Additional file 2: of Toxoplasma gondii alters NMDAR signaling and induces signs of Alzheimer’s disease in wild-type, C57BL/6 mice

Figure S2. T. gondii induces Aβ immunoreactivity. A. Representative × 20 magnification images from control brains and brains from mice orally infected with 10 ME49 cysts for 30 (top) and 60 days (bottom). The inset shows a close-up image at × 63 magnification. Tissue sections were fixed and stained with an anti-T. gondii antibody BAG1 (red) and counterstained with DAPI (blue). A comparable brain section from the same animal was stained with a mouse anti-beta amyloid antibody (green) and counterstained with DAPI (blue). Scale bar: 50 μm. B. Representative × 20 magnification images from control brains and brains from mice orally infected with 10 ME49 cysts for 60 days. Tissue sections from the same animals used in A were fixed and stained with a human anti-beta amyloid antibody, 6E10 (green) and an anti-T. gondii antibody BAG1 (red) and counterstained with DAPI (blue). A separate set of sections from T. gondii-infected brains were stained with isotype controls (mouse IgG and Rabbit IgG) to control for the specificity of the 6E10 and BAG-1 antibodies, respectively. A different set of sections from T. gondii-infected brains were stained for fluorescent secondary antibodies (anti-mouse 488 and Texas-Red anti-rabbit) to control for their specificity. Scale bar: 50 μm. (JPEG 303 kb

Sequestration of synaptic proteins by alpha-synuclein aggregates leading to neurotoxicity is inhibited by small peptide

<div><p>α-Synuclein (α-syn) is a major component of Lewy bodies found in synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). Under the pathological conditions, α-syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells and able to transmit across cells. However, mechanisms by which α-syn aggregates affect cytotoxicity in neurons have not been fully elucidated. Here we report that α-syn aggregates preferentially sequester specific synaptic proteins such as vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 (SNAP25) through direct binding which is resistant to SDS. The sequestration effect of α-syn aggregates was shown in a cell-free system, cultured primary neurons, and PD mouse model. Furthermore, we identified a specific blocking peptide derived from VAMP2 which partially inhibited the sequestration by α-syn aggregates and contributed to reduced neurotoxicity. These results provide a mechanism of neurotoxicity mediated by α-syn aggregates and suggest that the blocking peptide interfering with the pathological role of α-syn aggregates could be useful for designing a potential therapeutic drug for the treatment of PD.</p></div

α-Syn aggregates from PD mouse model directly bind to VAMP2 <i>in vivo</i>.

<p>(A) Immunohistochemistry of basal ganglia from 22 months old SNCA mouse brain. Multiple α-syn aggregates (distinct red signals in the upper right panel) are shown in the old SNCA mouse brain and one of the aggregates (white boxed inset) is shown at a high magnification 3-dimensional image in the middle panels. Two lateral section images crossed in white dotted lines (a and b from the middle right panel) are displayed in the bottom panels. The arrowheads in white indicate the clustered VAMP2 in the α-syn aggregates. Scale bars (upper panels; 100 µm, middle and lower panels; 20 µm) (B) Young (3 months) and old (22 months) SNCA mice brain tissue lysates were subjected to immunoprecipitation using VAMP2 antibody followed by immunoblot using α-syn antibody. Oligomer species were increased in old SNCA mice brain (marked in bracket) and high molecular weight oligomer (marked an asterisk) was detected only in the old mice. Immunoblot of VAMP2 was used as loading control. Immunoblot is a representative blot from two independent experiments.</p

α-Syn aggregates reduce selectively synaptic protein levels in cell-free systems.

<p>(A) α-Syn aggregates were generated by incubation with dopamine at 37°C for 3 days followed by size-exclusion chromatography. (B) Transmission electron micrograph of α-syn aggregates showed the spherical shape and 10–20 nm in diameter. Scale bar, 100 nm. (C) Recombinant synaptic proteins (1 µM) were incubated with α-syn aggregates (100 nM) at 37°C for 2 h and subjected to western blot analysis or SyproOrange staining to detect protein levels. (D) Recombinant VAMP2 was incubated with α-syn aggregates at 37°C for 2 h. Western blot analysis using anti-VAMP2 (left) or anti-α-syn antibody (right), respectively showed a strong binding of VAMP2 to α-syn aggregates.</p

α-Syn aggregates sequester VAMP2 through direct binding <i>in vitro</i>.

<p>(A) VAMP2 was incubated with α-syn aggregates (100 nM) at 37°C for the indicated time points. ΔI/I₀ values indicate the relatively bound VAMP2 protein levels, where I₀ is the intensity of control free VAMP2 at time 0 and I is the intensity of each free VAMP2 at the different time point (ΔI = I₀-I). (B) VAMP2 was incubated with different concentration of α-syn aggregates at 37°C for 2 h. I₀ is the intensity of control free VAMP2 and I is the intensity of each free VAMP2 at different concentration of α-syn aggregates. (C-D) The binding assay was performed at different temperature and pH. I₀ and I are the intensity of free VAMP2 in the absence and presence of α-syn aggregates, respectively, at the indicated temperature and pH. Each blot is a representative immunoblot from two or three independent experiments. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by two-tailed, unpaired <i>t</i>-test. Values indicate mean ± SEM.</p

VAMP2 peptide partially abolishes VAMP2 sequestration by α-syn aggregates in the cell-free conditions.

<p>(A) Five different VAMP2 peptides designated P1 to P5 in red boxes were designed based on the sequence of a soluble part in VAMP2. SNARE motif was underlined. (B) α-Syn aggregates and the indicated peptides (1mM) were pre-incubated for 1 h at room temperature and then VAMP2 binding assay was performed. The bar graph shows the free VAMP2 levels in the presence of α-syn aggregates and each peptide. Error bars were obtained from four independent experiments. ****, p < 0.0001, **, p < 0.01 by ANOVA with Dunnett’s multiple comparisons test. Values indicate mean ± SEM. (C) The VAMP2 binding assay was performed in the presence of α-syn aggregates pre-incubated with the indicated concentrations of peptide P5. VAMP2 sequestration by α-syn aggregates was inhibited by peptide P5 in a dose-dependent manner (left). Free VAMP2 levels seemed to reach the saturation with a minor increase. Immunoblot is a representative blot from three independent experiments.</p

Additional file 1: of Toxoplasma gondii alters NMDAR signaling and induces signs of Alzheimer’s disease in wild-type, C57BL/6 mice

Figure S1. Specificity of the 6E10 and BAG-1 antibodies. Representative × 20 magnification images from control brains and brains from mice orally infected with 10 ME49 cysts for 60 days. Brains were collected after perfusion with ice-cold PBS. Control and infected brains were fixed and stained with a human anti-beta amyloid antibody, 6E10 (green) and an anti-T. gondii antibody BAG1 (red) and counterstained with DAPI (blue). A separate set of sections from T. gondii-infected brains were stained with isotype controls (mouse IgG and Rabbit IgG) to control for the specificity of the 6E10 and BAG-1 antibodies, respectively. A different set of sections from T. gondii-infected brains were stained for fluorescent secondary antibodies (anti-mouse 488 and Texas-Red anti-rabbit) to control for their specificity. Scale bar: 50 μm. (JPEG 114 kb

VAMP2 does not bind to α-syn monomer and Aβ aggregates.

<p>Recombinant VAMP2 was incubated with the increasing concentration (0.1, 1, 10 µM) of α-syn monomer (A) or 100 nM Aβ aggregates (B) at 37°C for 2 h. Western blot analysis shows no binding of VAMP2 to α-syn monomer or Aβ aggregates. The weak band intensity of bound VAMP2 to α-syn aggregates was due to the saturated signals from free VAMP2.</p

VAMP2 sequestration by α-syn aggregates in cultured primary neurons results in synaptic dysfunction as well as cytotoxicity.

<p>(A) Immunocytochemistry in rat cortical primary neurons shows a direct binding of VAMP2 to α-syn aggregates. (B) Western blot analysis of cultured neurons shows selectively reduced VAMP2 and SNAP25 levels in α-syn aggregates-treated cells for 3 days consecutively. To analyze bound VAMP2, the blot used for α-syn aggregates detection was re-probed, marked in the red box. (C) Cortical neuronal cells were treated with α-syn aggregates for 3 days consecutively and glutamate release was determined. ****, p < 0.0001, by two-tailed, unpaired <i>t</i>-test (n = 10). (D) Cell viability was determined 14 days after addition of α-syn aggregates in cortical neurons. ***, p < 0.001, by two-tailed, unpaired <i>t</i>-test (n = 3). Values indicate mean ± SEM.</p

VAMP2-derived peptide reduces cytotoxicity induced by α-syn aggregates in SH-SY5Y cells and cultured primary neurons.

<p>Neuroblastoma SH-SY5Y cells were treated with α-syn aggregates pre-incubated with or without peptide P5. After 5 h treatment, (A) FM1-43 staining was performed (Scale bar, 50 µm) or (B) intracellular ROS levels were measured. (C) Pre-incubated α-syn aggregates with or without peptide P5 were treated cortical neuronal cells at DIV7 for 2 days consecutively and LDH cytotoxicity assay was performed. α-Syn aggregates toxicity was also evaluated by immunostaining of (D) cleaved caspase-3 and (E) NeuN in cortical neuronal cells at DIV7 after 3 days consecutive treatment. Scale bar, 50 µm. ****, p < 0.0001, ***, p < 0.001, *, p < 0.05, by ANOVA with Tukey's multiple comparisons test. Values indicate mean ± SEM.</p