and IL6 measured by ELISA (n = 2; 7 pups/GT/expt +/− s.e.m *P≤0.005 when α-syn TG samples were compared with non-TG). (<b>B</b>) Microglia isolated from three independent human α-syn BAC TG mouse lines, (line 422; 26; and 3) and their corresponding non-TG littermate controls. Cells were stimulated as above and measurements were made for TNFα by ELISA (n = 2; 3 pups/GT/expt +/− s.e.m *P≤0.05 when α-syn TG samples were compared with non-TG). (<b>C</b>) Line 26 TG mice and their non-TG littermates received an injection of low dose LPS for 6 months and serum inflammatory cytokines (TNFα and IL6) were measured by luminex multiplex analysis (n = 1; 7–10 mice/GT/expt +/− s.e.m* P≤0.05 when α-syn TG samples were compared with non-TG). (<b>D</b>) Microglia from line 26/syn ^null or α-syn ^null littermates mice were stimulated with LPS and cytokine expression for IL6 and TNFα assessed at the mRNA level by multiplex analysis (n = 2; 5 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). (<b>E+F</b>) Microglia isolated from line 26/syn ^null or α-syn ^null littermates were stimulated with LPS in the presence or absence of Brefeldin A. Tissue culture supernatant (<b>E</b>) or cell lysate (<b>F</b>) was assessed for TNFα production by ELISA (n = 2; 5 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG).</p

Increased levels of α-syn affect microglial phagocytosis.

<p>(<b>A</b>) Microglia from line 26 TG or non-TG littermates were incubated with 10 µ beads or apoptotic Jurkat T-cells for 90 minutes. A phagocytic index was calculated by microscopic visualization (n = 3; 3 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). (<b>B</b>) Microglia from line 26/syn ^null or α-syn ^null littermates were incubated with 10µ beads and a phagocytic index calculated (n = 3; 3 pups/GT/expt +/− s.e.m *p≤0.002 when α-syn TG samples were compared with non-TG). (<b>C</b>) Peritoneal macrophages isolated from line 26 TG or non-TG littermates were cultured with apoptotic Jurkat T-cells and a phagocytic index was calculated (n = 2; 5 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). (<b>D</b>) Microglia isolated from line 26 TG or non-TG littermates were left unfed or fed beads followed by FM1-43 addition on ice for 10 minutes, fluorescence was assessed by flow cytometry under resting and stimulated (plus bead) conditions, (histogram is representative of 3 independent expts). (<b>E</b>) Geometric mean fluorescence of FM1-43 incorporation in resting and stimulated cell was calculated (n = 3; 3 pups/GT/expt +/− s.e.m *p≤0.001 when FM1-43 incorporation between wild type and line 3 microglia stimulated with beads was compared).</p

Fibroblasts isolated from PD patients display defective phagocytosis.

<p>(<b>A</b>) Fibroblast from 10 independent patients with sporadic PD, 10 age-matched normal donors, or an individual carrying the SNCA triplication were fed 1 μ beads and a phagocytosis index determined (n = 3+/− s.e.m. *p≤0.001 when the phagocytic index between the SNCA triplicant and sporadic PD patients fibroblasts were compared with the phagocytic index for control patient fibroblasts). (<b>B</b>) Fibroblasts from sporadic PD patient, age-matched control, and patient carrying the SNCA triplication were subject to immunoblot and flow cytometry analysis (image and histogram are representative of n = 3).</p

Alpha-synuclein drives decreased phagocytosis in α-syn BAC transgenic mice.

<p>(<b>A</b>) Peritoneal macrophages from line 26 mice were cultured in Accell media+/− human α-syn siRNA, or non- targeting siRNA. Human α-syn mRNA and protein levels were assessed by RT-PCR and immunoblot analysis (n = 2; 4 pups/GT/expt +/−s.e.m *p<0.01 when α-syn TG samples treated with α-syn siRNA were compared with α-syn TG or NT siRNA). (<b>B</b>) Following siRNA treatment macrophages were fed 10µ beads and a phagocytic index calculated (n = 2; 4 animals/GT/expt +/− s.e.m *p≤0.001 when the phagocytic index between α-syn TG microglia treated with α-syn siRNA and α-syn TG microglia alone or treated with NT siRNA were compared). (<b>C</b>) H4 cells were transfected with α- or β-syn expression vectors followed by addition of 4 µ beads and a phagocytic index calculated (n = 4 +/− s.e.m *p≤0.001 when the phagocytic index of α-syn transfected H4 cells was compared with vector of β-syn transfected cells). (<b>D</b>) α-syn or vector transfected H4 cells were fed beads followed by FM1-43 labeling and FACS analysis. Data is presented at geometric mean fluorescence (n = 4 +/− s.e.m *p≤0.001 when FM1-43 incorporation was compared between vector treated cells fed beads and cells transfected with α-syn fed beads). (<b>E</b>) H4 cells were transfected with wild type, A53T, A30P, or E46K α-syn expression vectors. After 2 days 4 µ beads were added for and the phagocytic index was measured (n = 4 +/− s.e.m *p≤0.05 when vector transfected cells were compared with cells transfected with wild type or the various familial mutations of α-syn).</p

Altered phagocytosis in-vivo in α-syn BAC transgenic animals.

<p>(<b>A</b>) Representative images (left) and quantified phagocytic index (right) of peritoneal macrophages exposed in vivo to apoptotic Jurkat T-cells in line 26 and non-TG littermate mice (n = 2; 5 mice/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). Scale bars are equal to 1 micron. Arrows point towards ingested apoptotic cells and asterisked arrows towards bound/non-ingested cells. (<b>B</b>) Representative images (left) and quantified phagocytic index (right) of peritoneal macrophages exposed in vivo to fluorescent CD47−/− RBC's (red) in line 3 and non-TG littermates (n = 2; 5 mice/GT/expt+/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). Scale bars are equal to 1 micron. Arrows point towards ingested fluorescent RBC, which appear yellow as they are digested within the green labeled cells (FITC-phalloidin). (<b>C</b>) Stereotactic injection of red fluorescent RBC's into the hippocampus of line 3 and non-TG littermate mice. Fluorescence was visualized (upper image) and quantified (bar graph). Particle removal is represented as reduced fluorescence. RBC uptake and degradation was followed as the appearance of hemosiderin (lower image), (n = 3; 3 mice/GT/expt +/− s.e.m * p≤.0.001 compared to 1 hr non-TG ** compared to non-TG at 48hrs). Scale bars are equal to 10 microns. (<b>D</b>) Kidneys from line 26 and littermate non-TG female mice were stained for C3 and IgM. (<b>E</b>) Intensity of antibody was quantified by a certified pathologist in non-TG and α-syn TG kidneys (n = 5 animals/GT +/−s.e.m *p<0.01 when α-syn TG samples were compared with non-TG).</p

α-synuclein alters SNARE recycling.

<p>(<b>A</b>) H4 cells transfected with mock vector or α-syn and left unfed or fed 4 μ beads for 90 minutes, cells were processed for SNAP23 immunofluorescence (green), and polymerized actin (red.) Scale bars are equal to 1 micron image is representative of n = 3. Arrow points to area of bead contact and shows SNAP23 exclusion from the phagocytic cup with α-syn over expression. (<b>B</b>) Vector or α-syn transfected H4 cells were fed beads for various times. Cells were lysed and lysates were left unboiled or boiled at 100°C for 15 min. Samples were subjected to immunoblot analysis for SNAP23 containing SNARE complexes (two independent experiments are shown (top and bottom) (n = 3). (<b>C</b>) H4 cells transfected with α-syn or empty vector were fed beads and α-syn immunoprecipitated followed by immunoblotting for SNAP23 or VAMP2 (representative of n = 3). (<b>D</b>) Line 3 TG or non-TG microglia were fed and SNAP23 or α-syn immunoprecipitated followed by immunoblotting for α-syn, SNAP23, or VAMP2 respectively (n = 3) (E). Striatum from normal donors or PD patients were homogenized, α-syn immunoprecipitated followed by immunoblotting for SNAP23 or VAMP2. Three independent donors are shown here (representative of n = 2).</p

Monocytes isolated from PD patients display elevated α-syn levels and defective phagocytosis.

<p>(<b>A</b>) PBMCs isolated from a normal donor and an individual carrying the SNCA triplication were subject to immunoblot and flow cytometry analysis for α-syn levels (gel and histogram is representative of n = 1). (<b>B</b>) PBMCs from 2 normal donors and an individual carrying the SNCA triplication were fed fluorescently labeled RBCs. Percent monocytes positive for phagocytosis was determined by flow cytometry. Representative fluorescent images of 488 phalloidin stained PMBCs (green) with ingested RBCs (red) are shown. (<b>C</b>) PBMCs were stained for α-syn, and monocyte and lymphocyte populations were identified in the parent PBMC sample, using specific cell surface markers, we did not sort the cell populations. Intracellular α-syn levels are shown as the geo-mean of α-syn minus isotype control (n = 14 for normal donors and 34 sporadic PD +/− s.e.m *p≤001 when the α-syn geo mean for control donors was compared with the geo mean for sporadic PD PMBCs). PBMCs from 7 normal donors and 7 sporadic PD patients were subject to immunoblot analysis for α-syn. Specific PBMC samples 1–7 were selected for immunoblot analysis and marked in red for control donors and blue for sporadic PD donors. (<b>D</b>) Isolated PBMC's were fed fluorescently labeled RBCs. were subjected to flow cytometry analysis and percent monocytes positive for phagocytosis determine. Individual samples chosen for immunoblot analysis are marked out in red for control donors and blue for sporadic PD donors, (n = 14 for normal donors and 34 sporadic PD). The difference between the percent responses from control and PD was statistically significant with *p<0.001. (<b>E</b>). A correlation between α-syn levels, calculated as geo-mean and percent monocytes positive for phagocytosis was calculated.</p

Metadata of donors from control and PD blood used for phagocytosis and α-syn quantification.

<p>Demographic and disease progression information on control donor and PD patients who donated blood for our analysis of phagocytosis and intracellular α-syn levels.</p

Alpha-synuclein translocates to the phagocytic cup.

<p>(<b>A</b>) H4 cells were stimulated with 4 µ beads for 0 or 90 minutes. Samples were stained with anti-α-syn antibodies: 5C12, Syn1 or a rabbit polyclonal (detected with secondary antibody, shown in red; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071634#s4" target="_blank">Methods</a> for details of antibodies). Cells were counterstained with 488-phalloidin (green). Scale bars are equal to 1 micron. Arrows point to site of bead contact, and area of α-syn translocation, (beads are not visible by fluorescence; image is representative of n = 3). (<b>B</b>) Separate experiment from those in “A”, and shown at higher power. H4 cells were incubated +/− 4 µ beads for 90 minutes, and then stained for α-syn (red) with the antibody 5C12 (n = 5). Scale bars are equal to 1 micron. Arrows point to site of bead. (<b>C</b>) Line 3 α-syn TG microglia were incubated with 6µ beads for 0, 15, or 90 minutes. Samples were fixed, stained with an α-syn antibody (5C12; red) and counterstained with 488-phalloidin (green). Scale bars are equal to 1 micron. Arrows point towards site of bead contact (image is representative of n = 3).</p