Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) The 25 a.a. peptide used to immunize the mice and location in the full-length protein. B) Western blot showing the specificity of each hybridoma clone selected on HEK cells lysate. Cells were transfected with either an aSyn encoding plasmid alone or in combination with a PLK2 kinase encoding plasmid. A plasmid encoding the mutated aSyn-S129G that cannot be phosphorylated at the serine 129 was also used. A condition with no transfection was used as a negative control. Commercial antibodies against total and pS129-aSyn were used as comparison (ab51253 against pS129-aSyn, and CST2628 against total aSyn). C) Representative images of immunofluorescence on HEK cells that were transfected with the plasmids described above using each hybridoma clone media as a primary antibody solution. Antibody-specific signal is shown in white, DAPI is shown in blue. Scale bar = 50μm.

Article Snippet: aSyn monomers (Proteos #RP-003), pS129-aSyn monomers (Proteos #RP-004) and BSA (Sigma #A9647) were manually blotted on nitrocellulose membranes.

Techniques: Western Blot, Transfection, Plasmid Preparation, Negative Control, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Representative images of immunofluorescence using our purified monoclonal antibodies on human Parkinson’s disease (n=3), and control (n=2) substantia nigra sections. Antibody-specific signal is shown in white while TH is shown in red. Commercial antibodies were used as a comparison (CST2628 against total aSyn, and BioLegend 825701 against pS129-aSyn). B) Representative images of immunofluorescence using our purified monoclonal antibodies on human MSA (n=3), DLB (n=3) and control (n=2) cortex sections. SN = Substantia nigra; MSA = multiple system atrophy; DLB = dementia with Lewy bodies. Scale bar = 100μm.

Article Snippet: aSyn monomers (Proteos #RP-003), pS129-aSyn monomers (Proteos #RP-004) and BSA (Sigma #A9647) were manually blotted on nitrocellulose membranes.

Techniques: Immunofluorescence, Purification, Control, Comparison

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A-D) Real-time binding curves of 833D4 and 73B3 IgGs to aSyn and pS129-aSyn measured using surface plasmon resonance (SPR). aSyn monomers were coated on the sensor chip, and the antibodies were flown over at different concentrations. The response is shown in resonance units, which is proportional to the number of bound antibodies to the chip. E) Association (Ka), dissociation (Kd) and equilibrium (KD) constants of both antibodies towards aSyn and pS129-aSyn. F) and G) A Thioflavin T (ThT) aggregation assay was performed by incubating aSyn monomers in the presence of ThT with agitation for seven days at 37°C (n=3). F) Relative fluorescence units (RFU) of ThT correlating to the level of β-sheets structures of aSyn monomers in the presence of 833D4 or an unspecific IgG. G) RFU of ThT with pS129-aSyn monomers in the presence of 73B3 or an unspecific IgG.

Article Snippet: aSyn monomers (Proteos #RP-003), pS129-aSyn monomers (Proteos #RP-004) and BSA (Sigma #A9647) were manually blotted on nitrocellulose membranes.

Techniques: Binding Assay, SPR Assay, Fluorescence

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Illustration of the structure of a scFv generated from a monoclonal IgG. B) Illustration of the scFv construct cloned inside a scAAV expression plasmid. C) Dot blot performed by blotting aSyn or pS129 aSyn monomers (and BSA as a negative control) on a nitrocellulose membrane. Membranes were either incubated with cell media from HEK cells previously transfected with each pscFvs, or with the purified monoclonal IgGs. Results show that both scFvs maintained the same specificity as their parental IgG. D) ELISA performed by coating the plate with purified monomers of aSyn or pS129-aSyn (or BSA as a negative control) showing the specificity of scFv-833D4 and scFv-73B3. Again, the cell media from HEK cells previously transfected with each pscFvs was used, and commercial antibodies to total (CST2628) and pS129-aSyn (Wako 015-25191) were used as positive controls (n=2).

Article Snippet: aSyn monomers (Proteos #RP-003), pS129-aSyn monomers (Proteos #RP-004) and BSA (Sigma #A9647) were manually blotted on nitrocellulose membranes.

Techniques: Generated, Construct, Clone Assay, Expressing, Plasmid Preparation, Dot Blot, Negative Control, Membrane, Incubation, Transfection, Purification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Representative images of pS129-aSyn immunostaining in the midbrain, PAG/pons and medulla of M83 mice injected with PFFs and with scAAVs encoding scFv-833D4, scFv-73B3, and scFv-control. B), C), D) and E) Quantification of the pS129-aSyn staining as a percentage of the area measured (n=6/group). One-way ANOVA with Dunnett’s multiple comparison test *p<0.05. **p<0.01. F) Representative images of pS129-aSyn and total aSyn in western blot on medulla tissue lysate. G) Quantification of pS129-aSyn normalized on total aSyn. One-way ANOVA with Dunnett’s multiple comparison test *p<0.05. H) Quantification of total aSyn normalized on actin (n=3-4/group). One-way ANOVA with Dunnett’s multiple comparison test. (n=3-4/group).

Article Snippet: aSyn monomers (Proteos #RP-003), pS129-aSyn monomers (Proteos #RP-004) and BSA (Sigma #A9647) were manually blotted on nitrocellulose membranes.

Techniques: Immunostaining, Injection, Control, Staining, Comparison, Western Blot

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A-C) Aged M83 homozygous mice were injected intravenously with an AAV-CapB10-scFv-IRES-GFP. This construct allows the expression of a secreted HA-tagged anti-aSyn scFv (833D4) and a non-secreted GFP protein. Mice were sacrificed 3 weeks post-injection. B) Representative confocal image taken in the cerebellum showing HA (scFv) in white and GFP in green. Red arrows point to neurons that are HA + /GFP - and thus have internalized the scFv. C) Confocal image showing the colocalization of the scFv (HA) and pS129-aSyn in a GFP-negative neuron from the medulla. D) Confocal images showing the colocalization of the scFv (HA, white) and Iba1 (red), indicating the internalization of scFvs by microglia. E) Representative images of human iPSCs-derived dopaminergic neurons (identified with a TH staining) infected with a scAAV2/retro encoding either scFv-833D4, scFv-73B3 or control scFv (visualized with Myc tag staining) and exposed for 2 hours to human aSyn PFFs tagged with Alexa Fluor 488. Scale bar = 10μm. F) Quantification with corrected total cell fluorescence (CTCF) of Alexa Fluor 488 intensity inside TH+ neurons (n=6 coverslips/condition, from 3 independent differentiations). One-way ANOVA with Dunnett’s multiple comparison test, *p<0.05, **p<0.01.

Article Snippet: aSyn monomers (Proteos #RP-003), pS129-aSyn monomers (Proteos #RP-004) and BSA (Sigma #A9647) were manually blotted on nitrocellulose membranes.

Techniques: Injection, Construct, Expressing, Derivative Assay, Staining, Infection, Control, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Post-mortem evidence for a reciprocal relationship between genomic DNA damage and alpha-synuclein pathology in dementia with Lewy bodies

doi: 10.1101/2024.04.24.590825

Figure Lengend Snippet:

Article Snippet: Primary antibodies: mouse anti-γH2A.X IgG1 (Clone JBW301, Cat# 05-636, Sigma, 1:500); rabbit anti-XRCC1 (Cat# HPA0061717, Sigma, 1:250) or rabbit anti-pS129-aSyn (EP1536Y, Cat# ab15253, abcam, 1:500) and mouse anti-NeuN IgG2b (Cat# ab104223, abcam, 1:250), were applied to slides in 10 % NGS containing TBST overnight at 4°C.

Techniques: Western Blot, Mass Spectrometry, Immunohistochemical staining

Journal: bioRxiv

Article Title: Post-mortem evidence for a reciprocal relationship between genomic DNA damage and alpha-synuclein pathology in dementia with Lewy bodies

doi: 10.1101/2024.04.24.590825

Figure Lengend Snippet: Example confocal images (x63 objective) from control (Con) and cases of dementia with Lewy bodies (DLB) showing alpha-synuclein phosphorylated at serine 129 (pS129), double strand break (γH2A.X) immuno-fluorescence in neurons (NeuN) and non-neuronal cells. Nuclei are co-stained with DAPI (a). Quantification of nuclear pS129 (pS129 aSyn Nuc ) (b) and γH2A.X (c) fluorescence from Con (n=13 cases) and DLB (n=12 cases) are presented. Frequency distribution of ps129 (d.i-ii) per nuclei are shown as correlative levels of ps129 aSyn Nuc with levels of γH2A.X, per case, with spearman’s correlation (r) reported (e.i-ii). Measures are reported for neuronal (NeuN+) and non-neuronal (NeuN-) populations. Data are expressed in scatterplots with mean±SEM (in b,c,e) and as percentage frequency of immunofluorescence intensity per bins of 10 units width (in d). *=p<0.05, **=p<0.01 and **=p<0.001.

Article Snippet: Primary antibodies: mouse anti-γH2A.X IgG1 (Clone JBW301, Cat# 05-636, Sigma, 1:500); rabbit anti-XRCC1 (Cat# HPA0061717, Sigma, 1:250) or rabbit anti-pS129-aSyn (EP1536Y, Cat# ab15253, abcam, 1:500) and mouse anti-NeuN IgG2b (Cat# ab104223, abcam, 1:250), were applied to slides in 10 % NGS containing TBST overnight at 4°C.

Techniques: Fluorescence, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Post-mortem evidence for a reciprocal relationship between genomic DNA damage and alpha-synuclein pathology in dementia with Lewy bodies

doi: 10.1101/2024.04.24.590825

Figure Lengend Snippet: Example widefield images (x20 objective) cases of dementia with Lewy bodies (DLB) showing Lewy bodies (LBs) as detected via alpha-synuclein phosphorylated at serine 129 (pS129), γ H2.AX (a) and 53BP-1 (b) immuno-fluorescence, with nuclei are co-stained with DAPI. Note the colocalization of γH2.AX with extranuclear pS129 Lewy bodies (a; white arrow heads) and absence of colocalization of 53BP-1 with Lewy bodies (b; white arrow heads). Confocal image of perinuclear rounded LB (c) and peri nuclear aSyn accumulation (d) with pS129-aSyn and γH2A.X colocalised. Quantification of γH2.AX positive (γH2A.X +) and negative (γH2A.X -) LBs in ten examined cases (e). Data shown as percentage LBs positive and negative for γH2A.X.

Article Snippet: Primary antibodies: mouse anti-γH2A.X IgG1 (Clone JBW301, Cat# 05-636, Sigma, 1:500); rabbit anti-XRCC1 (Cat# HPA0061717, Sigma, 1:250) or rabbit anti-pS129-aSyn (EP1536Y, Cat# ab15253, abcam, 1:500) and mouse anti-NeuN IgG2b (Cat# ab104223, abcam, 1:250), were applied to slides in 10 % NGS containing TBST overnight at 4°C.

Techniques: Fluorescence, Staining