Parkinson's disease (PD) starts decades before symptoms appear, usually in the later decades of life, when age-related changes are occurring. To identify molecular changes early in the disease course and distinguish PD pathologies from aging, we generated Drosophila expressing alpha-synuclein (αSyn) in neurons and performed longitudinal bulk transcriptomics and proteomics on brains at six time points across the lifespan and compared the data to healthy control flies as well as human post-mortem brain datasets. We found that translational and energy metabolism pathways were downregulated in αSyn flies at the earliest timepoints; comparison with the aged control flies suggests that elevated αSyn accelerates changes associated with normal aging. Unexpectedly, single-cell analysis at a mid-disease stage revealed that neurons upregulate protein synthesis and nonsense-mediated decay, while glia drive their overall downregulation. Longitudinal multi-omics approaches in animal models can thus help elucidate the molecular cascades underlying neurodegeneration vs. aging and co-pathologies.
© 2025. The Author(s).
Product Citations: 471
In NPJ Parkinson's Disease on 11 March 2025 by Moore, J., Wu, T., et al.
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Drosophila melanogaster (Fruit fly)
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Neuroscience
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In Communications Biology on 22 February 2025 by Tullo, S., Park, J., et al.
Despite known sex differences in human synucleinopathies such as Parkinson's disease, the impact of sex on alpha-synuclein pathology in mouse models has been largely overlooked. To address this need, we examine sex differences in whole brain signatures of neurodegeneration due to aSyn toxicity in the M83 mouse model using longitudinal magnetic resonance imaging (MRI; T1-weighted; 100 μm3 isotropic voxel; -7, 30, 90 and 120 days post-injection [dpi]; n ≥ 8 mice/group/sex/time point). To initiate aSyn spreading, M83 mice are inoculated with recombinant human aSyn preformed fibrils (Hu-PFF) or phosphate buffered saline in the right striatum. We observe more aggressive neurodegenerative profiles over time for male Hu-PFF-injected mice when examining voxel-wise trajectories. However, at 90 dpi, we observe widespread patterns of neurodegeneration in the female Hu-PFF-injected mice. These differences are not accompanied by any differences in motor symptom onset between the sexes. However, male Hu-PFF-injected mice reached their humane endpoint sooner. These findings suggest that post-motor symptom onset, despite accelerated disease trajectories for male Hu-PFF-injected mice, neurodegeneration may appear sooner in the female Hu-PFF-injected mice (prior to motor symptomatology). These findings suggest that sex-specific synucleinopathy phenotypes urgently need to be considered to improve our understanding of neuroprotective and neurodegenerative mechanisms.
© 2025. The Author(s).
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Mus musculus (House mouse)
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Pathology
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Transgenic A53T mice have astrocytic α-synuclein aggregates in dopamine and striatal regions
Preprint on BioRxiv : the Preprint Server for Biology on 12 January 2025 by Peat, C., Prasad, A., et al.
Parkinson’s disease is considered biologically a neuronal α-synuclein disease, largely ignoring the more widespread α-synuclein deposition that occurs in astrocytes. Recent single cell transcriptomics have identified early astrocytic differences in both Parkinson’s disease and mouse models with an increase in reactive astrocytes associated with proteostasis. To identify whether astrocytes accumulate α-synuclein before or after neurons, the present study histologically assessed astrocytes and α-synuclein accumulation in the M83 A53T transgenic mouse model of Parkinson’s disease prior to significant neuronal α-synuclein accumulation. The brains of M83 A53T transgenic ( n =5) and wild-type ( n =4) mice were perfusion fixed and serial sections of the midbrain and striatum processed for multiplex labelling. Digital images were captured from standardised sampling regions and astrocyte quantitation performed using QuPath software. Multivariate linear region models with Turkey posthoc tests were used to evaluate the effects of genotype on regional astrocyte morphology and numbers. The density of astrocytes within the substantia nigra pars compacta was approximately 30% greater compared with other sampled regions ( P <0.005). Small aggregates of α-synuclein were observed in astrocytic processes, including in wild-type mice where a quarter of all astrocytes had an obvious α-synuclein aggregate. Compared to wild-type, A53T transgenic astrocytes had significantly enlarged somas ( P <0.001) with more processes ( P <0.001) consistent with a reactive phenotype. The expression of vascular endothelial growth factor A was present in analysed astrocytes, but not the synthesising enzyme for vitamin D CYP27B1. The A53T transgenic mice had more than double the numbers of astrocytes ( P <0.001) and 2.5 times more astrocytes with α-synuclein aggregates compared to wild-type mice ( P <0.001). These data suggest that α-synuclein is normally cleared by astrocytes and that the substantia nigra pars compacta requires more astrocytic support than other midbrain dopaminergic regions or the striatum. This adds another vulnerability factor to those already known for the substantia nigra. In the A53T transgenic mouse model, astrocytes have an early upregulation of their clearance of α-synuclein aggregates. While speculative, a loss of this ability to take up α-synuclein in these regions may precipitate the selective neuronal degeneration and pathologies observed in Parkinson’s disease. As we move to a biological definition for this disease, understanding this early role astrocytes needs to be considered further.
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In GLIA on 1 January 2025 by Niskanen, J., Peltonen, S., et al.
Microglia are brain resident immune cells that maintain proteostasis and cellular homeostasis. Recent findings suggest that microglia dysfunction could contribute to the pathogenesis of Parkinson's disease (PD). One of the hallmarks of PD is the aggregation and accumulation of alpha-synuclein (αSyn) into Lewy bodies inside nerve cells. Microglia may worsen the neuronal microenvironment by persistent inflammation, resulting in deficient clearing of aggregated αSyn. To model microglial behavior in PD, we utilized human induced pluripotent stem cells to generate functionally active microglia. We studied the microglial uptake of alpha-synuclein preformed fibrils (PFFs) and the effect of pro-inflammatory stimulation by interferon gamma. We demonstrate that combined exposure disrupts the phagosome maturation pathway while inflammatory stimuli suppress chaperone mediated autophagy and mitochondrial function. Furthermore, inflammatory stimulation impairs PFF uptake in microglia and increases cytokine production. Moreover, excessive PFF uptake by microglia results in induction of inducible nitric oxide synthase. Taken together, we demonstrate that this model is valuable for investigating the behavior of microglia in PD and provide new insights on how human microglia process aggregated αSyn.
© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.
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Homo sapiens (Human)
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Immunology and Microbiology
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Neuroscience
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In Brain Sciences on 25 December 2024 by Jung, H. & Kim, S.
α-Synuclein (α-syn) protein is a major pathological agent of familial Parkinson's disease (PD), and its levels and aggregations determine neurotoxicity in PD pathogenesis. Although the pathophysiological functions of α-syn have been extensively studied, its biological functions remain elusive, and there are reports of wild-type (WT) α-syn and two missense mutations of α-syn (A30P and A53T) inducing protective neuritogenesis through neurite outgrowth. However, the function of another α-syn mutation, E46K, has not been fully elucidated. Thus, we compared the effect of E46K α-syn with other types to identify the mechanisms underlying neurite outgrowth.
We transfected SK-N-SH cells with WT and mutant (A53T and E46K) α-syn to investigate the effects of their overexpression on neurite outgrowth. Then, we compared the differential effects of α-syn on neurite outgrowth using microscopic analysis, including confocal microscopy. We also analyzed the differential regulation of cell division control 42 effector protein 2 (Cdc42EP2) using real-time quantitative polymerase chain reaction and western blot analysis. Finally, to confirm the implication of neurite outgrowth, we knocked down Cdc42EP2 using small interfering RNA.
Unlike WT and A53T α-syn, E46K α-syn failed to promote neurite outgrowth by not inducing Cdc42EP2 and subsequent βIII-tubulin expression. Cdc42EP2 knockdown impaired neurite outgrowth in WT and A53T α-syn transfectants.
Our findings suggest that WT and mutant α-syn are linked to Cdc42EP2 production in neuritogenesis, implying α-syn involvement in the physiological function of axon growth and synapse formation. Thus, α-syn may be a potential therapeutic target for PD.
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In Cell Death Dis on 18 June 2024 by Masato, A., Andolfo, A., et al.
Fig.7.A
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αSynuclein buildup enhances DOPAL-induced p62 oligomerization.A Immunoblot of stable BE(2)-M17 cells with inducible dox-dependent αSyn overexpression, both in untreated and 100 µM DOPAL-treated for 18 h conditions. Relative quantification of B p62 monomer and C p62 oligomers after normalization t...
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αSynuclein buildup enhances DOPAL-induced p62 oligomerization.A Immunoblot of stable BE(2)-M17 cells with inducible dox-dependent αSyn overexpression, both in untreated and 100 µM DOPAL-treated for 18 h conditions. Relative quantification of B p62 monomer and C p62 oligomers after normalization to Actin used as loading control. Data are pooled together from four independent experiments normalized to each untreated sample, and analyzed by two-way ANOVA with Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, ****P < 0.0001). D Immunofluorescence of p62 (magenta) and αSyn (green) in the same cellular model. Scale bar: 20 µm and 5 µm in the inset. Relative quantification of E αSyn mean fluorescence/cell and F p62 mean fluorescence/cell. Data are pooled together from four independent experiments normalized to each untreated sample, and analyzed by two-way ANOVA with Sidak’s multiple comparison test (****P < 0.0001). G Correlation of αSyn and p62 mean fluorescence in each cell. The blue lines are centered on the reference level (normalization to 1) for each axis. On the bottom part, frequency distribution of αSyn fluorescence in overexpressing cells by 100 ng/ml dox, showing the effect of DOPAL on αSyn accumulation. On the left side of the graph, frequency distribution of p62 fluorescence in DOPAL-treated cells, showing the synergistic effect of DOPAL and αSyn overexpression on p62 buildup. H Immunoblot of non-transfected and EGFP-transfected BE(2)-M17 cells, both in untreated and 100 µM DOPAL-treated for 18 h conditions. Relative quantification of I EGFP, J p62 monomer, and K p62 oligomers after normalization to Actin used as loading control. Data are pooled together from three independent experiments normalized to each non-transfected untreated sample, and analyzed by two-way ANOVA with Tukey’s multiple comparison test (**P < 0.001, ****P < 0.0001).
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In MicroPubl Biol on 29 March 2024 by Dimopoulou, A., Panagiotakopoulou, V., et al.
Fig.1.B
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Mus musculus (House mouse)
A) Scatter plot graph presenting the phosphorylation state of α-synuclein at Serine 129 (S129) as measured by western blot, using the phospho α-syn antibody. A significant increase of phosphorylated (S129) α-synuclein (p-α-Syn) levels was observed in wv/wv compared to +/+ animals, in both Triton ...
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A) Scatter plot graph presenting the phosphorylation state of α-synuclein at Serine 129 (S129) as measured by western blot, using the phospho α-syn antibody. A significant increase of phosphorylated (S129) α-synuclein (p-α-Syn) levels was observed in wv/wv compared to +/+ animals, in both Triton X and SDS soluble fractions, across all three brain areas. The phosphorylated α-synuclein monomer (14 kDa) and Tubulin (55 kDa), serving as a loading control, are shown in cropped blots. [bars show averages and error bars standard deviations. * p<0.05, **p<0.01, ***p<0.001; using student’s t-test analysis]B) Scatter plot graph presenting the levels of total α-synuclein as measured by western blot, assessed using the Syn1 antibody. No difference in α-synuclein levels was detected between wv/wv and +/+ animals in either brain area, in either fraction. [bars show averages and error bars standard deviations. Statistical analysis was performed with student’s t-test analysis]C) SDS soluble fraction of MB of wv/wv and +/+ animals. The phosphorylated (S129) α-synuclein monomer (14kDa) can be seen in the cropped gel/blot. No species of higher molecular weight could be detected.D) Representative images of SN in “weaver” mice P100 (right) and age-matched wild-type mice (left).E) Corresponding quantification of TH+ immunolabelled neurons in SN of wild-type (+/+) P100 and “weaver” (wv/wv) animals. [bars show averages and error bars standard deviations. Statistical analysis was performed with student’s t-test analysis, *** p<0.001].
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Collected and cropped from MicroPubl Biol by CiteAb, provided under a CC-BY license
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In Sci Rep on 30 January 2024 by Perez-Villalba, A., Sirerol-Piquer, M. S., et al.
Fig.4.E
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Mus musculus (House mouse)
Neuronal loss in the SN and LC of P8 α-SynTg mice. (A) Representative images of the SN in 16-month-old R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice stained with anti-TH antibody. Scale bar = 500 μm. (B) Graph displaying mean ± SEM values for the number of TH + neurons in SN of 16-month-old ...
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Neuronal loss in the SN and LC of P8 α-SynTg mice. (A) Representative images of the SN in 16-month-old R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice stained with anti-TH antibody. Scale bar = 500 μm. (B) Graph displaying mean ± SEM values for the number of TH + neurons in SN of 16-month-old R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice (n = 4) mice per group. (C) Representative images of the LC in 16-month-old R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice stained with anti-TH antibody. Scale bar = 200 μm. (D) Graph shows mean ± SEM values for the number of TH + neurons in the LC of 16-month-old R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice (n = 3 mice per group). (E) Representative immunoblots with anti-p-Ser129-α-Syn, total α-Syn and β-actin antibodies using whole brain lysates from R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice. Western blots were cropped to focus on protein(s) of interest. Uncropped Western blots are shown in Supplementary Fig. S1. (F) Graph shows mean ± SEM of the p-Ser129-α-Syn immunoblot signal normalized to total synuclein (n = 3 mice per group). (G) Representative images of the SN in 16-month-old R1 nonTg, R1 α-SynTg, P8 nonTg and P8 α-SynTg mice stained with anti-p-Ser129-α-Syn antibody. Scale bar = 45 μm. SN substantia nigra, LC locus coeruleus.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 30 January 2024 by Perez-Villalba, A., Sirerol-Piquer, M. S., et al.
Fig.1.B
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Mus musculus (House mouse)
Experimental design and model validation. (A) Schematic representation of experimental design for generating distinct mouse groups. (B) Representative immunoblots using whole brain lysates from R1 nonTg, R1 α-SynTg, P8 nonTg, and P8 α-SynTg mice, probed with anti-human α-Syn (LB509), α-Syn, and G...
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Experimental design and model validation. (A) Schematic representation of experimental design for generating distinct mouse groups. (B) Representative immunoblots using whole brain lysates from R1 nonTg, R1 α-SynTg, P8 nonTg, and P8 α-SynTg mice, probed with anti-human α-Syn (LB509), α-Syn, and GAPDH antibodies. Western blots were cropped to focus on protein(s) of interest. Uncropped Western blots are shown in Supplementary Fig. S1. (C) Representative images of striatum in R1 α-SynTg, P8 nonTg and P8 α-SynTg mice stained with anti-TH and LB509 antibodies. DAPI shows cellular nuclei. Scale bar = 20 μm. (A) was created using Biorender.com.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Front Mol Neurosci on 28 August 2023 by Huh, E., Choi, J. G., et al.
Fig.1.H
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Phenotypes of P.mirabilis-induced PD mouse. Graphs of motor functions evaluated by the rotarod test (A), open-field test (B), and pole test (C). Representative photomicrographs (D) and graphs of TH-positive fibers in ST (E) and TH-positive cells in SNpc (F). Scale bar = 200 μm. Representative pho...
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Phenotypes of P.mirabilis-induced PD mouse. Graphs of motor functions evaluated by the rotarod test (A), open-field test (B), and pole test (C). Representative photomicrographs (D) and graphs of TH-positive fibers in ST (E) and TH-positive cells in SNpc (F). Scale bar = 200 μm. Representative photomicrographs (G) and western blotting images and graphs of α-syn expression (H). Scale bar = 100 μm. (A–C)N = 8 per group, (D–H)N = 4 per group. Values are given as the mean ± SEM. Significant differences were determined by unpaired student’s t-test. *p < 0.05 and **p < 0.01 compared with the normal group. NOR, Normal group, PM, P.mirabilis group.
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Collected and cropped from Front Mol Neurosci by CiteAb, provided under a CC-BY license
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In Nat Commun on 6 April 2023 by Rosado-Ramos, R., Poças, G. M., et al.
Fig.2.G
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Saccharomyces cerevisiae (Yeast)
Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells wi...
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Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells with αSyn-GFP inclusions (A, 1659 cells; AG, 2044 cells) cells for were counted. A vs AG, **p = 0.0014 (n = 4) d αSyn-GFP protein levels of cells subjected to the conditions indicated (Pgk1 was used as loading control), assessed by western blot (n = 3). e Filter trap assay of total yeast protein extract. Quantification of αSyn signal retained in the membrane by western blot (n = 3). f Total yeast protein extract was resolved using size exclusion-chromatography (SEC). Fractions were collected and the αSyn-GFP species present were analyzed by SDS-PAGE followed by western blot (n = 3). g Clearance of αSyn-GFP was evaluated by western blotting at the indicated time points. Pgk1 was used as loading control (n = 3). h Proteasome impairment evaluated by FCM using uGFP, Median Fluorescence Intensity (MFI) (n = 4, E vs A, *p = 0.0121 (n = 4). Control cells with empty vector – E, Control cells + 10 μM genipin – G, αSyn-GFP cells – A, αSyn-GFP cells + 10 μM genipin – AG.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 6 April 2023 by Rosado-Ramos, R., Poças, G. M., et al.
Fig.2.F
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Saccharomyces cerevisiae (Yeast)
Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells wi...
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Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells with αSyn-GFP inclusions (A, 1659 cells; AG, 2044 cells) cells for were counted. A vs AG, **p = 0.0014 (n = 4) d αSyn-GFP protein levels of cells subjected to the conditions indicated (Pgk1 was used as loading control), assessed by western blot (n = 3). e Filter trap assay of total yeast protein extract. Quantification of αSyn signal retained in the membrane by western blot (n = 3). f Total yeast protein extract was resolved using size exclusion-chromatography (SEC). Fractions were collected and the αSyn-GFP species present were analyzed by SDS-PAGE followed by western blot (n = 3). g Clearance of αSyn-GFP was evaluated by western blotting at the indicated time points. Pgk1 was used as loading control (n = 3). h Proteasome impairment evaluated by FCM using uGFP, Median Fluorescence Intensity (MFI) (n = 4, E vs A, *p = 0.0121 (n = 4). Control cells with empty vector – E, Control cells + 10 μM genipin – G, αSyn-GFP cells – A, αSyn-GFP cells + 10 μM genipin – AG.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 6 April 2023 by Rosado-Ramos, R., Poças, G. M., et al.
Fig.2.E
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Saccharomyces cerevisiae (Yeast)
Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells wi...
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Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells with αSyn-GFP inclusions (A, 1659 cells; AG, 2044 cells) cells for were counted. A vs AG, **p = 0.0014 (n = 4) d αSyn-GFP protein levels of cells subjected to the conditions indicated (Pgk1 was used as loading control), assessed by western blot (n = 3). e Filter trap assay of total yeast protein extract. Quantification of αSyn signal retained in the membrane by western blot (n = 3). f Total yeast protein extract was resolved using size exclusion-chromatography (SEC). Fractions were collected and the αSyn-GFP species present were analyzed by SDS-PAGE followed by western blot (n = 3). g Clearance of αSyn-GFP was evaluated by western blotting at the indicated time points. Pgk1 was used as loading control (n = 3). h Proteasome impairment evaluated by FCM using uGFP, Median Fluorescence Intensity (MFI) (n = 4, E vs A, *p = 0.0121 (n = 4). Control cells with empty vector – E, Control cells + 10 μM genipin – G, αSyn-GFP cells – A, αSyn-GFP cells + 10 μM genipin – AG.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 6 April 2023 by Rosado-Ramos, R., Poças, G. M., et al.
Fig.2.D
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Saccharomyces cerevisiae (Yeast)
Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells wi...
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Genipin reduces αSyn aggregation and toxicity.a αSyn-GFP toxicity was assessed by FCM. E vs A, *p = 0.0181; A vs AG, *p = 0.0159 (n = 3). b Fluorescence microscopy imaging of αSyn-GFP-overexpressing cells supplemented or not with 10 μM of genipin (scale bar 5 μm) (n = 4). c Percentage of cells with αSyn-GFP inclusions (A, 1659 cells; AG, 2044 cells) cells for were counted. A vs AG, **p = 0.0014 (n = 4) d αSyn-GFP protein levels of cells subjected to the conditions indicated (Pgk1 was used as loading control), assessed by western blot (n = 3). e Filter trap assay of total yeast protein extract. Quantification of αSyn signal retained in the membrane by western blot (n = 3). f Total yeast protein extract was resolved using size exclusion-chromatography (SEC). Fractions were collected and the αSyn-GFP species present were analyzed by SDS-PAGE followed by western blot (n = 3). g Clearance of αSyn-GFP was evaluated by western blotting at the indicated time points. Pgk1 was used as loading control (n = 3). h Proteasome impairment evaluated by FCM using uGFP, Median Fluorescence Intensity (MFI) (n = 4, E vs A, *p = 0.0121 (n = 4). Control cells with empty vector – E, Control cells + 10 μM genipin – G, αSyn-GFP cells – A, αSyn-GFP cells + 10 μM genipin – AG.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Front Cell Dev Biol on 1 November 2022 by Pilliod, J., Gélinas-Faucher, M., et al.
Fig.1.D
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Overexpression of VAMP8 increases the cleavage of Tau by Caspase 3 and increases the amount of active caspase-3 in the cell lysate. For all the figures, GFP alone corresponds to GFP-empty vector. (A) Schematic representation of Tau constructs and Tau antibodies. All the experiments were carried o...
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Overexpression of VAMP8 increases the cleavage of Tau by Caspase 3 and increases the amount of active caspase-3 in the cell lysate. For all the figures, GFP alone corresponds to GFP-empty vector. (A) Schematic representation of Tau constructs and Tau antibodies. All the experiments were carried out with 0N4R Tau isoform. The constructs TauNT and TauMBD-CT were produced from 0N4R. For the antibodies, the isoform 2N4R was used for consistency with the literature. For B and C, N2a cells were transfected either with Flag-Tau and GFP-empty or Flag-Tau and GFP-VAMP8 plasmids. (B) Representative Western blot with the anti-Tau antibody A0024 recognizing total intracellular Tau (InTau) and the antibody TauC3 directed against Tau cleaved by caspase-3 of the cell lysate revealing that the overexpression of VAMP8 decreased InTau but increased InTau cleaved by caspase-3 (TauC3). For the densitometry analysis of the TauC3/total Tau ratio, InTau (total Tau) was normalized with the actin signal, the loading reference. (C) Representative Western blot with the anti-Tau antibody A0024, the anti-caspase-3 antibody (C3) and the anti-cleaved caspase-3 antibody (CC3) of the cell lysate showing that the C3 signal is similar for cells overexpressing either Tau alone or Tau and VAMP8 while the co-expression of Tau and VAMP8 increased intracellular CC3. CC3/C3 ratio was analyzed by densitometry. n = 6. Data represent scatter plot and mean±SEM. **p < 0.01. (D) N2a cells were transfected either with Flag-empty and GFP-empty (Flag + GFP), α-synuclein and GFP-empty, Flag-Tau and GFP-empty, Flag-Tau and GFP-VAMP8 plasmids. Representative Western blot of the cell lysate revealed with the antibodies recognizing Tau (A0024), VAMP8, GFP, α-synuclein, caspase-3, cleaved caspase-3 and actin. No difference of total caspase-3 levels was noted between the experimental conditions. In the case of cleaved by caspase-3, the condition Tau and VAMP8 presented the highest levels. For the densitometry analysis of the signal of caspase-3 and cleaved caspase-3 was normalized with the actin signal, the loading reference. Black frames were used to mark the splice sites of immunoblot images. n = 5. Data represent scatter plot and mean±SEM. **p < 0.01.
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Collected and cropped from Front Cell Dev Biol by CiteAb, provided under a CC-BY license
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In Biomolecules on 12 August 2022 by Pieger, K., Schmitt, V., et al.
Fig.3.A
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(a) Dot blot analysis of enriched cytosolic and nuclear fractions from H4 cells using different anti-aSyn antibodies, including Nu-aSyn-C, Syn1, EP1536Y (specific for phosphorylated aSyn on serine 129), and LB 509 (recognizing aggregated aSyn). GAPDH and histone 3 serve as protein markers of the ...
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(a) Dot blot analysis of enriched cytosolic and nuclear fractions from H4 cells using different anti-aSyn antibodies, including Nu-aSyn-C, Syn1, EP1536Y (specific for phosphorylated aSyn on serine 129), and LB 509 (recognizing aggregated aSyn). GAPDH and histone 3 serve as protein markers of the cytosol and the nucleus, respectively. In H4 cells, nuclear aSyn exhibits a higher phosphorylated and aggregated status than cytosolic aSyn. (b) WB analysis of human recombinant aSyn (Rc-aSyn) and preformed aSyn oligomers. The Nu-aSyn-C antibody preferentially recognizes higher-molecular-weight oligomers.
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Collected and cropped from Biomolecules by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 27 July 2022 by Kang, S. J., Kim, S. J., et al.
Fig.5.D
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ApoE deficiency inhibits α-syn release in SH-SY5Y cells. (A) Differentiated NT and ApoE KD #1, #2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured in serum-free media for 1 day, and culture media were collected. The cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The ...
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ApoE deficiency inhibits α-syn release in SH-SY5Y cells. (A) Differentiated NT and ApoE KD #1, #2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured in serum-free media for 1 day, and culture media were collected. The cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The samples were then analyzed by Western blot. Values were derived from three independent experiments (n = 3). (B) The α-syn in culture media was analyzed by ELISA (n = 3). (C) Culture media were analyzed by α-syn RT-QUIC. Values were derived from triplicate and a representative of three independent experiments (n = 3). (D) A53T α-syn-EGFP OE SH-SY5Y cells were transfected with plasmids for ApoE isoforms (ApoE2, ApoE3, and ApoE4) and differentiated by 50 μM RA for 5 days. The cells were then cultured in serum-free media for 1 day and culture media were collected. Cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The samples were then analyzed by Western blot. Values were derived from three independent experiments (n = 3). (E) Culture media were analyzed by α-syn RT-QUIC. Values were derived from triplicate and a representative of three independent experiments (n = 3). (F) SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, ApoE KD #1, or #2/A53T α-syn-EGFP OE SH-SY5Y cells for 24 h. The cells in the lower chamber were immunostained with an anti-EGFP antibody. Values were derived from three independent experiments (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 against the control; one-way ANOVA. Blue indicates DAPI staining. Scale bar indicates 20 μm.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
Image 1 of 49
In Mol Brain on 1 December 2020 by Vanan, S., Zeng, X., et al.
Fig.6.A
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WB
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Mus musculus (House mouse)
Western blot analysis of PD related proteins in the striatum. a Striatal samples from 22-months-old NTg mice (n = 3) and Tg mice (n = 3) were tested for a variety of PD associated proteins: TH, α-synuclein, Phosphor-Tau (AT8), DAT and DD2R by Western blot. β-actin was used as a loading control. b...
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Western blot analysis of PD related proteins in the striatum. a Striatal samples from 22-months-old NTg mice (n = 3) and Tg mice (n = 3) were tested for a variety of PD associated proteins: TH, α-synuclein, Phosphor-Tau (AT8), DAT and DD2R by Western blot. β-actin was used as a loading control. b Quantification analysis of proteins detected (mean ± SEM). There was no significant difference in the relative expression of TH (P = 0.500), α-synuclein (P = 0.886), AT8 (P = 0.831), DAT (P = 0.407) and DD2R (P = 0.532). Student’s t-test. c Striatal samples were tested for the mitochondrial proteins Tom20, Mfn2 (Mitofusin-2), and Drp1, and the autophagy-related protein LC3-II by Western blot. β-actin was used as a loading control. d Quantification analysis of proteins detected (mean ± SEM). There was no significant difference in the relative expression of Tom20 (P = 0.662), Mfn2 (P = 0.927), Drp1 (P = 0.283) and LC3-II (P = 0.210). n = 3. Student’s t-test
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Collected and cropped from Mol Brain by CiteAb, provided under a CC-BY license
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In Eneuro on 6 September 2020 by Verma, D. K., Ghosh, A., et al.
Fig.9.G
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WB
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Rattus norvegicus (Rat)
Ubc9 overexpression enhances the levels of SUMO1 and ubiquitin on α-syn, while it prevents MPP+-induced SUMO-1 removal from α-syn. A, WB images show that the level of SUMO1 was increased with Ubc9 overexpression from total cell lysates. GAPDH was used as a loading control for total cell lysates. ...
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Ubc9 overexpression enhances the levels of SUMO1 and ubiquitin on α-syn, while it prevents MPP+-induced SUMO-1 removal from α-syn. A, WB images show that the level of SUMO1 was increased with Ubc9 overexpression from total cell lysates. GAPDH was used as a loading control for total cell lysates. B, MPP+ exposure significantly reduces the total amount of SUMO1 in N27-EGFP cells, while Ubc9-OE prevents MPP+-induced SUMO1 down-regulation in total cell lysates. C, In WB using IP’ed α-syn, MPP+ reduces the level of SUMO1 bound to α-syn in EGFP cells. α-Syn was used as a loading control after IPs. D, Ubc9 overexpression prevents MPP+-induced SUMO1 removal from α-syn in quantitative WB analysis. E, Examples of WB show that the total amount of ubiquitin (Ub) was reduced by MPP+ treatment. GAPDH was used as a loading control for total cell lysates. F, The levels of ubiquitin from total cell lysates were decreased by MPP+ exposure both in EGFP and Ubc9 cells. G, In WB, the levels of ubiquitin bound to α-syn were substantially reduced by MPP+ treatment. α-Syn was used as a loading control after IPs. H, MPP+ exposure results in a significant reduction of ubiquitin bound to α-syn in both EGFP and Ubc9 cells. MPP+ (640 μm) treatment for 24 h was applied to all experiments. Integrated density of WB images was measured using ImageJ. The ratios of SUMO1 or ubiquitin over respected loading control were presented as mean ± SEM in % of control. Statistical analysis was applied using two-way ANOVA, Tukey’s multiple comparison post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 5–8 each group).
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Collected and cropped from Eneuro by CiteAb, provided under a CC-BY license
Image 1 of 49
In Eneuro on 6 September 2020 by Verma, D. K., Ghosh, A., et al.
Fig.9.C
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WB
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Rattus norvegicus (Rat)
Ubc9 overexpression enhances the levels of SUMO1 and ubiquitin on α-syn, while it prevents MPP+-induced SUMO-1 removal from α-syn. A, WB images show that the level of SUMO1 was increased with Ubc9 overexpression from total cell lysates. GAPDH was used as a loading control for total cell lysates. ...
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Ubc9 overexpression enhances the levels of SUMO1 and ubiquitin on α-syn, while it prevents MPP+-induced SUMO-1 removal from α-syn. A, WB images show that the level of SUMO1 was increased with Ubc9 overexpression from total cell lysates. GAPDH was used as a loading control for total cell lysates. B, MPP+ exposure significantly reduces the total amount of SUMO1 in N27-EGFP cells, while Ubc9-OE prevents MPP+-induced SUMO1 down-regulation in total cell lysates. C, In WB using IP’ed α-syn, MPP+ reduces the level of SUMO1 bound to α-syn in EGFP cells. α-Syn was used as a loading control after IPs. D, Ubc9 overexpression prevents MPP+-induced SUMO1 removal from α-syn in quantitative WB analysis. E, Examples of WB show that the total amount of ubiquitin (Ub) was reduced by MPP+ treatment. GAPDH was used as a loading control for total cell lysates. F, The levels of ubiquitin from total cell lysates were decreased by MPP+ exposure both in EGFP and Ubc9 cells. G, In WB, the levels of ubiquitin bound to α-syn were substantially reduced by MPP+ treatment. α-Syn was used as a loading control after IPs. H, MPP+ exposure results in a significant reduction of ubiquitin bound to α-syn in both EGFP and Ubc9 cells. MPP+ (640 μm) treatment for 24 h was applied to all experiments. Integrated density of WB images was measured using ImageJ. The ratios of SUMO1 or ubiquitin over respected loading control were presented as mean ± SEM in % of control. Statistical analysis was applied using two-way ANOVA, Tukey’s multiple comparison post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 5–8 each group).
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Collected and cropped from Eneuro by CiteAb, provided under a CC-BY license
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In Sci Rep on 30 July 2020 by Dominguez-Meijide, A., Vasili, E., et al.
Fig.2.A
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WB
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Homo sapiens (Human)
Proteins are released to the cell culture media. (a) Western blot showing presence of different aSyn BiFC fragments in cell culture media. Full length blots are presented in Supplementary Fig. S6C. (b) Western blot showing presence of different tau BiFC fragments in cell culture media. Full lengt...
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Proteins are released to the cell culture media. (a) Western blot showing presence of different aSyn BiFC fragments in cell culture media. Full length blots are presented in Supplementary Fig. S6C. (b) Western blot showing presence of different tau BiFC fragments in cell culture media. Full length blots are presented in Supplementary Fig. S6D. (c) ELISA measurements of aSyn levels in cell culture media show presence of the protein in cell media (N = 4). (d) ELISA measurements of tau levels confirm the presence of the protein in the cell culture media (N = 5). *P < 0.05; **P < 0.005; ***P < 0.0005 in relation to empty vector. Error bars represent SD. ANOVA and subsequent paired t-test in relation to empty vector.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Invest Ophthalmol Vis Sci on 3 June 2020 by Kaehler, K., Seitter, H., et al.
Fig.1.A
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WB
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Mus musculus (House mouse)
α-Syn in mouse retinas. α-Syn expression was higher in Plp-α-Syn as compared to wild-type (WT) samples. (A) Retina of both WT and Plp-α-Syn mice compared to brain positive controls. The antibody (pan α-Syn; see Table 1) detected the murine and human forms of α-Syn. (B) The presence of the phospho...
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α-Syn in mouse retinas. α-Syn expression was higher in Plp-α-Syn as compared to wild-type (WT) samples. (A) Retina of both WT and Plp-α-Syn mice compared to brain positive controls. The antibody (pan α-Syn; see Table 1) detected the murine and human forms of α-Syn. (B) The presence of the phosphorylated α-Syn (S129) in the retina, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as loading control. In all experiments, the indicated amounts of retinal and brain tissue were loaded. The age of animals was 8 to 10 weeks. The figure shows representative examples of three experiments.
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Collected and cropped from Invest Ophthalmol Vis Sci by CiteAb, provided under a CC-BY license
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In Nat Commun on 13 March 2020 by Choi, I., Zhang, Y., et al.
Fig.4.K
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WB
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Mus musculus (House mouse)
α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number o...
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α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number of hα-Syn/ubiquitin-positive puncta was quantified (c). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In (a), F(3,16) = 3.381, p = 0.0443 for hα-Syn-genotype interaction; F(3, 6) = 11.46, p = 0.0003 for hα-Syn; F(1,16) = 43.09, p = 6.51E-06 for genotype. In b, F(3,40) = 3.199, p = 0.0334 for hα-Syn-genotype interaction; F(3,40) = 8.326, p = 0.0002 for hα-Syn; F(1,40) = 21.77, p = 3.41E-05 for genotype. In c, F(2,174) = 12.56, p = 8.03E-06 for hα-Syn-genotype interaction; F(2,174) = 91.61, p = 6.64E-28 for hα-Syn; F(1,174) = 24.89, p = 1.46E-06 for genotype. d, e Cells were pretreated with TAK-242 and treated with 250 nM hα-Syn protein. The protein levels of p62 and the number of hα-Syn/ubiquitin-positive puncta were determined by W.B (d, n = 4) and immunostaining (e, n = 35), respectively. p-values were calculated by unpaired two-tailed Student’s t test (d, p = 0.0001) and one-way ANOVA with Newman–Keuls post hoc test (e, F(2,92) = 156.7, p = 2.35E-30). f Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4-KO mice injected with AAV-hα-Syn brains. Scale bar, 10 µm. g The number of p62 puncta was quantified in microglia at striatum of WT mice (n = 5) and Tlr4-KO mice (n = 5). p-values were calculated by two-tailed Mann–Whitney U test. h Microglia were treated with either 250 nM hα-Syn protein (red, n = 6) or 50 ng/ml LPS (blue, n = 7 for 0–6 h, n = 6 for 9 h) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. In hα-Syn protein treatment, F(3,20)=35.43, p = 3.39E-08 for p62, F(3,20) = 6.655, p = 0.0027 for Il-1b, F(3,20) = 8.907, p = 0.0006 for Tnf, F(3,20) = 5.104, p = 0.0087 for Infb1; In LPS treatment, F(3,23) = 19.53, p = 1.61E-06 for p62, F(3,23) = 4.643, p = 0.0111 for Il-1b, F(3,23) = 12.09, p = 5.92E-05 for Tnf, F(3,23)=35.86, p = 7.68E-09 for Infb1. i After treating 250 nM hα-Syn protein or 50 ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. j After treating hα-Syn protein (n = 7) or LPS (n = 7) for 30 min to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(2,18) = 15.10, p = 0.0001. k Microglia were pretreated with Dynasore (n = 3) for indicated concentration and treated with 250 nM hα-Syn protein for 3 h. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 13 March 2020 by Choi, I., Zhang, Y., et al.
Fig.4.I
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WB
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Mus musculus (House mouse)
α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number o...
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α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number of hα-Syn/ubiquitin-positive puncta was quantified (c). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In (a), F(3,16) = 3.381, p = 0.0443 for hα-Syn-genotype interaction; F(3, 6) = 11.46, p = 0.0003 for hα-Syn; F(1,16) = 43.09, p = 6.51E-06 for genotype. In b, F(3,40) = 3.199, p = 0.0334 for hα-Syn-genotype interaction; F(3,40) = 8.326, p = 0.0002 for hα-Syn; F(1,40) = 21.77, p = 3.41E-05 for genotype. In c, F(2,174) = 12.56, p = 8.03E-06 for hα-Syn-genotype interaction; F(2,174) = 91.61, p = 6.64E-28 for hα-Syn; F(1,174) = 24.89, p = 1.46E-06 for genotype. d, e Cells were pretreated with TAK-242 and treated with 250 nM hα-Syn protein. The protein levels of p62 and the number of hα-Syn/ubiquitin-positive puncta were determined by W.B (d, n = 4) and immunostaining (e, n = 35), respectively. p-values were calculated by unpaired two-tailed Student’s t test (d, p = 0.0001) and one-way ANOVA with Newman–Keuls post hoc test (e, F(2,92) = 156.7, p = 2.35E-30). f Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4-KO mice injected with AAV-hα-Syn brains. Scale bar, 10 µm. g The number of p62 puncta was quantified in microglia at striatum of WT mice (n = 5) and Tlr4-KO mice (n = 5). p-values were calculated by two-tailed Mann–Whitney U test. h Microglia were treated with either 250 nM hα-Syn protein (red, n = 6) or 50 ng/ml LPS (blue, n = 7 for 0–6 h, n = 6 for 9 h) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. In hα-Syn protein treatment, F(3,20)=35.43, p = 3.39E-08 for p62, F(3,20) = 6.655, p = 0.0027 for Il-1b, F(3,20) = 8.907, p = 0.0006 for Tnf, F(3,20) = 5.104, p = 0.0087 for Infb1; In LPS treatment, F(3,23) = 19.53, p = 1.61E-06 for p62, F(3,23) = 4.643, p = 0.0111 for Il-1b, F(3,23) = 12.09, p = 5.92E-05 for Tnf, F(3,23)=35.86, p = 7.68E-09 for Infb1. i After treating 250 nM hα-Syn protein or 50 ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. j After treating hα-Syn protein (n = 7) or LPS (n = 7) for 30 min to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(2,18) = 15.10, p = 0.0001. k Microglia were pretreated with Dynasore (n = 3) for indicated concentration and treated with 250 nM hα-Syn protein for 3 h. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 13 March 2020 by Choi, I., Zhang, Y., et al.
Fig.4.D
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WB
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Mus musculus (House mouse)
α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number o...
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α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number of hα-Syn/ubiquitin-positive puncta was quantified (c). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In (a), F(3,16) = 3.381, p = 0.0443 for hα-Syn-genotype interaction; F(3, 6) = 11.46, p = 0.0003 for hα-Syn; F(1,16) = 43.09, p = 6.51E-06 for genotype. In b, F(3,40) = 3.199, p = 0.0334 for hα-Syn-genotype interaction; F(3,40) = 8.326, p = 0.0002 for hα-Syn; F(1,40) = 21.77, p = 3.41E-05 for genotype. In c, F(2,174) = 12.56, p = 8.03E-06 for hα-Syn-genotype interaction; F(2,174) = 91.61, p = 6.64E-28 for hα-Syn; F(1,174) = 24.89, p = 1.46E-06 for genotype. d, e Cells were pretreated with TAK-242 and treated with 250 nM hα-Syn protein. The protein levels of p62 and the number of hα-Syn/ubiquitin-positive puncta were determined by W.B (d, n = 4) and immunostaining (e, n = 35), respectively. p-values were calculated by unpaired two-tailed Student’s t test (d, p = 0.0001) and one-way ANOVA with Newman–Keuls post hoc test (e, F(2,92) = 156.7, p = 2.35E-30). f Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4-KO mice injected with AAV-hα-Syn brains. Scale bar, 10 µm. g The number of p62 puncta was quantified in microglia at striatum of WT mice (n = 5) and Tlr4-KO mice (n = 5). p-values were calculated by two-tailed Mann–Whitney U test. h Microglia were treated with either 250 nM hα-Syn protein (red, n = 6) or 50 ng/ml LPS (blue, n = 7 for 0–6 h, n = 6 for 9 h) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. In hα-Syn protein treatment, F(3,20)=35.43, p = 3.39E-08 for p62, F(3,20) = 6.655, p = 0.0027 for Il-1b, F(3,20) = 8.907, p = 0.0006 for Tnf, F(3,20) = 5.104, p = 0.0087 for Infb1; In LPS treatment, F(3,23) = 19.53, p = 1.61E-06 for p62, F(3,23) = 4.643, p = 0.0111 for Il-1b, F(3,23) = 12.09, p = 5.92E-05 for Tnf, F(3,23)=35.86, p = 7.68E-09 for Infb1. i After treating 250 nM hα-Syn protein or 50 ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. j After treating hα-Syn protein (n = 7) or LPS (n = 7) for 30 min to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(2,18) = 15.10, p = 0.0001. k Microglia were pretreated with Dynasore (n = 3) for indicated concentration and treated with 250 nM hα-Syn protein for 3 h. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
Image 1 of 49
In Nat Commun on 13 March 2020 by Choi, I., Zhang, Y., et al.
Fig.4.A
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WB
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Mus musculus (House mouse)
α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number o...
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α-synuclein-induced p62 upregulation requires TLR4.a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number of hα-Syn/ubiquitin-positive puncta was quantified (c). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In (a), F(3,16) = 3.381, p = 0.0443 for hα-Syn-genotype interaction; F(3, 6) = 11.46, p = 0.0003 for hα-Syn; F(1,16) = 43.09, p = 6.51E-06 for genotype. In b, F(3,40) = 3.199, p = 0.0334 for hα-Syn-genotype interaction; F(3,40) = 8.326, p = 0.0002 for hα-Syn; F(1,40) = 21.77, p = 3.41E-05 for genotype. In c, F(2,174) = 12.56, p = 8.03E-06 for hα-Syn-genotype interaction; F(2,174) = 91.61, p = 6.64E-28 for hα-Syn; F(1,174) = 24.89, p = 1.46E-06 for genotype. d, e Cells were pretreated with TAK-242 and treated with 250 nM hα-Syn protein. The protein levels of p62 and the number of hα-Syn/ubiquitin-positive puncta were determined by W.B (d, n = 4) and immunostaining (e, n = 35), respectively. p-values were calculated by unpaired two-tailed Student’s t test (d, p = 0.0001) and one-way ANOVA with Newman–Keuls post hoc test (e, F(2,92) = 156.7, p = 2.35E-30). f Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4-KO mice injected with AAV-hα-Syn brains. Scale bar, 10 µm. g The number of p62 puncta was quantified in microglia at striatum of WT mice (n = 5) and Tlr4-KO mice (n = 5). p-values were calculated by two-tailed Mann–Whitney U test. h Microglia were treated with either 250 nM hα-Syn protein (red, n = 6) or 50 ng/ml LPS (blue, n = 7 for 0–6 h, n = 6 for 9 h) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. In hα-Syn protein treatment, F(3,20)=35.43, p = 3.39E-08 for p62, F(3,20) = 6.655, p = 0.0027 for Il-1b, F(3,20) = 8.907, p = 0.0006 for Tnf, F(3,20) = 5.104, p = 0.0087 for Infb1; In LPS treatment, F(3,23) = 19.53, p = 1.61E-06 for p62, F(3,23) = 4.643, p = 0.0111 for Il-1b, F(3,23) = 12.09, p = 5.92E-05 for Tnf, F(3,23)=35.86, p = 7.68E-09 for Infb1. i After treating 250 nM hα-Syn protein or 50 ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. j After treating hα-Syn protein (n = 7) or LPS (n = 7) for 30 min to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(2,18) = 15.10, p = 0.0001. k Microglia were pretreated with Dynasore (n = 3) for indicated concentration and treated with 250 nM hα-Syn protein for 3 h. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
Image 1 of 49
In Sci Rep on 21 May 2019 by Uehara, T., Choong, C. J., et al.
Fig.6.C
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WB
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Mus musculus (House mouse)
Intracerebroventricular administration of ASOA19 reduced SNCA mRNA and protein levels in brains of TH-SNCA mice. (a,b) qPCR analysis of mRNA levels of hSNCA and mouse endogenous Snca, Sncb, and Sncg in mouse brains. ASOA19 or control was injected into the left lateral ventricle of TH-SNCA-140 m m...
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Intracerebroventricular administration of ASOA19 reduced SNCA mRNA and protein levels in brains of TH-SNCA mice. (a,b) qPCR analysis of mRNA levels of hSNCA and mouse endogenous Snca, Sncb, and Sncg in mouse brains. ASOA19 or control was injected into the left lateral ventricle of TH-SNCA-140 m mice expressing human SNCA with Ala53Thr mutation. Total RNA was extracted from left cerebral hemisphere samples collected 14 days after ASOA19 injection. ASOA19 significantly reduced the levels of SNCA mRNA. The levels of Snca, Sncb, and Sncg mRNA showed no reduction. Data are expressed as mean ± SEM (n = 5). *p < 0.05 by t-test. (c,d) Immunoblotting and densitometry analysis showing that ASOA19 reduced the levels of SNCA protein. Proteins were extracted from right cerebral hemisphere samples two weeks after administration of ASOA19 and subjected to immunoblotting. The images of blots were cropped from different parts of the same gel. Full-length gels are shown in Supplemental Fig. 4. Immunoblotting with anti-beta-actin was used as an internal control. ASOA19 significantly reduced the levels of SNCA protein. Densitometry data are expressed as mean ± SEM (n = 5).
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 49
In Nat Commun on 26 February 2018 by Ryan, T., Bamm, V. V., et al.
Fig.4.A
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WB
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Mus musculus (House mouse)
Cardiolipin is externalized to the mitochondrial surface in α-syn mutant hNs and transgenic mice in response to α-syn accumulation. a Western analysis of lysates from corrected and A53T cells at DIV 14 and DIV 60 labeled for total α-syn, PS129, TH (DA neuronal marker), nestin (NPC marker) or GAPD...
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Cardiolipin is externalized to the mitochondrial surface in α-syn mutant hNs and transgenic mice in response to α-syn accumulation. a Western analysis of lysates from corrected and A53T cells at DIV 14 and DIV 60 labeled for total α-syn, PS129, TH (DA neuronal marker), nestin (NPC marker) or GAPDH show that levels of PS129-modified α-syn are elevated in A53T hNs at DIV 14 and remain elevated at DIV 60 relative to corrected cells. b, c Mitochondria were purified from A53T cells and genetically corrected cells at both DIV 14 and DIV 60. Lysates were then separated based on TX-100 solubility (soluble) or urea solubility (insoluble). Western analysis of A53T cells shows elevated soluble α-syn at the mitochondria relative to corrected cells at DIV 14 and DIV 60 (denoted by ** and * respectively). Quantification of soluble and insoluble α-syn levels in mitochondrial fractions normalized to Coomassie (c). Data represent mean ± s.e.m. *P < 0.05, **P < 0.01 by ANOVA followed by Tukey’s post hoc test, n = 4. d, e Micrographs of corrected and A53T-mutant NPCs expressing the negative charge probe RPRE-RFP and CL-GFP show RPRE-RFP translocation from a primarily plasma membrane localization in isogenic-corrected hNs to a punctate intracellular localization in A53T hNs in tight cellular localization with CL-GFP (d). Quantification of percent total cell number with colocalized RPRE-RFP and CL-GFP (e). Data represent mean ± s.e.m. **P < 0.0001 by t-test, n = 3 coverslips over 2 independent differentiations, DIV: 14; scale bar: 10 μm. f, g Fluorescence resonance energy transfer (FRET) from CL-GFP to RPRE-RFP in hiPSC-derived A53T and corrected hNs or hESC-derived WT, A53T and E46K hNs was assessed (f) and mean FRET intensity was quantified (g). Data represent mean ± s.e.m. **P < 0.01 by ANOVA followed by Tukey’s post hoc test, for hiPSCs, n = 6, for hESCs, n = 8 coverslips over two independent differentiations, DIV: 14. Scale bar: 10 µm. h Mitochondria were isolated from the SNpc of 6-month-old WT and A53T transgenic animals and labeled with Annexin V to measure the abundance of cardiolipin on the mitochondrial surface. Controls were rotenone-exposed WT samples. Animals were not randomized, nor was the analysis blinded. Data represent mean ± s.e.m. **P < 0.01 by ANOVA followed by Dunnett post hoc test, n = 4. Clipart was obtained at clker.com
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
Image 1 of 49
In Sci Rep on 28 November 2017 by Karampetsou, M., Ardah, M. T., et al.
Fig.8.B
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WB
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Mus musculus (House mouse)
Increased uptake and faster seeding of the endogenous α-Synuclein in primary neurons following P-PFF treatment (a) Mouse primary cultures (6div) were treated with PFF for 8 and 24 hours. Following Triton-X extraction, internalized fibrils were visualized with the human specific α-Synuclein antibo...
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Increased uptake and faster seeding of the endogenous α-Synuclein in primary neurons following P-PFF treatment (a) Mouse primary cultures (6div) were treated with PFF for 8 and 24 hours. Following Triton-X extraction, internalized fibrils were visualized with the human specific α-Synuclein antibody 4B12. Increased uptake of P-PFF was observed as early as 8 h. The uptake was quantified by densitometry and found to be significantly increased for P-PFF in both time points compared to wt- or S129A- PFF. P-PFF uptake was further verified using the phospho Ser 129 antibody. β-Actin was used as a loading control (cropped gel/blot is shown). (b) Sarcosyl-soluble α-Synuclein species were also increased following P-PFF treatment of primary cortical neurons. γ-tubulin was used as a loading control (cropped gel/blot is shown). (c) Immunocytochemistry with the rodent specific α-Synuclein antibody (D37A6) in primary mouse cortical cultures treated with the three fibrilar types. P-PFF seed the endogenous α-Synuclein more effectively compared to the other fibrilar types and PBS control treated neurons for 5 days. However (d) 8-day-treated neurons exhibit similar levels of endogenous α-Synuclein signal for both the wt- and P-PFF treatments, in contrast to the significant lower levels of S129A-treated neurons. Scatter plots present the mean fluorescence intensity/cell of three independent experiments (n≈90 single cells/condition per replicate). β Tubulin III (Tuj 1) was used as a neuronal marker. TO-PRO-3 (blue) was used as a cell nuclear marker. All data represent mean values ± SEM from three independent experiments. Statistical significance was obtained by ANOVA followed by Tukey’s post-hoc test for (a) and non parametric Kruskal-Wallis test followed by Dunn’s post-hoc test for (c and d). (a) For 8 h p < 0,0001 and for 24 h p = 0,002, (c) p < 0,0001 (d) p < 0,0001.Scale bar in (c and d) represent 25 μm.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 49
In Sci Rep on 28 November 2017 by Karampetsou, M., Ardah, M. T., et al.
Fig.8.A
-
WB
-
Mus musculus (House mouse)
Increased uptake and faster seeding of the endogenous α-Synuclein in primary neurons following P-PFF treatment (a) Mouse primary cultures (6div) were treated with PFF for 8 and 24 hours. Following Triton-X extraction, internalized fibrils were visualized with the human specific α-Synuclein antibo...
more
Increased uptake and faster seeding of the endogenous α-Synuclein in primary neurons following P-PFF treatment (a) Mouse primary cultures (6div) were treated with PFF for 8 and 24 hours. Following Triton-X extraction, internalized fibrils were visualized with the human specific α-Synuclein antibody 4B12. Increased uptake of P-PFF was observed as early as 8 h. The uptake was quantified by densitometry and found to be significantly increased for P-PFF in both time points compared to wt- or S129A- PFF. P-PFF uptake was further verified using the phospho Ser 129 antibody. β-Actin was used as a loading control (cropped gel/blot is shown). (b) Sarcosyl-soluble α-Synuclein species were also increased following P-PFF treatment of primary cortical neurons. γ-tubulin was used as a loading control (cropped gel/blot is shown). (c) Immunocytochemistry with the rodent specific α-Synuclein antibody (D37A6) in primary mouse cortical cultures treated with the three fibrilar types. P-PFF seed the endogenous α-Synuclein more effectively compared to the other fibrilar types and PBS control treated neurons for 5 days. However (d) 8-day-treated neurons exhibit similar levels of endogenous α-Synuclein signal for both the wt- and P-PFF treatments, in contrast to the significant lower levels of S129A-treated neurons. Scatter plots present the mean fluorescence intensity/cell of three independent experiments (n≈90 single cells/condition per replicate). β Tubulin III (Tuj 1) was used as a neuronal marker. TO-PRO-3 (blue) was used as a cell nuclear marker. All data represent mean values ± SEM from three independent experiments. Statistical significance was obtained by ANOVA followed by Tukey’s post-hoc test for (a) and non parametric Kruskal-Wallis test followed by Dunn’s post-hoc test for (c and d). (a) For 8 h p < 0,0001 and for 24 h p = 0,002, (c) p < 0,0001 (d) p < 0,0001.Scale bar in (c and d) represent 25 μm.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 49