Cytoplasmic alpha-synuclein (αSyn) aggregates are a typical feature of Parkinson's disease (PD). Extracellular insoluble αSyn can induce pathology in healthy neurons suggesting that PD neurodegeneration may spread through cell-to-cell transfer of αSyn proteopathic seeds. Early pro-homeostatic reaction of microglia to toxic forms of αSyn remains elusive, which is especially relevant considering the recently uncovered microglial molecular diversity. Here, we show that periventricular microglia of the subependymal neurogenic niche monitor the cerebrospinal fluid and can rapidly phagocytize and degrade different aggregated forms of αSyn delivered into the lateral ventricle. However, this clearing ability worsens with age, leading to an increase in microglia with aggregates in aged treated mice, an accumulation also observed in human PD samples. We also show that exposure of aged microglia to aggregated αSyn isolated from human PD samples results in the phosphorylation of the endogenous protein and the generation of αSyn seeds that can transmit the pathology to healthy neurons. Our data indicate that while microglial phagocytosis rapidly clears toxic αSyn, aged microglia can contribute to synucleinopathy spreading.
© 2025. The Author(s).
Product Citations: 191
In Molecular Neurodegeneration on 5 March 2025 by Sirerol-Piquer, M. S., Perez-Villalba, A., et al.
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IHC
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Homo sapiens (Human)
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Neuroscience
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In Neuron on 19 February 2025 by Lam, I., Ndayisaba, A., et al.
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Neuroscience
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Stem Cells and Developmental Biology
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In Acta Neuropathologica Communications on 11 February 2025 by Otero-Jimenez, M., Wojewska, M. J., et al.
Alpha-synucleinopathies are neurodegenerative diseases characterized by the spread of alpha-synuclein (α-syn) aggregates throughout the central nervous system in a stereotypical manner. These diseases include Lewy body disease (LBD), which encompass Dementia with Lewy bodies (DLB), Parkinson's Disease (PD), and Parkinson's Disease Dementia (PDD), and Multiple System Atrophy (MSA). LBD and MSA chiefly contain α-syn aggregates in neurons and oligodendrocytes, respectively, although glial α-syn pathology in LBD is increasingly being recognized. Semi-quantitative and machine learning-based quantifications of neuronal, oligodendrocytic and astrocytic α-syn pathology were implemented on a cohort of LBD and MSA post-mortem tissue samples. The neuroanatomical distribution of each cell-type specific α-syn pathology was evaluated using conditional probability matrices and Subtype and Stage Inference (SuStaIn) algorithm. We revealed extensive glial α-syn pathology in LBD, emphasizing the disease- and region-specific profile of astrocytic α-syn pathology, which was absent in MSA and minimal in the substantia nigra of LBD. Furthermore, we have described distinct morphologies of astrocytic α-syn pathology, which were found to correlate with the density of astrocytic α-syn inclusions. Astrocytic α-syn pathology was mainly centered in the amygdala and exhibited a unique stereotypical progression whilst oligodendrocytes displayed a distribution akin to the established neuronal progression pattern. SuStaIn modeling was further used to test for heterogeneity in the spatiotemporal progression, revealing that a subset of cases might follow an alternative pattern. Based on these findings, we introduce a novel multimodal progression framework that integrates, for the first time, the temporal and spatial progression of astrocytic and oligodendrocytic α-syn pathology alongside neuronal pathology in PD, providing further information regarding the role of neurons and glia in disease pathogenesis.
© 2025. The Author(s).
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IHC
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Homo sapiens (Human)
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Neuroscience
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Pathology
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Lewy-MSA hybrid fold drives distinct neuronal α-synuclein pathology
Preprint on BioRxiv : the Preprint Server for Biology on 22 November 2024 by Enomoto, M., Martinez-Valbuena, I., et al.
ABSTRACT The ordered assembly of α-synuclein protein into filaments encoded by SNCA characterizes neurodegenerative diseases called synucleinopathies. Lewy body disease (LBD) shows predominantly neuronal α-synuclein pathology and multiple system atrophy (MSA) predominantly oligodendrocytic α-synuclein pathology affecting subcortical brain structures. Based on cryo-electron microscopy, it was reported that structures of α-synuclein filaments from LBD differ from MSA and juvenile onset synucleinopathy (JOS) caused by a 21-nucleotide duplication in the second exon of one allele of SNCA gene 1-3 . Importantly, a rare subtype of MSA, called atypical MSA 4 shows abundant neuronal argyrophilic α-synuclein inclusions in the limbic system. Current concepts indicate that disease entities are characterized by unique protofilament folds. Here we demonstrate that in addition to the MSA fold, α-synuclein can form a new Lewy-MSA hybrid fold in the same brain region, leading to the atypical histopathological form of MSA. Distinct biochemical characteristics of α-synuclein, as demonstrated by protease-sensitivity digestion assay, seed amplification assays (SAAs) and conformational stability assay (CSA), are also linked to cytopathological differences (e.g. neuronal or oligodendroglial). We expand the current structure-based classification of α−synucleinopathies and propose that cell-specific protein pathologies can be associated with distinct filament folds.
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Homo sapiens (Human)
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Pathology
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Novel tools to quantify total, phospho-Ser129 and aggregated alpha-synuclein in the mouse brain.
In NPJ Parkinson's Disease on 8 November 2024 by Trist, B. G., Wright, C. J., et al.
Assays for quantifying aggregated and phosphorylated (S129) human α-synuclein protein are widely used to evaluate pathological burden in patients suffering from synucleinopathy disorders. Many of these assays, however, do not cross-react with mouse α-synuclein or exhibit poor sensitivity for this target, which is problematic considering the preponderance of mouse models at the forefront of pre-clinical α-synuclein research. In this project, we addressed this unmet need by reformulating two existing AlphaLISA® SureFire® Ultra™ total and pS129 α-synuclein assay kits to yield robust and ultrasensitive (LLoQ ≤ 0.5 pg/mL) quantification of mouse and human wild-type and pS129 α-synuclein protein. We then employed these assays, together with the BioLegend α-synuclein aggregate ELISA, to assess α-synuclein S129 phosphorylation and aggregation in different mouse brain tissue preparations. Overall, we highlight the compatibility of these new immunoassays with rodent models and demonstrate their potential to advance knowledge surrounding α-synuclein phosphorylation and aggregation in synucleinopathies.
© 2024. The Author(s).
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In PLoS Pathog on 1 September 2024 by So, R. W. L., Amano, G., et al.
Fig.2.F
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WB
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PrPC does not affect the biochemical attributes of the S and NS strains following intracerebral inoculation of M83 mice.A) Immunoblots of detergent-insoluble α-syn in brain extracts from symptomatic M83-Prnp+/+ and M83-Prnp0/0 mice at the indicated days post-inoculation (DPI) with S or NS strain ...
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PrPC does not affect the biochemical attributes of the S and NS strains following intracerebral inoculation of M83 mice.A) Immunoblots of detergent-insoluble α-syn in brain extracts from symptomatic M83-Prnp+/+ and M83-Prnp0/0 mice at the indicated days post-inoculation (DPI) with S or NS strain compared with 18-month-old uninoculated (—) mice, probed for total α-syn and PSyn. In the left blots, one mouse is shown for each experimental condition. In the right blots, two distinct mice per experimental condition are shown, along with their respective inocula. B) Quantification of detergent-insoluble PSyn levels in brain extracts from the 4 experimental groups (n = 5–9 mice per experimental condition). The graph displays mean ± s.e.m. Statistical significance was assessed using a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. C) Pooled detergent-insoluble PSyn data for M83-Prnp+/+ and M83-Prnp0/0 mice inoculated with either S (n = 13) or NS (n = 17) strain. The graph displays mean ± s.e.m. Statistical significance was assessed using a Mann-Whitney test. D) Immunoblots of detergent-insoluble PSyn in brain extracts from uninoculated, S-inoculated, and NS-inoculated M83-Prnp+/+ and M83-Prnp0/0 mice after thermolysin (TL) digestion, as well as detergent-insoluble total α-syn after proteinase K (PK) digestion. E) Immunoblots for detergent-insoluble TL-resistant PSyn and PK-resistant total α-syn in the S and NS inocula used for the intracerebral inoculations. F) Immunoblots for detergent-insoluble TL-resistant PSyn and PK-resistant total α-syn in brain extracts from symptomatic S- or NS-inoculated M83-Prnp+/+ and M83-Prnp0/0 mice at the indicated DPI. Two distinct mice are shown per experimental condition.
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Collected and cropped from PLoS Pathog by CiteAb, provided under a CC-BY license
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In Acta Neuropathol Commun on 3 May 2023 by Lau, H. H. C., Martínez-Valbuena, I., et al.
Fig.2.A
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WB
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Biochemical detection of α-syn aggregates in the brains of inoculated TgM83+/- mice. a Immunoblots of total α-syn, detergent-soluble α-syn, and detergent-insoluble PSyn species in brain homogenates from G51D PD-1 and G51D PD-2 mice at 540–543 DPI, clinically ill MSA mice at 155–257 DPI, as well a...
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Biochemical detection of α-syn aggregates in the brains of inoculated TgM83+/- mice. a Immunoblots of total α-syn, detergent-soluble α-syn, and detergent-insoluble PSyn species in brain homogenates from G51D PD-1 and G51D PD-2 mice at 540–543 DPI, clinically ill MSA mice at 155–257 DPI, as well as uninoculated TgM83+/- mice at 580 days of age. The blot of total α-syn was reprobed with an antibody against actin. b Quantification of detergent-insoluble PSyn levels (mean ± s.e.m.) in the brains of G51D PD-1 (n = 6) and G51D PD-2 (n = 5) mice at 540–543 DPI compared to age-matched uninoculated TgM83+/- mice (n = 8). P values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. c Immunoblot of detergent-insoluble, thermolysin (TL)-resistant PSyn and total α-syn levels in brain homogenates from G51D PD-1 mice at 543 DPI or age-matched uninoculated TgM83+/- mice (n = 6 each). d Immunoblot of detergent-insoluble, TL-resistant PSyn levels in brain homogenates from G51D PD-1 mice at either 21 (n = 9) or 543 (n = 2) DPI. In panels a, c, and d, PSyn was detected using the antibody EP1536Y and α-syn was detected using the antibody Syn-1
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Collected and cropped from Acta Neuropathol Commun by CiteAb, provided under a CC-BY license
Image 1 of 20
In Int J Mol Sci on 5 February 2022 by Seebauer, L., Schneider, Y., et al.
Fig.5.B
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WB
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Homo sapiens (Human)
Interaction of aSyn and bTubIII and their reorganization in SNCADupl mDANs. (A) WB analysis of immunoprecipitation of aSyn from hiPSC-derived mDANs differentiated for 10 days by using an anti-aSyn antibody (Syn1). bTubIII is co-precipitated with aSyn. (B) In-cell fractionation of control and SNCA...
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Interaction of aSyn and bTubIII and their reorganization in SNCADupl mDANs. (A) WB analysis of immunoprecipitation of aSyn from hiPSC-derived mDANs differentiated for 10 days by using an anti-aSyn antibody (Syn1). bTubIII is co-precipitated with aSyn. (B) In-cell fractionation of control and SNCADupl mDANs and WB analysis of aSyn and bTubIII in soluble (S) and insoluble, microtubule-associated (IS-MT) fractions. Representative WB images of three experiments (#1, #2, #3) with mDANs from one control and one SNCADupl hiPSC cell line are shown. Note, due to a strong dilution effect after immunoprecipitation and in-cell fractionation, transferred aSyn on the blots shown in (A,B) was only visible by loading the maximum volume onto SDS-PAGE gels and using the SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific). (C) For quantification, the ratio of bTubIII or aSyn in the IS-MT versus the S fraction was calculated. Values from one control line as well as one SNCADupl line and three independent differentiation rounds per cell line were used for the quantification (n = 3/line). aSyn overexpression in hiPSC-neurons leads to a shift of aSyn and bTubIII into the IS-MT pool. Statistics: unpaired t-test; * p < 0.05; ** p < 0.01.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
Image 1 of 20
In Int J Mol Sci on 5 February 2022 by Seebauer, L., Schneider, Y., et al.
Fig.5.A
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WB
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Homo sapiens (Human)
Interaction of aSyn and bTubIII and their reorganization in SNCADupl mDANs. (A) WB analysis of immunoprecipitation of aSyn from hiPSC-derived mDANs differentiated for 10 days by using an anti-aSyn antibody (Syn1). bTubIII is co-precipitated with aSyn. (B) In-cell fractionation of control and SNCA...
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Interaction of aSyn and bTubIII and their reorganization in SNCADupl mDANs. (A) WB analysis of immunoprecipitation of aSyn from hiPSC-derived mDANs differentiated for 10 days by using an anti-aSyn antibody (Syn1). bTubIII is co-precipitated with aSyn. (B) In-cell fractionation of control and SNCADupl mDANs and WB analysis of aSyn and bTubIII in soluble (S) and insoluble, microtubule-associated (IS-MT) fractions. Representative WB images of three experiments (#1, #2, #3) with mDANs from one control and one SNCADupl hiPSC cell line are shown. Note, due to a strong dilution effect after immunoprecipitation and in-cell fractionation, transferred aSyn on the blots shown in (A,B) was only visible by loading the maximum volume onto SDS-PAGE gels and using the SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific). (C) For quantification, the ratio of bTubIII or aSyn in the IS-MT versus the S fraction was calculated. Values from one control line as well as one SNCADupl line and three independent differentiation rounds per cell line were used for the quantification (n = 3/line). aSyn overexpression in hiPSC-neurons leads to a shift of aSyn and bTubIII into the IS-MT pool. Statistics: unpaired t-test; * p < 0.05; ** p < 0.01.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
Image 1 of 20
In Int J Mol Sci on 5 February 2022 by Seebauer, L., Schneider, Y., et al.
Fig.1.B
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WB
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Homo sapiens (Human)
Interaction of aSyn and tubulin in aSyn overexpressing H4 cells. (A) Dot blot analysis of total aSyn and aggregated aSyn in aSyn overexpressing H4 cell models (high aSyn: H4-aSyn and H4-aSyn tet-off cells) and their respective low aSyn expressing cell lines (low aSyn, naïve H4 cells and H4-aSyn t...
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Interaction of aSyn and tubulin in aSyn overexpressing H4 cells. (A) Dot blot analysis of total aSyn and aggregated aSyn in aSyn overexpressing H4 cell models (high aSyn: H4-aSyn and H4-aSyn tet-off cells) and their respective low aSyn expressing cell lines (low aSyn, naïve H4 cells and H4-aSyn tet-off+Dox). Total aSyn levels were determined by using a pan aSyn antibody Syn 1, while the levels of aggregated aSyn conformers (aggr. conformer) were assessed by using a conformation-specific antibody MJFR-14-6-4-2. Both aSyn overexpressing cell lines (high aSyn) exhibit higher levels of aSyn aggregates when comparted to their respective low aSyn counterparts. Total protein loaded was controlled by direct blue staining (shown in Supplementary Figure S1). For quantification, the fold change in aSyn level in a high aSyn cell line was calculated by normalization against total protein (direct blue) and the average level in the corresponding low aSyn cells (n = 4). Statistics: Mann–Whitney test; * p < 0.05. (B) Immunoprecipitation of aSyn from H4-aSyn cells using a pan aSyn antibody Syn1. bTubIII and Tau are co-precipitated. For immunoprecipitation, a buffer without detergents (TBS, a), buffers containing non-ionic milder detergents (TBS + 1% Triton X 100, b or TBS + 1% NP40, c), or a buffer containing stronger detergents (RIPA, d) were used. bTubIII and Tau are detectable in all conditions. (C) Mass spectrometric identification of proteins co-precipitated with aSyn in H4-aSyn tet-off cells. Co-immunoprecipitation was performed by using the pan aSyn antibody Syn211 or the conformation-specific anti-aSyn antibody MJFR-14-6-4-2. Volcano plots of co-precipitated proteins identified by mass spectrometry are shown (left: proteins co-precipitated with Syn211, right: proteins co-precipitated with MJFR-14-6-4-2). Significant proteins from two independent experiments are over the solid lines. Identified microtubule-associated proteins are highlighted by their gene ID and listed in the table. A two-tailed t-test was performed in Perseus (1.6.10.43) using a permutation-based FDR to account for the multiple testing hypothesis (technical injection replicates were preserved during randomization). Candidates were filtered using an FDR of 1% and a fold change (s0) of 2.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Acta Neuropathol Commun on 9 March 2021 by Latina, V., Giacovazzo, G., et al.
Fig.4.A
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WB
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Mus musculus (House mouse)
Immunotherapy with 12A12mAb mitigates the AD-associated synaptic and apoptotic changes and prevents the microtubule destabilization in retinas from diseased animals. Western blotting analyses (a) were carried out on equal amounts of total protein extract (50 µg) from retinas of animals of three e...
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Immunotherapy with 12A12mAb mitigates the AD-associated synaptic and apoptotic changes and prevents the microtubule destabilization in retinas from diseased animals. Western blotting analyses (a) were carried out on equal amounts of total protein extract (50 µg) from retinas of animals of three experimental groups (wild-type, Tg2576 and Tg2576 + mAb). Immunoblots were probed with antibodies against several pre- and postsynaptic proteins—including the N-Methyl-D-aspartate (NMDA) receptor subunits 1 (NR1), synapsin I, synaptophysin, syntaxin 1, SNAP25, α-synuclein and the active (cleaved) form of caspase-6 (Asp162). Data were quantified for molecular weight size ranges for each antibody and normalized to β-actin which was used as loading control. Relative intensity of each protein was calculated and semi-quantitative densitometric analysis (n = 7) is shown (b). Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Statistically significant differences (see details in the main text) were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test for multiple comparison among more than two groups. p < 0.05 was accepted as statistically significant (*p < 0.05; **p < 0.01; ***p < 0.0005; ****p < 0.0001). The functional integrity of axonal track was evaluated by probing the immunoblots with antibodies against the acetyl- and tyrosinylated-α-tubulin, as markers for stable and unstable/dynamic microtubule respectively (c). Data were quantified for molecular weight size ranges for each antibody and normalized to β-actin which was used as loading control. Relative intensity of each protein was calculated and semi-quantitative densitometric analysis (n = 6) is shown (d). Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Statistically significant differences (see details in the main text) were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test for multiple comparison among more than two groups. p < 0.05 was accepted as statistically significant (*p < 0.05; **p < 0.01; ***p < 0.0005; ****p < 0.0001)
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Collected and cropped from Acta Neuropathol Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 7 March 2019 by Carballo-Carbajal, I., Laguna, A., et al.
Fig.7.C
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IHC-P-IF
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Rattus norvegicus (Rat)
Dispensability of aSyn for PD-like inclusion formation and neurodegeneration linked to NM accumulation. a Schematic representation of the site of AAV-hTyr unilateral stereotaxic injection above the SNpc of aSyn knockout (KO) and wild-type (WT) mice. b Top, representative photomicrographs of NM-la...
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Dispensability of aSyn for PD-like inclusion formation and neurodegeneration linked to NM accumulation. a Schematic representation of the site of AAV-hTyr unilateral stereotaxic injection above the SNpc of aSyn knockout (KO) and wild-type (WT) mice. b Top, representative photomicrographs of NM-laden neurons in hematoxylin-eosin (H&E)-stained ipsilateral SNpc brain sections from AAV-hTyr-injected aSyn KO and WT mice at 6 m post-AAV injection. Scale bar, 12.5 μm. Bottom, quantification of intracellular NM optical density in ipsilateral SNpc DA neurons of AAV-hTyr-injected aSyn KO and WT mice. p = 0.879 (two-tailed t-test). c Top, ipsilateral SNpc sections from AAV-hTyr-injected aSyn KO and WT mice exhibiting NM-laden neurons with p62-positive (red) PB (arrowhead) and LB-like inclusions (arrow) at 2 m post-AAV injection. aSyn immunofluorescence is shown in green. Scale bar, 12.5 μm. Bottom, quantification of NM-laden neurons with p62-positive PB or LB-like inclusions in AAV-hTyr-injected aSyn KO and WT mice at 2 m post-AAV injection. p = 0.866 (two-way ANOVA). d Top, optical densitometry of striatal TH-positive fibers in AAV-hTyr-injected aSyn KO and WT mice at 6 m post-AAV injection. *p < 0.05, compared to respective contralateral (non-injected) side (two-way ANOVA; Student–Newman–Keuls post-hoc test). Bottom, stereological cell counts of SNpc TH-positive neurons in AAV-hTyr-injected aSyn KO and WT mice at 6 m post-AAV injection. *p < 0.05, compared to respective contralateral (non-injected) side (two-way ANOVA; Student–Newman–Keuls post-hoc test). In all panels, values are mean ± SEM. In b, n = 76 neurons from n = 4 mice (WT), n = 112 neurons from n = 4 mice (aSyn KO). In c, n = 3 (WT), n = 4 (aSyn KO) mice. In d (top), n = 9 (WT), n = 5 (aSyn KO) mice. In d (bottom), n = 8 (WT), n = 6 (aSyn KO) mice. BF bright-field. In all panels, unstained NM appears as brown. Photomicrographs correspond to 5-μm-thick sections
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Aging Cell on 1 August 2018 by Stankova, T., Piepkorn, L., et al.
Fig.4.B
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WB
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Mus musculus (House mouse)
Western blot analysis of SUMO1 conjugates in vivo. Representative detergent extract input sample (Input) from 8‐week‐old (young, Y) and 36‐week‐old (old, O) WT/WT, WT/AD, KI/WT, and KI/AD animals and specific HA‐peptide eluates (Eluate) of HA immunoaffinity‐purified samples were analyzed by SDS/P...
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Western blot analysis of SUMO1 conjugates in vivo. Representative detergent extract input sample (Input) from 8‐week‐old (young, Y) and 36‐week‐old (old, O) WT/WT, WT/AD, KI/WT, and KI/AD animals and specific HA‐peptide eluates (Eluate) of HA immunoaffinity‐purified samples were analyzed by SDS/PAGE and Western blotting (WB) with antibodies to the indicated proteins. Black arrows indicate RanGAP1, stars indicate nonspecific bands, arrowhead indicates free SUMO1 peptide, and bracket indicates His6‐HA‐SUMO1 conjugates
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Collected and cropped from Aging Cell by CiteAb, provided under a CC-BY license
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In PLoS One on 7 September 2017 by Park, H. J., Ryu, D., et al.
Fig.4.D
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WB
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Homo sapiens (Human)
Proteasome inhibition rescues αSyn levels.(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with ...
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Proteasome inhibition rescues αSyn levels.(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with its effects on macroautophagy and the lysosome. (B) Chloroquine treatment blocks autophagy activity. Cells were treated with 10–100 μM Chlorquine for 24 h. Lipidated and sequesterted LC3-II increased by chloroquine treatment (top panel). Cells were transfected with GFP-LC3 and 24 h post transfection, cells were treated with 100 μM Chloroquine for 24 h. Diffused pattern of LC3-I decreased and punctated pattern of LC3-II increased by Chloroquine treatment (bottom panel; scale bar = 10 μm). (C) Cells were co-transfected with αSyn and either EGFP or RER1, and then 24h post transfection treated with DMSO, 100 nM Bafilomycin (Baf), 10 μM MG132 (MG), or 10 μM Eeyarestatin1 (Eer1). In cells co-transfected with EGFP control, MG132 did not increase αSyn levels compared to cells exposed to DMSO. In cells co-transfected with RER1, MG132 showed a similar partial recovery of RER1-mediated αSyn reduction (mean, 69.2%), whereas the macroautophagy and ERAD inhibitors, Bafilomycin and Eeyarestatin1, had no apparent effect (**p<0.01, ***p<0.001; F2,6 = 28, p = 0.0009; n = 3/group). The right 2 lanes (separated by dotted line) show lysates from cells co-transfection of αSyn with RER1Δ25 for comparison. (D) RER1 decreases the levels of αSyn K80R mutant significantly. αSyn wild type or K80R mutant was co-transfected with RER1 or EGFP into HEK293 cells. R = RER1 transfected; G = EGFP. (E) RER1 interacts with NEDD4. RER1 is co-immunoprecipitated with NEDD4 in HEK293 cells co-expressing RER1 and NEDD4. N.C. = negative control using nonreactive serum.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 7 September 2017 by Park, H. J., Ryu, D., et al.
Fig.4.C
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WB
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Homo sapiens (Human)
Proteasome inhibition rescues αSyn levels.(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with ...
more
Proteasome inhibition rescues αSyn levels.(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with its effects on macroautophagy and the lysosome. (B) Chloroquine treatment blocks autophagy activity. Cells were treated with 10–100 μM Chlorquine for 24 h. Lipidated and sequesterted LC3-II increased by chloroquine treatment (top panel). Cells were transfected with GFP-LC3 and 24 h post transfection, cells were treated with 100 μM Chloroquine for 24 h. Diffused pattern of LC3-I decreased and punctated pattern of LC3-II increased by Chloroquine treatment (bottom panel; scale bar = 10 μm). (C) Cells were co-transfected with αSyn and either EGFP or RER1, and then 24h post transfection treated with DMSO, 100 nM Bafilomycin (Baf), 10 μM MG132 (MG), or 10 μM Eeyarestatin1 (Eer1). In cells co-transfected with EGFP control, MG132 did not increase αSyn levels compared to cells exposed to DMSO. In cells co-transfected with RER1, MG132 showed a similar partial recovery of RER1-mediated αSyn reduction (mean, 69.2%), whereas the macroautophagy and ERAD inhibitors, Bafilomycin and Eeyarestatin1, had no apparent effect (**p<0.01, ***p<0.001; F2,6 = 28, p = 0.0009; n = 3/group). The right 2 lanes (separated by dotted line) show lysates from cells co-transfection of αSyn with RER1Δ25 for comparison. (D) RER1 decreases the levels of αSyn K80R mutant significantly. αSyn wild type or K80R mutant was co-transfected with RER1 or EGFP into HEK293 cells. R = RER1 transfected; G = EGFP. (E) RER1 interacts with NEDD4. RER1 is co-immunoprecipitated with NEDD4 in HEK293 cells co-expressing RER1 and NEDD4. N.C. = negative control using nonreactive serum.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
Image 1 of 20
In PLoS One on 7 September 2017 by Park, H. J., Ryu, D., et al.
Fig.4.A
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WB
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Homo sapiens (Human)
Proteasome inhibition rescues αSyn levels.(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with ...
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Proteasome inhibition rescues αSyn levels.(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with its effects on macroautophagy and the lysosome. (B) Chloroquine treatment blocks autophagy activity. Cells were treated with 10–100 μM Chlorquine for 24 h. Lipidated and sequesterted LC3-II increased by chloroquine treatment (top panel). Cells were transfected with GFP-LC3 and 24 h post transfection, cells were treated with 100 μM Chloroquine for 24 h. Diffused pattern of LC3-I decreased and punctated pattern of LC3-II increased by Chloroquine treatment (bottom panel; scale bar = 10 μm). (C) Cells were co-transfected with αSyn and either EGFP or RER1, and then 24h post transfection treated with DMSO, 100 nM Bafilomycin (Baf), 10 μM MG132 (MG), or 10 μM Eeyarestatin1 (Eer1). In cells co-transfected with EGFP control, MG132 did not increase αSyn levels compared to cells exposed to DMSO. In cells co-transfected with RER1, MG132 showed a similar partial recovery of RER1-mediated αSyn reduction (mean, 69.2%), whereas the macroautophagy and ERAD inhibitors, Bafilomycin and Eeyarestatin1, had no apparent effect (**p<0.01, ***p<0.001; F2,6 = 28, p = 0.0009; n = 3/group). The right 2 lanes (separated by dotted line) show lysates from cells co-transfection of αSyn with RER1Δ25 for comparison. (D) RER1 decreases the levels of αSyn K80R mutant significantly. αSyn wild type or K80R mutant was co-transfected with RER1 or EGFP into HEK293 cells. R = RER1 transfected; G = EGFP. (E) RER1 interacts with NEDD4. RER1 is co-immunoprecipitated with NEDD4 in HEK293 cells co-expressing RER1 and NEDD4. N.C. = negative control using nonreactive serum.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 7 September 2017 by Park, H. J., Ryu, D., et al.
Fig.2.C
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WB
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Homo sapiens (Human)
RER1 expression significantly reduces αSyn levels.(A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expressi...
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RER1 expression significantly reduces αSyn levels.(A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expression. (B) Wild type RER1 expression significantly decreased αSyn levels (mean, 87.8 ±7.1%), but expression of RER1Δ25 resulted in significantly smaller effects on levels of αSyn (mean, 24.3 ±10.6%) (*p<0.05, **p<0.01; F2,6 = 17.31, p = 0.0032) (n = 5/control, n = 7/RER1, n = 3/ RER1Δ25) in HEK293. RER1 also decreased αSyn levels in human neuroglioma H4 cells (middle panel). (C) αSyn levels in both 1% Triton X-100 and 2% SDS fractions were decreased by RER1 overexpression (*p<0.05, **p<0.01; F2,9 = 14.02, p = 0.0017) (n = 4/group). C = control; R = RER1 transfected; Δ25 = RER1Δ25. Scale bar is 40 μm.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 7 September 2017 by Park, H. J., Ryu, D., et al.
Fig.2.B
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WB
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Homo sapiens (Human)
RER1 expression significantly reduces αSyn levels.(A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expressi...
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RER1 expression significantly reduces αSyn levels.(A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expression. (B) Wild type RER1 expression significantly decreased αSyn levels (mean, 87.8 ±7.1%), but expression of RER1Δ25 resulted in significantly smaller effects on levels of αSyn (mean, 24.3 ±10.6%) (*p<0.05, **p<0.01; F2,6 = 17.31, p = 0.0032) (n = 5/control, n = 7/RER1, n = 3/ RER1Δ25) in HEK293. RER1 also decreased αSyn levels in human neuroglioma H4 cells (middle panel). (C) αSyn levels in both 1% Triton X-100 and 2% SDS fractions were decreased by RER1 overexpression (*p<0.05, **p<0.01; F2,9 = 14.02, p = 0.0017) (n = 4/group). C = control; R = RER1 transfected; Δ25 = RER1Δ25. Scale bar is 40 μm.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In Dis Model Mech on 1 May 2017 by Lahut, S., Gispert, S., et al.
Fig.1.C
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WB
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Mus musculus (House mouse)
Large kindred with autosomal dominant PD inheritance attributable to PARK4 mutation with 12 presymptomatic PARK4 heterozygotes. (A) Pedigree structure with genotype of SNCA gene duplication. Squares and circles denote male and female individuals, respectively. Black filling versus the black symbo...
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Large kindred with autosomal dominant PD inheritance attributable to PARK4 mutation with 12 presymptomatic PARK4 heterozygotes. (A) Pedigree structure with genotype of SNCA gene duplication. Squares and circles denote male and female individuals, respectively. Black filling versus the black symbol with ‘+’ inside illustrates clinically manifested PD versus presymptomatic PARK4 heterozygote status, respectively. The letter ‘N’ indicates the individuals genotyped and found not to carry the PARK4 mutation. Samples from the rest of the individuals were unavailable to us; therefore, their status was indicated according to the information gained from the family. (B) DNA from the Turkish PARK4 family exhibits duplications at the SNCA locus. The analysis of the chromosome 4q22.1-22.2 region by molecular karyotyping using Agilent array CGH revealed two adjacent copy number gains on chromosome 4 (left chromosome view, right gene view). The first duplication, 833-862 kb in size, carries GPRIN3, SNCA and MMRN1 in their entirety. The second duplication, in close proximity, affects exons 7-13 of the GRID2 gene. The genomic position coordinates as annotated by hg19 nomenclature are indicated above for both chromosome 4 fragments that exist in three copies. (C) PARK4 blood shows upregulation of α-synuclein monomer, but no high molecular weight aggregates in the limited mobility zone of gels. Whole blood protein extracts were depleted in hemoglobin, analyzed on polyacrylamide blots subjected to delipidation, then studied for α-synuclein (above) and β-actin (below, as a loading control) immunoreactivity.
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Collected and cropped from Dis Model Mech by CiteAb, provided under a CC-BY license
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In Dis Model Mech on 1 May 2017 by Lahut, S., Gispert, S., et al.
Fig.5.A
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Homo sapiens (Human)
Accumulation of complexin 1 protein and downregulation of CPLX1 mRNA levels after overexpression of WT SNCA in SH-SY5Y cells. Human SH-SY5Y neuroblastoma cells were transiently transfected with plasmid pcDNA3.1(+) as an empty vector control or WT SNCA. (A) Immunoblot confirmation of successful SN...
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Accumulation of complexin 1 protein and downregulation of CPLX1 mRNA levels after overexpression of WT SNCA in SH-SY5Y cells. Human SH-SY5Y neuroblastoma cells were transiently transfected with plasmid pcDNA3.1(+) as an empty vector control or WT SNCA. (A) Immunoblot confirmation of successful SNCA overexpression and of the effect on complexin 1 protein levels on the second day after transfection, using GAPDH and β-actin as the loading controls (n=3 versus n=3 versus n=3 in independent experiments). (B) Densitometric quantification of complexin 1 (antibody from Synaptic systems) versus ACTB ratios, normalized against control, in a scattergram analysis with t-test. (C) Analysis of corresponding CPLX1 mRNA levels at 2 days after transfection. Asterisks indicate statistical significance versus control group (n=6 versus n=3 versus n=12). *P<0.05, **P<0.01. ACTB, β-actin; α-Syn, α-synuclein; WT, wild type.
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Collected and cropped from Dis Model Mech by CiteAb, provided under a CC-BY license
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In Sci Rep on 13 November 2015 by Ninkina, N., Connor-Robson, N., et al.
Fig.2.A
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WB
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Mus musculus (House mouse)
Restoration of α-synuclein expression in the cerebral cortex of floxed mice following germline deletion of neo-cassette.Western blot analysis of total protein samples extracted from the cerebral cortex of wild type mice (SNCA+/+), α-synuclein knockout mice (SNCA−/−, described previously19), floxe...
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Restoration of α-synuclein expression in the cerebral cortex of floxed mice following germline deletion of neo-cassette.Western blot analysis of total protein samples extracted from the cerebral cortex of wild type mice (SNCA+/+), α-synuclein knockout mice (SNCA−/−, described previously19), floxed mice before deletion of neo-cassette (SNCAflox(neo)/flox(neo)) and floxed mice after deletion of neo-cassette (SNCAfloxΔneo/floxΔneo).
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In PLoS One on 8 July 2014 by Aldrin-Kirk, P., Davidsson, M., et al.
Fig.10.A
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IHC-IF
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Rattus norvegicus (Rat)
Morphological changes and evidence of α-synuclein uptake in IBA-1 positive microglia.Immunohistochemical staining of IBA-1 using DAB precipitation coloring in the cerebral cortex revealed a substantial change in the microglial morphology in the cortex of AAV6|a-Syn animals compared to AAV6|GFP an...
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Morphological changes and evidence of α-synuclein uptake in IBA-1 positive microglia.Immunohistochemical staining of IBA-1 using DAB precipitation coloring in the cerebral cortex revealed a substantial change in the microglial morphology in the cortex of AAV6|a-Syn animals compared to AAV6|GFP and Intact ctrl animals at 10 months post AAV injection (A–D). In the regions overlapping with the most intense human α-synuclein over-expression, most microglial cells appeared with very few visible processes and majority of the IBA-1 positive staining localized to the nucleus (A), this stood in stark contrast to the normal IBA-1 staining of microglia in the corresponding cortical regions of AAV6|GFP animals (B). In surrounding cortical regions, the microglial morphology in AAV6|a-Syn animals showed a second distinct morphology, with elongated cell body and fewer processer, suggesting a migratory profile (C). Using double-labeled, fluorescence with IBA-1 and human α-synuclein/GFP antibodies in a scanning confocal microscope, the presence of α-synuclein in microglia was investigated (E–F″). At the core of the human α-synuclein over-expression in the AAV6|a-Syn animals, microglia were found with significantly changes morphology and intra-cellular α-synuclein (E–E″). Interestingly, two localization patterns were observed, both in the cell soma in circular, vesicular structures and as small accumulations in distal processes of the microglia. No such patterns were observed in AAV6|GFP (F–F″) or intact controls.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 8 July 2014 by Aldrin-Kirk, P., Davidsson, M., et al.
Fig.9.C
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WB
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Rattus norvegicus (Rat)
Quantification of cholinergic neurons regions with high expression of human α-synuclein.Immunohistochemical staining of ChAT positive Cholinergic interneurons using DAB precipitation coloring of the striatum and cerebral cortex revealed a significant, progressive loss in both the striatum and the...
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Quantification of cholinergic neurons regions with high expression of human α-synuclein.Immunohistochemical staining of ChAT positive Cholinergic interneurons using DAB precipitation coloring of the striatum and cerebral cortex revealed a significant, progressive loss in both the striatum and the medial lateral Cerebral cortices starting later than 8(8 wks) no loss of cholinergic neurons was apparent in either the striatum (A) or the cerebral cortices (B). However, ChAT + interneurons were qualitatively lighter stained with fewer processes clearly visible (C–D) relative to GFP injected animals (E–F). At 10 months post viral injection, however, there was a significant reduction on Cholinergic interneurons in both Striatum (G) and Cortex (H) of AAV6|a-Syn animals compared to both AAV6|GFP and Intact ctrl animals. At this time point, ChAT staining revealed a high degree of clearance of cholinergic neurons within the striatum (I) and medial lateral cerebral cortices (J), relative to GFP injected animals (K–L). (* = p<0.05, one-way ANOVA with Newman-Keuls post hoc test). Data is expressed as mean ± SEM.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 17 November 2011 by Maarouf, C. L., Daugs, I. D., et al.
Fig.6.A
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WB
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Homo sapiens (Human)
Western blots of tau, ApoE, α-synuclein and ApoJ.A total of 20 µg of total protein was loaded into each lane for the ApoE blot, while all other blots contained 40 µg of total protein. Unpaired, 2-tailed t-tests were used for statistical analysis. The primary and secondary antibody descriptions an...
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Western blots of tau, ApoE, α-synuclein and ApoJ.A total of 20 µg of total protein was loaded into each lane for the ApoE blot, while all other blots contained 40 µg of total protein. Unpaired, 2-tailed t-tests were used for statistical analysis. The primary and secondary antibody descriptions and sources are given in Table 2. The relative abundance of the proteins were densitometrically estimated. Each blot was reprobed with actin as a loading control, as shown at the bottom of each primary antibody Western blot. The numbers to the left of the Western blots are the molecular weight in kDa. HPC = non-demented high pathology controls; AD = Alzheimer's disease; exp = exposure.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 17 November 2011 by Maarouf, C. L., Daugs, I. D., et al.
Fig.7.A
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WB
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Homo sapiens (Human)
Western blots of VEGF, PEDF, BDNF, TDP-43, synaptophysin and S100B.Forty µg of total protein was loaded onto VEFG, BDNF and TDP-43 acrylamide gels. The PEDF and synpaptophysin blots contained 20 µg and 10 µg of total protein, respectively. Unpaired, 2-tailed t-tests were used for statistical anal...
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Western blots of VEGF, PEDF, BDNF, TDP-43, synaptophysin and S100B.Forty µg of total protein was loaded onto VEFG, BDNF and TDP-43 acrylamide gels. The PEDF and synpaptophysin blots contained 20 µg and 10 µg of total protein, respectively. Unpaired, 2-tailed t-tests were used for statistical analysis. The primary and secondary antibody descriptions and sources are given in Table 2. The relative abundance of the proteins were densitometrically estimated. Each blot was reprobed with actin as a loading control, as shown at the bottom of each primary antibody Western blot. The numbers to the left of the Western blots are the molecular weight in kDa. HPC = non-demented high pathology controls; AD = Alzheimer's disease; exp = exposure.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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