asyn epitope 1 20  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a. Seeding model in primary hippocampal neurons. 70 nM of mouse PFFs were added to neurons at DIV 5 (day in vitro ). Control neurons were treated with Tris buffer used to prepare PFFs. After 4 days of treatment, positive pS129-aSyn aggregates were detected in the extension of the neurons. After 7 days of treatment, the aggregates appeared in the cytosol of the neurons. The number of LB-like inclusions increased over time, as shown at 10 days of treatment. Scale bars = 20 μm. b-e. ICC analysis of the LB-like inclusions that formed at 10 days after adding mouse PFFs to aSyn KO neurons (b) or to WT neurons (c-e). Aggregates were detected using pS129 (MJFR13) in combination with total aSyn (SYN-1), p62, or ubiquitin antibodies. Neurons were counterstained with microtubule-associated protein (MAP2) antibody, and the nucleus was counterstained with DAPI staining. Scale bars = 5 μm. f-g. WB analyses of the insoluble fraction of PFFs-treated WT neurons ( f ) or PFFs-treated KO neurons ( g ). Control neurons were treated with Tris buffer (Tris). After sequential extractions of the soluble and insoluble fractions, cell lysates were analyzed by immunoblotting. Total aSyn, pS129 and actin were respectively detected by SYN-1, pS129 (MJFR13), and actin antibodies. Levels of total aSyn (15 kDa, indicated by a double red asterisk; 12 kDa indicated by a single red asterisk or HMW) or pS129-aSyn were estimated by measuring the WB band intensity and normalized to the relative protein levels of actin. Purple arrows indicate the intermediate aSyn-truncated fragments. The graphs represent the mean +/-SD of 3 independent experiments. ( f ) *p<0.01, **p<0.001, ***p<0.0001* (ANOVA followed by Tukey HSD post-hoc test, Tris vs. PFFs-treated neurons) and # p<0.01, ## p<0.001 (ANOVA followed by Tukey HSD post-hoc test, PFFs-treated neurons D10 vs. D7 or D4 or D1). ( g ) *p<0.01, ***p<0.0001 (ANOVA followed by Tukey HSD post-hoc test, level of aSyn 15 kDa at 1 hour vs. other time-points or levels of aSyn 12 kDa at 1 hour vs. other time-points or Tris vs. PFFs-treated neurons). h-i. Insoluble fractions of aSyn KO primary neurons treated with 70 nM of mouse PFFs for 4 or 14 hours were separated on a 16% Tricine gel. After Coomassie staining, two bands at ∼15 (indicated by a black dashed box) and 12 kDa (indicated by a purple dashed box) were extracted from 16% Tricine gels (See Figure S4). Isolated bands were selected based on the size of the proteolytic fragments observed by WB ( h ) and subjected to proteolytic digestion followed by LC-MS/MS analysis. Proteomic analysis showed that aSyn fragments produced in KO neurons transduced with PFFs result from C-terminal truncation but not from N-terminal cleavage of the PFF seeds. The diagram in i shows the different aSyn fragments generated upon C-terminal truncation and their relative position in a WB. Three fragments (1-135, 1-129, and 1-119) were detected in the upper band sliced, and one main fragment (1-114) was found in the lower band. j. Epitope mapping of antibodies raised against the NAC, N-terminal, or C-terminal domains of aSyn. k. N-terminal antibodies raised against residues 1-5 or residues 1-20 could detect full-length (15 kDa, indicated by a double red asterisk) or truncated (∼12 kDa indicated by a single red asterisk) aSyn in the insoluble fraction of KO neurons treated for 14 hours, confirming that the N-terminal region of aSyn PFF seeds is intact after internalization into the neurons. l. Mapping of the C-terminal cleaved product using antibodies raised against the NAC and the C-terminal domains of aSyn. Immunoblots of insoluble fractions of KO neurons treated with aSyn PFFs showed that the fragment 1-114 generated in these neurons was well recognized by the NAC antibodies [(FL-140; 61-95) and (SYN-1; 91-99)] and a C-terminal antibody raised against the residues 108-120. However, it was not recognized by antibodies raised against peptides bearing residues after 116 in the C-terminal domain [(ab6162; 116-131); (ab131508; 134-138) and (ab52168; 131-135)]. Altogether, our data demonstrate that after internalization, aSyn PFF seeds are efficiently C-terminally truncated before the initiation of the intracellular seeding mechanisms.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: In Vitro, Control, Staining, Western Blot, Isolation, Liquid Chromatography with Mass Spectroscopy, Produced, Transduction, Generated

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a-b. aSyn KO neurons were treated for up to 72 hours with WT fluorescently labelled PFFs 488 . The internalization and the truncation of the seeds were evaluated by confocal imaging. a. One hour after addition to the KO neurons, we observed that most of the intracellular PFFs 488 were co-stained by an antibody raised against the extremity of aSyn C-terminal domain (epitope: 134-138, yellow arrows). C-terminal truncation of the seeds over time was confirmed by the loss of detection of the seeds by the C-terminal aSyn antibody (134-138, red; green arrows). b. The internalization of the seeds via the endo-lysosomal pathway was confirmed by the detection of the fluorescently labelled PFF 488 seeds in LAMP1-positive (late endosome, red) compartments overtime. a-b. Neurons were counterstained with MAP2 antibody and the nucleus with DAPI stain. Scale bars = 10 μm. c. Cathepsin B activity was measured in KO neurons treated with 70 nM of WT PFFs seeds for up to 48 hours. Control neurons were treated with Tris buffer. The graphs represent the mean +/-SD of 3 independent experiments. *p<0.01, **p<0.001, ***p<0.0001 (ANOVA followed by Tukey HSD post-hoc test, Tris vs. PFFs-treated neurons). d. Truncation of aSyn PFFs in the cytosol is confirmed by microinjection. The diagram on the left-hand side shows the experimental approach used to microinject WT PFFs 488 in KO neurons. Cells were fixed after 24 hours and immunostained using the N-terminus antibody (aSyn 1-20) or C-terminus antibody (aSyn 134-138). Confocal imaging showed that WT PFFs 488 were detected by the N-terminus antibody (yellow arrows, merge), but not the C-terminus antibody (green arrows, merge). Neurons were counterstained with MAP2 antibody and the nucleus with DAPI stain. Scale bars = 40 μm.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: Imaging, Staining, Activity Assay, Control, Microinjection

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a-g. Identification of C-terminal truncated fragments by proteomic analysis ( a-b ), WB ( c and ) or confocal imaging ( d-g ). WT neurons were treated with 70 nM of mouse PFFs for 10 days. a-b. The insoluble fractions of PFFs-treated neurons were separated on a 16% Tricine gel. After Coomassie staining, 8 bands were extracted from the Tricine gel ( a ). Isolated bands were subjected to proteolytic digestion using trypsin for C-terminal truncation identification , followed by LC-MS/MS analysis. b. Proteomic analyses showed the presence of the 1-114 and 1-119 C-terminal truncated fragments in the HMW species. c. Table summarizing the capacity of NAC domain, N-terminal, and C-terminal antibodies to detect full-length aSyn (15 kDa), the C-terminally cleaved fragment of aSyn (∼12 kDa), and the HMW formed in WT neurons after 10 days of treatment with PFFs (see WB in ). d-g . Antibody mapping of the newly formed inclusions by confocal imaging using pS129 antibody (81a clone) in combination with N-terminal ( d , epitope 1-20) or C-terminal ( e-g , respective epitopes 108-120, 116-131, or 134-138) antibodies revealed the presence of aSyn-positive aggregates that were not pS129 positive or only partially phosphorylated at S129 residue ( e-f ). Neurons were counterstained with MAP2 antibody and the nucleus with DAPI stain. The white arrows indicate the sub-populations of aggregates localized near the pS129-positive inclusions, and the white asterisk those inside the pS129-positive filamentous structures. Scale bars = 10 μm.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: Imaging, Staining, Isolation, Liquid Chromatography with Mass Spectroscopy, Residue

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a. WB analyses of the truncation pattern of aSyn in human brain tissue from MSA patients and healthy controls. After sequential extractions of the soluble and insoluble fractions, cell lysates were analyzed by immunoblotting. The levels of total aSyn (1-20, SYN-1 or 134-138 antibodies) or pS129 aSyn were estimated by measuring the WB band intensity and normalized to the relative protein levels of actin. The 15 kDa band is indicated by a double red asterisk, the 12 kDa band by a single red asterisk, and the purple arrows indicate the intermediate aSyn-truncated fragments. b-c. Serial sections from the midbrains of PDD (pars compacta) and SNCA duplication (tegmentum) cases were stained with aSyn antibodies raised specifically against the N-terminal (epitope: 1-20), the NAC (91-99), the C-terminal (epitopes: 110-115 and 134-138) or pS129 (EP1536Y) regions. Scale bars = 50 μm.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: Western Blot, Staining

Journal: Acta Neuropathologica Communications

Article Title: Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders

doi: 10.1186/s40478-022-01468-8

Figure Lengend Snippet: The star-like astrocytic aSyn accumulations in the DLB entorhinal cortex (EC). a The EC of DLB and CTR brains were immunohistochemically stained using aSyn antibodies with epitopes against the N-terminus (LASH-EGTNter 1–20 and LASH-BL 34–45), the NAC region (LASH-BL 80–96 and BD SYN-1 91–99) and the C-terminus (2F10-E12 110–115 and AB 134–138) of aSyn. The extreme N-terminal antibody LASH-EGTNter as well as the C-terminal antibodies 2F10-E12 and AB 134–138 showed neuronal pathology in the soma and neurites. The late N-terminal antibody LASH-BL 34–45 as well as the two NAC region antibodies LASH-BL 80–96 and BD SYN-1 were positive for LBs and LNs, but also distinctively detected star-shaped glial aSyn species (insets). Representative images taken from the cortical deep grey matter (layers V–VI) of DLB2 and CTR1. b The aSyn PTM antibodies against phosphorylation and nitration at Tyrosine 39 (Y39) were also reactive to the star-like astroglial pattern (insets). Representative images taken from the cortical deep grey matter (layers V–VI) of DLB2 and CTR2. c Star-like aSyn species are associated with the GFAP-positive astrocytes in the DLB brains as shown by IF using antibodies for astrocytic and neuronal markers GFAP and NF, and LASH-BL 34–45 antibody against aSyn. The star-like aSyn species (arrows) appeared in and around the GFAP-positive astrocytes, and not in the LNs. Representative images from DLB1 cingulate cortex. Images on the upper panel taken using Olympus slide scanner at 40 × magnification, and the lower panel on Zeiss LSM700 confocal microscope. Scale bar for Fig. 1a, b is 20 µm for the main images and 40 µm for the insets. aSyn = alpha-synuclein; CTR = control; DLB = dementia with Lewy bodies; EC = entorhinal cortex; GFAP = glial fibrillary acidic protein; IF = immunofluorescence; LB = Lewy body; LN = Lewy neurite; NAC = non-amyloid component; NF = neurofilament; PTM = post-translational modification

Article Snippet: Fig. 1 The star-like astrocytic aSyn accumulations in the DLB entorhinal cortex (EC). a The EC of DLB and CTR brains were immunohistochemically stained using aSyn antibodies with epitopes against the N-terminus (LASH-EGTNter 1–20 and LASH-BL 34–45), the NAC region (LASH-BL 80–96 and BD SYN-1 91–99) and the C-terminus (2F10-E12 110–115 and AB 134–138) of aSyn.

Techniques: Staining, Phospho-proteomics, Nitration, Microscopy, Control, Immunofluorescence, Modification

Journal: Acta Neuropathologica Communications

Article Title: Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders

doi: 10.1186/s40478-022-01468-8

Figure Lengend Snippet: Astrocytic aSyn accumulations occur across LB disorders and may be truncated in the N- and C-termini. a CTR, PD, PDD, DLB, SNCA G51D mutation and SNCA duplication cingulate cortices were immunohistochemically stained using three aSyn antibodies, LASH-BL 34–45, LASH-BL 80–96 and BD SYN-1, and astrocytic accumulations (insets) were revealed across these LB disorders. b To further map the C-terminal truncation region of the astrocytic aSyn, the same cingulate cortex sections were stained using the C-terminal BL 4B12 antibody with an epitope 103–108 of aSyn. Neuronal inclusions were revealed, but the astrocytic aSyn accumulation was not detected, suggesting that the aSyn species associated with the astrocytes are truncated at residues 21–33 in the N-terminus, and at residues 100–102 in the C-terminus. Representative images in Fig. 3a, b taken from the cortical deep grey matter (layers V–VI) of CTR1, PD2, PDD2, DLB1, SNCA G51D3 and SNCA duplication. c A diagram to show the antibodies that are positive and negative for astrocytic aSyn, and their epitopes. The areas in stripes denote the potential truncation regions in the N- and C-termini. Schematic created with BioRender.com (agreement no: DJ23GJF70T ). Scale bar for Fig. 3a is 20 µm for the main images and 40 µm for the insets. aSyn = alpha-synuclein; CTR = control; DLB = dementia with Lewy bodies; LB = Lewy body; PD = Parkinson’s disease; PDD = Parkinson’s disease with dementia

Article Snippet: Fig. 1 The star-like astrocytic aSyn accumulations in the DLB entorhinal cortex (EC). a The EC of DLB and CTR brains were immunohistochemically stained using aSyn antibodies with epitopes against the N-terminus (LASH-EGTNter 1–20 and LASH-BL 34–45), the NAC region (LASH-BL 80–96 and BD SYN-1 91–99) and the C-terminus (2F10-E12 110–115 and AB 134–138) of aSyn.

Techniques: Mutagenesis, Staining, Control