Journal: Investigative Ophthalmology & Visual Science

Article Title: Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases

doi: 10.1167/iovs.61.6.12

Figure Lengend Snippet: Primary Antibodies

Article Snippet: Anti-pan synuclein (pan α-Syn, mouse) , 1:1000 , BD Biosciences (San Jose, CA, USA) , 610787.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases

doi: 10.1167/iovs.61.6.12

Figure Lengend Snippet: α-Syn immunoreactivity in retinas of different ages. Human α-Syn immunoreactivity ( green ) and 4′,6-diamidino-2-phenylindole (DAPI) ( blue ) of wild-type (WT) and Plp-α-Syn retinal sections. Distinct α-Syn immunoreactivity was obvious in the IPL and GCL in both age groups of Plp-α-Syn but was absent in WT mice. ( A ) Central and peripheral retina of adult (8 weeks) and aged (12 months) Plp-α-Syn animals. The antibody exclusively detected human α-Syn (anti-human-α-synuclein; see ). The staining intensity increased in aged animals ( arrows ). ( B ) Central retina of adult and aged WT mice. Scale bar : 50 µm.

Article Snippet: Anti-pan synuclein (pan α-Syn, mouse) , 1:1000 , BD Biosciences (San Jose, CA, USA) , 610787.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: The disordered protein SERF promotes α-Synuclein aggregation through liquid–liquid phase separation

doi: 10.1016/j.jbc.2024.105667

Figure Lengend Snippet: Characterization of SERF LLPS in vitro and in vivo . A , EGFP-SERF sequence shows the α-Syn binding and RNA-binding region. Letters in blue indicate negative charge distribution and the positive charge in red . Gray shading represents disorder domain. B , representative confocal microscopy images of EGFP-SERF ( green ) at different protein concentrations in the presence and absence of the molecular crowder PEG-8000. C , droplet fusion experiment of 30 μM EGFP-SERF with 5% PEG. The scale bar in the top row represents 1 μm. D , FRAP of the droplets formed by EGFP-SERF in vitro in the presence of 5% PEG. The scale bar represents 2 μm. E , confocal microscopy images of EGFP-SERF (30 μM) droplets in the presence of 5% PEG demonstrate the reversible nature of the droplets on heating (37 °C) and cooling (4 °C). Representative images are shown. F , confocal microscopy images of the expression of endogenous SERF ( green ) distribution in HeLa cells, respectively. Arrows , spot of SERF in cell nucleus. Nuclei are stained with DAPI ( blue ). G , confocal microscopy images of the transiently overexpressed EGFP-SERF ( green ) distribution in living HeLa cells, respectively. Arrows , spot of SERF in cell nucleus. Nuclei are stained with Hoechst 33342 ( blue ). H , FRAP of the condensates formed by SERF in cells. The scale bar represents 1 μm. I , left : confocal microscopy images of EGFP-SERF (30 μM) at different salt concentrations in the presence of 5% PEG. Right: turbidity measurement of SERF with different salt concentrations. ns, not significant, ns = 0.5488. Comparisons among groups were performed using one-way ANOVA with Kruskal–Wallis test or unpaired two-tailed Student’s t tests with GraphPad Prism 9.0.0. J and K , confocal microscopy images of liquid droplets formed by EGFP-SERF (30 μM) in the presence of ploy U at different concentrations ( J ), and in the presence or absence RNase of ploy U (91.8 μg/ml) ( K ). The image ( K , right ) reuse to show the RNase was added in the same sample. L , fusion of two EGFP-SERF assemblies upon contact. The scale bar represents 4 μm. M , confocal microscopy images of the droplets formed by WT and mutated-type EGFP-SERF (30 μM) with 91.8 μg/ml ploy U. All the experiments were performed three times with similar observations ( B – L ). Conditions: 20 mM PB pH 7.5. All scale bars represent 10 μm except where noted. α-Syn, α-Synuclein; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; EGFP, enhanced green fluorescent protein; FRAP, fluorescence recovery after photobleaching; HSQC, heteronuclear single quantum coherence; LLPS, liquid–liquid phase separation; SERF, small EDRK-rich factor.

Article Snippet: Purified mouse α-Syn antibody clone 42 (BD Biosciences) was used for the detection of α-Syn monomer. horse radish peroxidase-labeled Goat Anti-Rabbit IgG and horse radish peroxidase-labeled Goat Anti-mouse IgG (Beyotime Biotechnology) were used as secondary antibody.

Techniques: In Vitro, In Vivo, Sequencing, Binding Assay, RNA Binding Assay, Confocal Microscopy, Expressing, Staining, Two Tailed Test, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The disordered protein SERF promotes α-Synuclein aggregation through liquid–liquid phase separation

doi: 10.1016/j.jbc.2024.105667

Figure Lengend Snippet: Solution-state NMR characterization of LLPS of WT SERF and K17E SERF. A , primary sequence of SERF used in the experiments. Five amino acids were added in the N terminal of the recombinant SERF due to the PreScission protease digestion of GSH-Sepharose affinity chromatography. B and C , overlaid 2D 1 H- 15 N HSQC spectra of WT SERF in buffers with different NaCl concentrations. D , overlaid 2D 1 H- 15 N HSQC spectra of WT SERF and K17E SERF in buffers with 300 mM NaCl. E and F , overlaid 2D 1 H- 15 N HSQC spectra of K17E SERF in buffers with different NaCl concentrations. G , the ratio of normalized 1 H- 15 N HSQC peak intensities of WT SERF at 0 and 300 mM NaCl ( upper ). The ratio of normalized 1 H- 15 N HSQC peak intensities of K17E SERF at 0 and 300 mM NaCl ( lower ). A horizontal line is draw to guide visualization. SERF are 15 N labeled in the samples. Cyan arrows in C and D mark those showing chemical shift perturbations in the spectrum of K17E mutant in comparison with that of WT SERF. Arrows in B mark peaks that exhibit significantly attenuated intensities at 0 M NaCl in comparison with that at 300 mM NaCl, among which cyan arrows mark the same peaks as displayed in C . Green arrows mark the peaks that are new in the spectrum of K17E mutant in comparison with that of WT SERF. In G, cyan and green arrows mark the same peaks as shown in D . In G , the ID number of peaks is defined as displayed in Fig. S2 , and the profile of peak intensity ration at different NaCl concentrations are presented in Fig. S2 . Spectra acquired at 298 K and a protein concentration of 250 μM. α-Syn, α-Synuclein; EGFP, enhanced green fluorescent protein; HSQC, heteronuclear single quantum coherence; LLPS, liquid–liquid phase separation; SERF, small EDRK-rich factor.

Article Snippet: Purified mouse α-Syn antibody clone 42 (BD Biosciences) was used for the detection of α-Syn monomer. horse radish peroxidase-labeled Goat Anti-Rabbit IgG and horse radish peroxidase-labeled Goat Anti-mouse IgG (Beyotime Biotechnology) were used as secondary antibody.

Techniques: Sequencing, Recombinant, Affinity Chromatography, Labeling, Mutagenesis, Comparison, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: The disordered protein SERF promotes α-Synuclein aggregation through liquid–liquid phase separation

doi: 10.1016/j.jbc.2024.105667

Figure Lengend Snippet: SERF cophase separate with α-Syn in vitro . A , confocal microscopy images of assembling status of EGFP-SERF, α-Syn, and EGFP-SERF/α-Syn complex liquid droplets (50 μM each). Liquid droplets were imaged immediately. B and C , confocal microscopy images of EGFP-SERF/α-Syn complex in the PEG concentration (2% and 5%) of liquid droplets ( B ); the ThT fluorescence traces for α-Syn aggregation kinetic under the same condition at 37 °C ( C ), error bars represent SD (n = 3). D and E , confocal microscopy images of EGFP-SERF/α-Syn complex at different salt concentrations in the presence of 5% PEG ( D ); the ThT fluorescence traces for α-Syn aggregation kinetic under the same condition at 37 °C ( E ), error bars represent SD (n = 3). F and G , confocal microscopy images of EGFP-SERF/α-Syn complex with Poly P at different ratios ( F ) and the ThT fluorescence traces for α-Syn aggregation kinetic under the same condition at 37 °C ( G ), error bars represent SD (n = 3). H and I , confocal microscopy images of the complex of WT EGFP-SERF and three mutants combining with α-Syn in the presence of 5% PEG ( H ); the ThT fluorescence traces for α-Syn aggregation kinetic under the same condition at 37 °C ( I ), error bars represent SD (n = 3). All the experiments were performed in the PB buffer pH 7.5. The protein complex system is 50 μM in which the ratio of SERF/α-Syn is 1:1 (50 μM protein, 1:10 ratio of Cy3-labeled α-Syn to unlabeled protein), and all of THT samples was composed of unlabeled SERF. All the image scale bars represent 10 μm. α-Syn, α-Synuclein; EGFP, enhanced green fluorescent protein; SERF, small EDRK-rich factor; ThT, thioflavin T.

Article Snippet: Purified mouse α-Syn antibody clone 42 (BD Biosciences) was used for the detection of α-Syn monomer. horse radish peroxidase-labeled Goat Anti-Rabbit IgG and horse radish peroxidase-labeled Goat Anti-mouse IgG (Beyotime Biotechnology) were used as secondary antibody.

Techniques: In Vitro, Confocal Microscopy, Concentration Assay, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The disordered protein SERF promotes α-Synuclein aggregation through liquid–liquid phase separation

doi: 10.1016/j.jbc.2024.105667

Figure Lengend Snippet: SERF promotes the liquid-to-solid transition of α-Syn to forming amyloid aggregation. A , confocal microscopy images of the EGFP-SERF/α-Syn complex (50 μM protein, 1:10 ratio of Cy3-labeled α-Syn to unlabeled protein) incubated for 0, 3, and 7 days at 37 °C. The scale bar represents 10 μm. B , FRAP of the condensate formed in condition ( A ). The scale bar represents 1 μm (0 days); and the scale bar represents 500 nm (3 and 7 days). C , confocal microscopy images of HeLa cells overexpressing EGFP-SERF ( green )/mcherry–α-Syn ( red ) complex for 12 h after transfection in the presence or in the absence of 500 nM rotenone or 24 h of 20 μM copper sulfate. Arrows point out the spot of SERF, α-Syn, or the merge of them in cell. The scale bar represents 10 μm. D , the fluorescence intensity profiles along the indicated lines across the complex in ( C ). α-Syn, α-Synuclein; EGFP, enhanced green fluorescent protein; FRAP, fluorescence recovery after photobleaching; SERF, small EDRK-rich factor.

Article Snippet: Purified mouse α-Syn antibody clone 42 (BD Biosciences) was used for the detection of α-Syn monomer. horse radish peroxidase-labeled Goat Anti-Rabbit IgG and horse radish peroxidase-labeled Goat Anti-mouse IgG (Beyotime Biotechnology) were used as secondary antibody.

Techniques: Confocal Microscopy, Labeling, Incubation, Transfection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The disordered protein SERF promotes α-Synuclein aggregation through liquid–liquid phase separation

doi: 10.1016/j.jbc.2024.105667

Figure Lengend Snippet: SERF accelerates the step of α-Syn oligomer formation. A , Western blot showing α-Syn monomer ( bottom ) and oligomer ( middle ) at different time with or without SERF transfected into the HeLa cells. Then cells were stimulated in the presence of 500 nM rotenone or 20 μM copper sulfate. B , quantification of α-Syn monomer in the presence or absence of SERF in ( A ). Comparisons among groups were performed using one-way ANOVA with Kruskal–Wallis test or unpaired two-tailed Student’s t tests with GraphPad Prism 9.0.0. Error bars represent SD (n = 3). C , TEM images of α-Syn with or without the presence of SERF and confocal microscopy image of SERF/α-Syn complex combining with ThT. The scale bar of TEM represents 0.5 μm, and the scale bar of ThT represents 10 μm. All of the samples were incubated without agitation in 20 mM PB, pH 7.5 and 37 °C for 14 days. D , viability of HeLa cells measured by CCK8 assay. Fibrils (50 μM) were incubated in the presence or absence of SERF for 24 h at 37 °C and diluted into the cell culture media at the indicated concentrations. Error bars = SD (n = 3). All protein samples were incubated in 20 mM PB, pH 7.5. α-Syn, α-Synuclein; CCK8, cell counting kit-8; SERF, small EDRK-rich factor; TEM, transmission electron microscopy; ThT, thioflavin T.

Article Snippet: Purified mouse α-Syn antibody clone 42 (BD Biosciences) was used for the detection of α-Syn monomer. horse radish peroxidase-labeled Goat Anti-Rabbit IgG and horse radish peroxidase-labeled Goat Anti-mouse IgG (Beyotime Biotechnology) were used as secondary antibody.

Techniques: Western Blot, Transfection, Two Tailed Test, Confocal Microscopy, Incubation, CCK-8 Assay, Cell Culture, Cell Counting, Transmission Assay, Electron Microscopy

Journal: The Journal of Biological Chemistry

Article Title: The disordered protein SERF promotes α-Synuclein aggregation through liquid–liquid phase separation

doi: 10.1016/j.jbc.2024.105667

Figure Lengend Snippet: Schematic illustration of the process and function for the SERF/α-Syn cophase separation in the cell .

Article Snippet: Purified mouse α-Syn antibody clone 42 (BD Biosciences) was used for the detection of α-Syn monomer. horse radish peroxidase-labeled Goat Anti-Rabbit IgG and horse radish peroxidase-labeled Goat Anti-mouse IgG (Beyotime Biotechnology) were used as secondary antibody.

Techniques:

Journal: bioRxiv

Article Title: GDNF/RET signaling pathway activation eliminates Lewy Body pathology in midbrain dopamine neurons

doi: 10.1101/752899

Figure Lengend Snippet: (A) Selected images depicting progressive accumulation of phosphorylated (at Ser129) α-synuclein (pαSyn) in midbrain neuronal cultures. α-synuclein aggregation was seeded with pre-formed fibrils (PFFs) at Day In Vitro (DIV) 8 and assessed by immunofluorescent staining on cultures fixed 4h, 3d, 5d and 7d after seeding. First pαSyn aggregates (green) can be seen at DIV11 outside of dopamine neurons’ soma (immunostained with anti-tyrosine hydroxylase (TH, red) antibody). Large intrasomal aggregates of pαSyn in fraction of dopamine neurons can be seen at DIV15 (7 days post PFF addition). (B) Neither PFFs no GDNF treatment (started at DIV8) affected dopamine neuron survival. (C) GDNF added 1h after PFFs significantly reduced number of dopamine neurons harboring Lewy Body-like intrasomal pαSyn aggregates (ratio paired t test). (D) Selected images of control (top) and PFF treated cells at DIV15 (7 days post PFF addition) from vehicle (middle) or GDNF treated (bottom) groups. ***p<0.001, n=5 independent experiments. Data are mean ±SD. Scale bars, 50 µm.

Article Snippet: Recombinant mouse α-syn PFFs (5 mg/ml) were diluted 1:50 in PBS and sonicated at high power (10 cycles, 30 seconds on/ 30 seconds off) with Bioruptor sonicator (Diagenode, Liege, Belgium).

Techniques: In Vitro, Staining

Journal: bioRxiv

Article Title: GDNF/RET signaling pathway activation eliminates Lewy Body pathology in midbrain dopamine neurons

doi: 10.1101/752899

Figure Lengend Snippet: (A) Compared to control (PFFs only) and LV-GFP (control vector), LV-GDNF reduces number of dopamine cells with misfolded α-syn, detected by α-syn filament antibody. (B-E) All the groups shown at graphs had PFF treatment on DIV8. (B) Quantification of TH-positive cells with aggregated α-syn shows that the number of cells with aggregates were significantly lower at the group transduced with LV-GDNF, 3 days before PFFs (ratio paired t test). (C) Dopamine cell survival assessed by number of TH-positive cells was not affected by either lentivirus vector. (D) LV-GDNF added 8 or 3 days before, or 1 day after PFFs significantly reduces number of dopamine cells with pαSyn in their soma (mixed effect ANOVA, Holm-Sidak’s multiple comparison test). (E) Dopamine cell survival assessed by number of TH-positive cells was not affected by either vector or different time points of transduction. **p<0.01, ***p<0.001, n=2-6 independent experiments. Data are mean ±SD. Scale bar, 50 µm.

Article Snippet: Recombinant mouse α-syn PFFs (5 mg/ml) were diluted 1:50 in PBS and sonicated at high power (10 cycles, 30 seconds on/ 30 seconds off) with Bioruptor sonicator (Diagenode, Liege, Belgium).

Techniques: Plasmid Preparation, Transduction