Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a The diagram depicts the workflow of our study to assess the background and specificity of commercial and homemade pS129 antibodies using recombinant aSyn monomers and fibrils (WT or carrying specific PTMs such as nitration, phosphorylation and/or truncation), mammalian cell lines overexpressing aSyn, aSyn KO primary neurons, WT neurons treated with aSyn PFFs or WT mice injected with aSyn PFFs. The background and specificity of the selected pS129 antibodies were evaluated by Western blot analyses or ICC or HTS combined with confocal imaging. Panel A was created using BioRender.com. b The diagram depicts aSyn human sequence, the epitopes of each of the pS129 antibodies (whenever available), and the post-translational modifications that can occur concomitantly in the C-terminal region of aSyn. c The diagram depicts truncations that occurs in the different regions of aSyn in human brains and the appendix.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Recombinant, Nitration, Injection, Western Blot, Imaging, Sequencing

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: The capacity of human or mouse recombinant monomeric aSyn (WT or modified by specific PTMs) to be detected by pS129 commercial antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, and GTX) or the LASH-EGT homemade pS129 antibody was assessed by WB ( a , b , d ) or ELISA ( c ). a , b Forty nanograms of human recombinant monomeric aSyn unphosphorylated (WT) or phosphorylated at the S129 residue (pS129) or di-phosphorylated (pY125/pS129) were detected by WB using total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein) combined with either a commercial (MJF-R13 or pSyn#64) and homemade (LASH-EGT) pS129 antibodies or b commercial pY125 antibody (BD) or the homemade antibody LASH-EGT-pY125. c ELISA confirmed that MJF-R13, but not 81A, is no longer able to detect pS129-aSyn when aSyn was di-phosphorylated (pY125/pS129). d , e One-hundred nanograms of unmodified aSyn (WT) or phosphorylated aSyn at S129 and/or Y125 residues (respectively named pS129, pY125 or pS129/pY125) full length or truncated after residues 133 (1-133/pS129) or 135 (1-135/pS129) were detected by WB using pS129 antibodies ( d ) or total aSyn antibodies (SYN-1 or LASH-EGT 1-20) ( e ). All blots were derived from the same experiment and were processed in parallel.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Recombinant, Modification, Enzyme-linked Immunosorbent Assay, Residue, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a Preparation of aSyn fibrils carrying both phosphorylation and nitration modifications. aSyn fibrils were first site-specifically phosphorylated at S129 residue by being incubated with PLK3. Afterwards, TNM was added to induce the nitration of the phosphorylated aSyn fibrils. b The capacity of aSyn fibrils site-specifically phosphorylated at S129 and/or nitrated to be detected by the pS129 commercial antibodies (pSyn#64, MJF-R13, 81A, and EP1536Y) or the LASH-EGT homemade pS129 antibody was assessed by slot blot ( c ) and WB ( d ) analyses. a , b were created using BioRender.com. c , d Thirty-six nanograms of aSyn fibrils (PFFs) that were unmodified (WT), phosphorylated (P), nitrated (N), or nitrated/phosphorylated (N/P) were detected by slot blot ( c ) and WB ( d ) using pS129 antibodies (top panel) in combination with total aSyn antibodies (1–20 and 34–45, bottom panel). All blots were derived from the same experiment and were processed in parallel.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Nitration, Residue, Incubation, Dot Blot, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: After 14 days of treatment with 70 nM of mouse aSyn PFFs or PBS buffer (negative control), primary hippocampal neurons were fixed or lysed, and ICC ( a – c ) or WB ( d ) analyses were performed using pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, and GTX or LASH-EGT). a – c Newly formed fibrils were detected by confocal imaging ( a ) and quantified by a high-throughput wide-field cell imaging system as previously described . b , c Neurons were counterstained with MAP2 antibody and the nucleus with DAPI staining. Scale bar = 10 μM. b The left-hand side part of the histogram shows the background of each pS129 antibody in PBS-treated primary neurons, while the right-hand side part of the histogram shows the pS129 level detected by the pS129 antibodies in PFFs-treated primary neurons. c pS129 level in PFF-treated primary neurons was re-evaluated after subtracting the pS129 background level from the PBS-treated neurons. The graphs represent the mean +/− SD of three independent experiments. * p < 0.01, *** p < 0.0001 (ANOVA followed by Tukey HSD post hoc test, PBS vs. PFF-treated neurons). d WB analyses of the insoluble fractions of the PBS- and PFF-treated neurons. Membranes were then counterstained by total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein), and actin was used as a loading control. The red arrows indicate the pS129-aSyn positive bands. The blue arrows indicate the undefined bands. All blots were derived from the same experiment and were processed in parallel.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Negative Control, Imaging, High Throughput Screening Assay, Staining, Control, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a , b Sequential biochemical extraction was performed on aSyn KO and WT primary hippocampal neurons cultured for 14 days (DIV14), and the pS129 level was assessed in the nuclear (N), cytosolic (C), and membrane (M) fractions ( a ) or in the soluble (sol) and insoluble (insol) fractions ( b ) by WB using the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Forty nanograms of recombinant aSyn WT or phosphorylated at residue S129 (pS129, indicated by the red arrow) were used as positive controls. The pS129 antibodies were used at a dilution of 1/1000. Membranes were then counterstained by total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein). aTubulin, Laminin B, and E-Cadherin were used as loading control for the cytosolic, nuclear, and membrane fractions, respectively ( a ). Actin was used as a loading control for the soluble and insoluble fractions ( b ). All blots were derived from the same experiment and were processed in parallel.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Extraction, Cell Culture, Membrane, Recombinant, Residue, Control, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a , b After 21 days of treatment with 70 nM of human E83Q aSyn PFFs, primary cortical neurons were fixed, and ICC was performed. The seeded-aggregates were detected using the pS129 antibodies (MJF-R13, GTX, 81A, or pSyn#64). Neurons were counterstained with the MAP2 antibody and the nucleus with DAPI staining. Scale bar = 10 μM. All four pS129 antibodies were able to distinguish the seeded-aggregates with the filamentous-like morphology and the LB-like inclusions with the dense core and the ring-like morphology. However, only the MJF-R13 and GTX antibodies were able to detect the nuclear, granular or granular and filamentous-like aggregates ( a ). The yellow arrows indicate the filamentous-like aggregates imbricated between the granular-like aggregates. The orthogonal view from different planes ( x / y , x / z , and y / z ) of the “nuclear-like” pS129-aSyn aggregates stained with MJF-R13 or GTX antibodies as well as the Imaris 3D animations (see Videos in SI files) confirmed their intranuclear localization. The schematic representation of the different types of seeded-aggregates was created using BioRender.com.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Staining

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a Brain coronal sections (50 μM thick) from different brain areas of aSyn KO mice (Amygdala, Striatum, Motor Cortex, Hippocampus and Substantia Nigra) have been examined to assess the different staining patterns of the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Scale bar = 100 μM. b pS129 level of detection in the soluble and insoluble fractions extracted from the aSyn KO mice brains using the six pS129 antibodies (pSyn#64, MJF-R13, 81a, EP1536Y, GTX, and LASH-EGT). Sequential biochemical extraction was performed on different brain regions (cortical/CTX; Midbrain/MID and Striatum/STR) derived from aSyn KO mice, and the level of pS129 was assessed in the soluble (0.1% Triton-soluble fraction) and the insoluble fraction (2% SDS-soluble fraction). 40 ng of recombinant aSyn WT or phosphorylated at residue S129 (pS129, indicated by the red arrow) were used as positive controls. Membranes were counterstained for total aSyn (SYN-1 or D37A6), and actin was used as a loading control. The red arrows indicate the pS129-aSyn-positive bands. The blue arrows indicate the undefined bands. All blots were derived from the same experiment and were processed in parallel.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Staining, Extraction, Derivative Assay, Recombinant, Residue, Control

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a Brain coronal sections (50 μM thick) from different brain areas of C57BL6/J PFF-injected mice (Amygdala, Striatum, Motor Cortex), 3 months post-injection, have been examined to assess the different staining patterns of the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Scale bar = 100 μM. Quantification for each area has been carried out using Qupath and was expressed as a percentage of positive area stained of total ROI. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 according to one-way ANOVA followed by the Tukey test for multiple comparisons. b Microphotographs of the amygdala were stained with the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX and LASH-EGT) at higher magnification. Scale bar = 10 μM.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Injection, Staining

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a List of peptides used to search the Uniprot database to identify putative proteins that could cross-react against antibodies raised against the known aSyn phosphorylated residues highlighted in bold at the middle of each sequence. The number of candidate hits retrieved for human, mouse, or common to both organisms is indicated for each peptide. The asterisk (*) present in the last sequence indicates the C-terminal residue. b Sequence alignment of the region around human aSyn pS129 and Neurofilament light polypeptide (NFL) pS472, which is known to cross-react with antibodies raised against pS129-aSyn. c Sequence alignment of the region around human aSyn pY133 and Actin-binding LIM protein 1 pY439, illustrating the high level of similarity. d List of the top ten candidate proteins that can cross-react with pS129 antibody.

Article Snippet: Nuclear and the “nuclear-like” pS129-aSyn aggregates segmentation and visualization were performed using Imaris version 9.9.1.

Techniques: Sequencing, Residue, Binding Assay

Journal: PLoS Biology

Article Title: The mechanism of sirtuin 2–mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease

doi: 10.1371/journal.pbio.2000374

Figure Lengend Snippet: (A) AAV6-mediated delivery of EGFP and mutant aSyn (KQ or KR) into the SN of the rat brain. TH and GFP or aSyn expression was examined in brain sections 3 wk after injection by immunohistochemistry (TH, red; GFP or aSyn, green; DAPI, blue). Representative sections are shown. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm. (B) Stereological counting of the number of TH-positive neurons in the SN. The contralateral SN of the different groups of animals was used as a control (intact). Data in panels are average ± SD. (C) Brain sections stained for aSyn (green), pS129 aSyn (red), and DAPI (Blue). Representative sections are shown. Dashed square boxes delineate the magnification presented on the right. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm and 50 μm. *** p < 0.001, **** p < 0.0001, one-way ANOVA with Bonferroni correction used for statistical calculations. In (B), GFP was used as a control; n = 6–7 animals per condition; five sections from a one-in-six series were analyzed per brain. Data in .

Article Snippet: The following antibodies were used to detect specific synucleins: N-terminal rat aSyn (Mouse, Bd Transduction Laboratories, 610787), recognized amino acid 121–125 of human aSyn (Mouse, Santa-Cruz, SC-12767), pS129 aSyn (Rabbit, Abcam, ab51253), and aggregated forms of aSyn (Mouse, Millipore, clone 5G4, MABN389).

Techniques: Mutagenesis, Expressing, Injection, Immunohistochemistry, Isolation, Staining

Journal: NPJ Parkinson's Disease

Article Title: The small GTPase Rit2 modulates LRRK2 kinase activity, is required for lysosomal function and protects against alpha-synuclein neuropathology

doi: 10.1038/s41531-023-00484-2

Figure Lengend Snippet: a Representative images showing pS129-aSyn immunostaining in SH-SY5Y, WT-LRRK2 and G2019S-LRRK2 cells with or without Rit2 overexpression. b Quantification pS129-aSyn in cell overexpressing either G2019S-LRRK2 alone or G2019S-LRRK2 with Rit2 ( n = 4). c PLA was used to quantify phosphorylation of Serine 1292 of LRRK2 in neuroblastoma cell lines. d The G2019S-LRRK2 mutation increases Serine 1292 phosphorylation, which is reduced by Rit2 overexpression or treatment with PF-475 LRRK2 kinase inhibitor ( n = 4). e CO-immunoprecipitation of LRRK2 using Rit2 antibody in mouse brain lysates. LRRK2 can be precipitated while bound to Rit2 and therefore, the two proteins are physically interacting with each other also at endogenous protein levels in the mouse brain ( n = 3). Data are represented as median, boxes show the IQ and whiskers show min–max. In imaging experiments, analysis was conducted on 700–1000 cells per group in each experiment. * p < 0.05, ** p < 0.01, **** p < 0.0001 two-tailed Student’s t test.

Article Snippet: Cells or tissue lysates for Western blotting were prepared as previously described in ref. . Primary antibodies used: anti-LRRK2 1:20,000 (Abcam, ab172378), anti-pS935-LRRK2 1:1000 (Abcam, ab172382), anti-pS1292-LRRK2 1:1000 (Abcam, ab206035), anti-LC3 1:1000 (Cell Signaling Technologies, 3868), anti-β-actin 1:6000 (Sigma-Aldrich, A-5316), anti-aSyn (Abnova, MAB5383), anti-Myc (9E10 hybridoma supernatant, 1:100), total aSyn (CST 2628), pS129-aSyn (WAKO, 015-25191).

Techniques: Immunostaining, Over Expression, Mutagenesis, Immunoprecipitation, Imaging, Two Tailed Test

Journal: NPJ Parkinson's Disease

Article Title: The small GTPase Rit2 modulates LRRK2 kinase activity, is required for lysosomal function and protects against alpha-synuclein neuropathology

doi: 10.1038/s41531-023-00484-2

Figure Lengend Snippet: a Total aSyn levels in mice injected with AAV-A53T-aSyn alone or in combination with AAV-Rit2 were assessed by blotting for total and phosphorylated aSyn and β-actin. b Quantification of total aSyn levels, normalized on β-actin ( n = 4). Co-injection of AAV-Rit2 significantly reduces aSyn levels in the ipsilateral side. c Quantification of pS129-aSyn levels, normalized on β-actin (5 animals/group). Co-injection of AAV-Rit2 significantly reduces pS129-aSyn levels in the ipsilateral side. d Quantification of pS129-aSyn levels, normalized on total aSyn. Co-injection of AAV-Rit2 does not alter pS129-aSyn/aSyn ratio. e IHC staining of pS129-aSyn and TH in the midbrain of AAV-GFP, AAV-A53T-aSyn and AAV-A53T-aSyn + AAV-Rit2 injected mice. f Quantification of pS129-aSyn intensity. AAV-A53T-aSyn injection significantly increases the intensity of pS129-aSyn signal, which is reduced by the co-injection of AAV-Rit2 (5 animals/group). Scale bar = 20 um. Data represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by the Bonferroni’s post hoc test.

Article Snippet: Cells or tissue lysates for Western blotting were prepared as previously described in ref. . Primary antibodies used: anti-LRRK2 1:20,000 (Abcam, ab172378), anti-pS935-LRRK2 1:1000 (Abcam, ab172382), anti-pS1292-LRRK2 1:1000 (Abcam, ab206035), anti-LC3 1:1000 (Cell Signaling Technologies, 3868), anti-β-actin 1:6000 (Sigma-Aldrich, A-5316), anti-aSyn (Abnova, MAB5383), anti-Myc (9E10 hybridoma supernatant, 1:100), total aSyn (CST 2628), pS129-aSyn (WAKO, 015-25191).

Techniques: Injection, Immunohistochemistry