spr  (StressMarq)

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows high labeling accuracy for α-syn aggregates in vitro, compared to phosphorylated (pS129) α-syn antibody which was used as the gold standard reference staining for α-syn aggregates. (a) Structure of peptide-based probe P1 ; (b) Setup of primary hippocampal neuronal culture and induction of α-syn aggregation upon addition of PFFs; (c) Confocal microscopy of primary neuronal cells post 7 days of PFF addition. Nuclei are colored blue, total α-syn green, pS129 α-syn orange, and probe P1 in red. Scale bar represents 200 μm; (d) Probe P1 shows colocalized staining with the PS129 α-syn antibody staining. The probe labels most of the same focal locations, but with slightly larger surrounding area than the PS129 α-syn antibody hitsthis is still regarded as a positive colocalization. True positives (indicated by green arrows) are stained by both P1 and the pS129 α-syn antibody. False negatives (indicated by pink arrows) are not stained by P1 but stained by the pS129 α-syn antibody. Scale bar represents 100 μm; (e) Graph showing average positive predictive value (true positives/probe positives) of 87.4% and miss rate (false negatives/antibody positives) of 8.7%. Values on graph are given as the mean ± SEM calculated over 10 wells, with 9 fields of view each.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Labeling, In Vitro, Staining, Confocal Microscopy

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 exhibits preferential binding to α-syn fibrils over α-syn monomers. (a) Fluorescent excitation and (b) fluorescent emission spectra of P1 . 2 μM probe P1 was incubated with 10 μM of various α-syn species, including monomers, A* oligomers, B* oligomers, and PFFs. Concentrations of all α-syn species are expressed at their equivalent monomer concentration. Fluorescence intensity of P1 increases significantly upon interaction with PFFs (7.6× and 16× for excitation and emission respectively); (c) Fluorescence titration curve of P1 (0.5 μM) to PFFs, showing micromolar K D ; (d) Native-PAGE images of P1 with various α-syn species, showing that P1 preferentially binds to B* oligomers and PFFs, over α-syn monomers and A* oligomers. 2 μg of P1 was incubated with 2 μg of α-syn monomers and various α-syn aggregate species and run under native PAGE conditions. In-gel fluorescence was first visualized in the 647 channel, and then the gel was subsequently imaged in the brightfield channel after staining with Coomassie blue. Red bands correspond to P1 bound to the B* oligomers and PFFs; (e) Dot-blot images of P1 (0.5 μM) with various human α-syn species, showing concentration-dependent labeling of kinetically stable oligomers and PFFs.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Binding Assay, Incubation, Concentration Assay, Fluorescence, Titration, Clear Native PAGE, Staining, Dot Blot, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows specific staining of α-syn aggregates in tissues from PFF-injected mouse models. (a) Models used for ex vivo tissue labeling: PFF-injected mouse (C57BL6), where PFF was injected directly into the brain or duodenum, and transgenic mouse models overexpressing human α-syn; (b) Confocal microscopy of brain tissues after 30 days of PFF injection showing high levels of α-syn aggregation in the hippocampal and cortex regions, labeled specifically by both pS129 α-syn antibody and P1 ; (c) Confocal microscopy of duodenum tissues after 75 days of PFF injection showing low levels of α-syn aggregation in the mucosa region indicated by the white arrows. Images of tissues from control PBS-injected mice are shown in Figure S5 for comparison; (d) Confocal microscopy of brain tissue (substantia nigra) of a healthy control and of a PD patient (post-mortem), with pS129 α-syn antibody staining and P1 staining observed in the substantia nigra of the PD patient; (e) Confocal microscopy showing Lewy bodies (LB) and Lewy neurites (LN) highlighted by pS129 α-syn antibody staining in the substantia nigra of a PD patient. Higher magnification images of LB and LN show specific labeling by P1 that corresponds to the antibody staining. Nuclei in blue, α-syn in green, pS129 α-syn in orange and probe P1 in red.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Staining, Injection, Ex Vivo, Labeling, Transgenic Assay, Confocal Microscopy, Control, Comparison

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Specific α-syn antibody and probe P1 labeling of entire GI tract (esophagus, stomach, duodenum, jejunum, ileum, and colon) in transgenic mice (JAX004479). (a) Aggregated α-syn is observed in the mucosa layers and stained by both pS129 α-syn antibody and P1 (annotated with white arrows). Tissue layers are indicated by black arrows: muscularis externa (ME), submucosa (SM), and mucosa (M). Nuclei are in blue, α-syn in green, pS129 α-syn in orange, and probe in red. Scale bar represents 200 μm; (b) Total expression areas of α-syn aggregates labeled by pS129 α-syn antibody and P1 were quantified, with the highest expressions observed in the esophagus and colon; (c) High degree of colocalization (average of 0.91) was observed between pS129 α-syn antibody and probe P1 channels across all the GI tract tissues, determined by the Costes method. Manders colocalization and correlation coefficients are shown in Figure S7a ; (d) Probe positive predictive values were consistent across the respective GI tract tissues. Values on graphs are given as the mean ± SEM ( n = 3 per group); (e) P1 labeling is observed in the enteric neurons in colon tissue, marked by neuronal marker TUBB3 (green).

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Labeling, Transgenic Assay, Staining, Expressing, Marker

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Increased accumulation of α-syn aggregates across GI tissues in older transgenic mice (JAX010799). (a) Western blot showing abundance of total α-syn in brain, esophagus, duodenum, and colon of young (5-month) and old (15-month) transgenic mice (JAX010799), with higher molecular weight α-syn species observed in the older mice (indicated by red arrows); (b) Graph showing intensity of bands corresponding to α-syn, normalized to beta-actin, quantified from blots in Figure S7 . Older mice showed increased intensities of α-syn bands; Confocal images showing increased staining of aggregated α-syn in (c) esophagus and (d) colon of older transgenic mice. Tissue layers as indicated by black arrows: muscularis externa (ME), submucosa (SM), mucosa (M). Scale bar represents 200 μm; (e) Graphs showing degree of α-syn aggregation determined by pS129 α-syn antibody labeling and P1 labeling, with generally higher degrees of α-syn aggregation observed across all GI tissues in older mice. Values on graphs are given as the mean ± SEM ( n = 3 per group). Ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, determined by two-way ANOVA test.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Transgenic Assay, Western Blot, Molecular Weight, Staining, Antibody Labeling, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Longitudinal view of unrolled colon tissue of transgenic mouse showing differing levels of α-syn aggregation across tissue layers (mucosa, submucosa and muscularis externa). (a) Confocal microscopy of selected section of colon tissue from transgenic mouse, with the different tissue layers marked by white dashed lines. α-Syn aggregates on the mucosa surface are specifically labeled by pS129 α-syn antibody (orange) and P1 (red). Full view of section is shown in Figure S8 ; (b) Graph showing degree of α-syn aggregation (pS129 α-syn antibody or P1 positives, divided by α-syn positives) in the mucosa, submucosa, and muscularis externa regions. Mucosa shows lower but detectable levels of P1 staining compared to the other tissue layers; (c) Confocal microscopy of colon tissue from a transgenic mouse, flushed with P1 (15 μg/mL), followed by fixation and immunofluorescence staining with pS129 α-syn antibody (orange) and aggregated α-syn antibody, clone 5G4 (green). P1 labels α-syn aggregates in the surface mucosal layer.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Transgenic Assay, Confocal Microscopy, Labeling, Staining, Immunofluorescence