Journal: Molecular Neurodegeneration

Article Title: LRP1 is a neuronal receptor for α-synuclein uptake and spread

doi: 10.1186/s13024-022-00560-w

Figure Lengend Snippet: LRP1 regulates α-Syn uptake in iPSNs. a and b , α-Syn uptake in WT and LRP1 -KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). c Representative images of WT or LRP1 -KO iPSNs after α-Syn uptake. Scale bars, 20 μm. d and e , Transferrin (Tfn) uptake in WT and LRP1 -KO iPSNs measured by flow cytometry (300 nM, 3 h of treatment). f Uptake of α-Syn and Tfn in the presence of increasing concentrations of RAP. g EM images showing the structure of α-Syn oligomers and preformed fibrils (PFFs) used in panel h. Scale bars, 200 nm. h Uptake of α-Syn oligomers and PFFs in WT and LRP1 -KO iPSNs (100 nM monomer equivalent, 3 h of treatment). All experiments in ( a , b , d , e , f and h ) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The morphology of α-Syn species was confirmed by negative stain transmission electron microscopy. α-Syn oligomers (StressMarq, cat# SPR-484) and PFFs (StressMarq, cat# SPR-322-C) were prepared at 25 µM in water.

Techniques: Flow Cytometry

Journal: medRxiv

Article Title: Alpha-synuclein misfolding as a fluid biomarker for Parkinson’s disease and synucleinopathies measured with the iRS platform

doi: 10.1101/2024.09.02.24312694

Figure Lengend Snippet: iRS-surface characterization by synthetic αSyn antigens. Panel A : The secondary structure sensitive Amide-I band absorbance of capture-antibody-bound αSyn-monomers (green, Stressmarq Bioscience Inc SPR-321), αSyn-oligomers (orange, Stressmarq Bioscience Inc SPR-466), and αSyn-PPFs (red, Stressmarq Bioscience Inc SPR-322) in PBS at high concentration of 500 ng/ml and scaled (x1.5-4) for comparison. Their structural differences are indicated by the significant wavenumber shift (cm -1 ) ranging from 1652 cm -1 for α-helical/random-coil (green) over 1647 cm -1 (orange) to β-sheet dominated structures absorbing at 1624 cm -1 (red). Panel B : The inertness measures on the blocking solution (BS) layer at high concentrations (500-5000 ng/ml) without the capture antibody. No signal is observed without the antibody on the blocking layer demonstrating sufficient inertness for pg-ng/ml concentrations of αSyn in CSF. Abbreviations: αSyn, alpha-synuclein; BS, blocking solution; PFF, pre-formed fibril.

Article Snippet: Briefly, αSyn stocks (Stressmarq, Cat. No. SPR-321), stored at -80 °C, were slowly thawed on ice.

Techniques: Concentration Assay, Comparison, Blocking Assay

Journal: Molecular Neurodegeneration

Article Title: LRP1 is a neuronal receptor for α-synuclein uptake and spread

doi: 10.1186/s13024-022-00560-w

Figure Lengend Snippet: LRP1 regulates α-Syn uptake in iPSNs. a and b , α-Syn uptake in WT and LRP1 -KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). c Representative images of WT or LRP1 -KO iPSNs after α-Syn uptake. Scale bars, 20 μm. d and e , Transferrin (Tfn) uptake in WT and LRP1 -KO iPSNs measured by flow cytometry (300 nM, 3 h of treatment). f Uptake of α-Syn and Tfn in the presence of increasing concentrations of RAP. g EM images showing the structure of α-Syn oligomers and preformed fibrils (PFFs) used in panel h. Scale bars, 200 nm. h Uptake of α-Syn oligomers and PFFs in WT and LRP1 -KO iPSNs (100 nM monomer equivalent, 3 h of treatment). All experiments in ( a , b , d , e , f and h ) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The morphology of α-Syn species was confirmed by negative stain transmission electron microscopy. α-Syn oligomers (StressMarq, cat# SPR-484) and PFFs (StressMarq, cat# SPR-322-C) were prepared at 25 µM in water.

Techniques: Flow Cytometry

Journal: Molecular Neurodegeneration

Article Title: LRP1 is a neuronal receptor for α-synuclein uptake and spread

doi: 10.1186/s13024-022-00560-w

Figure Lengend Snippet: LRP1 regulates α-Syn uptake via lysine residues in the N-terminus of α-Syn. a Schematic diagram of α-Syn domains highlighting the lysine residues (K). b Uptake of α-Syn and lysine-capped α-Syn in WT iPSNs. c , Uptake of α-Syn-488, N-α-Syn-488 and ΔN-α-Syn-488 in WT and LRP1 -KO iPSNs. d Uptake of α-Syn-488 in the presence of excessive non-labeled α-Syn N-terminus (N-α-Syn) or α-Syn lacking N-terminus (ΔN-α-Syn) fragments. All experiments in ( b – d ) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data in ( b ) was analyzed by unpaired two-sided t-test. *** P < 0.001. Data in ( c and d ) were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The morphology of α-Syn species was confirmed by negative stain transmission electron microscopy. α-Syn oligomers (StressMarq, cat# SPR-484) and PFFs (StressMarq, cat# SPR-322-C) were prepared at 25 µM in water.

Techniques: Labeling

Journal: Molecular Neurodegeneration

Article Title: LRP1 is a neuronal receptor for α-synuclein uptake and spread

doi: 10.1186/s13024-022-00560-w

Figure Lengend Snippet: Neuronal Lrp1 knockout reduces α-Syn spread in vivo. a Schematic drawing for the stereotactic injection of AAV-synapsin-GFP-synapsin-h-α-Synuclein into the neuronal Lrp1 knockout ( Lrp1 -nKO) mice and wild type (WT) littermate controls, and the experimental workflow. b and c , Western blotting showing the endogenous Lrp1 protein in the cortex of WT ( n = 6) and Lrp1 -nKO mice ( n = 6). d Representative sections showing GFP and h-α-Syn signals in mouse brains. Dotted line marks the outline of each section. Scale bars, 500 μm. e Representative images showing GFP and h-α-Syn signals in the hippocampus region from WT and Lrp1 -nKO mice. Scale bars, 50 μm. f Quantitative analysis of GFP intensity in hippocampus from WT or Lrp1 -nKO mice. g Quantitative analysis of h-α-Syn immunofluorescence intensity in hippocampus from WT or Lrp1 -nKO mice. h Representative images showing h-α-Syn spreading to the cortex region from WT or Lrp1 -nKO mice. Scale bars, 50 μm. i Quantitative analysis of h-α-Syn immunofluorescence intensity in the cortex from WT or Lrp1 -nKO mice. Experiments in ( e – i ) n = 5 mice (3 males and 2 females) for WT and n = 6 mice (3 males and 3 females) for Lrp1 -nKO mice. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by unpaired two-sided t-test. NS, not significant; * P < 0.05, *** P < 0.001

Article Snippet: The morphology of α-Syn species was confirmed by negative stain transmission electron microscopy. α-Syn oligomers (StressMarq, cat# SPR-484) and PFFs (StressMarq, cat# SPR-322-C) were prepared at 25 µM in water.

Techniques: Knock-Out, In Vivo, Injection, Western Blot, Immunofluorescence

Journal: NPJ Parkinson's Disease

Article Title: Glycation modulates glutamatergic signaling and exacerbates Parkinson’s disease-like phenotypes

doi: 10.1038/s41531-022-00314-x

Figure Lengend Snippet: Wild-type littermate (WT) and Thy1-aSyn transgenic mice received an intracerebroventricular (ICV) injection of MGO or vehicle (PBS) and protein brain extracts from several regions analyzed 5 weeks post injection. Protein extracts were resolved by SDS-PAGE or loaded into membranes in a dot-blot system (see Supplementary Fig. ). Membranes were probed with anti-aSyn, anti-CEL, anti-pS129-aSyn and anti-β-actin for normalization. Representative blots showing two samples from each experimental group are shown for aSyn, and densitometric analysis represented for aSyn and CEL in a , b midbrain, c , d striatum, e , f cerebellum, g , h prefrontal cortex, and i , j hippocampus, respectively. k Representative blot showing four samples from vehicle- and MGO-injected Thy1-aSyn mice are shown for pS129-aSyn and aSyn total signal. Densitometric analysis of the ratio between pS129-aSyn and aSyn is presented. l Representative blot showing aSyn probing in soluble and insoluble fraction from vehicle- and MGO-injected Thy1-aSyn mice. Densitometric analysis of the ratio between the insoluble and the sum of both soluble and insoluble signals is presented. At least n = 5 in all groups, data in all panels are average with error bars representing standard deviation, Ordinary one-way ANOVA, * p < 0.05, ** p < 0.01, **** p < 0.0001; unpaired two-tailed t -test with equal SD, # p < 0.05.

Article Snippet: Membranes were incubated with blocking solution (5% bovine serum albumin) in 1× TBS (20 mM Tris, 136 mM NaCl, pH 7.6) at room temperature for 30 min. Primary antibody incubations were carried out overnight at 4 °C, using given concentrations in blocking solution: CEL (Mouse Anti-N ε -carboxyethyl lysine, in a dilution of 1:1000 in blocking solution, Cosmo-Bio, USA), aSyn (Purified Mouse Anti-α-Synuclein antibody, in a dilution of 1:1000 in blocking solution, BD Biosciences; San Jose, CA, USA), phosphorylated aSyn at residue 129 (pS129, [J18] SMC-600, StressMarq; Victoria, Canada), glyoxalase I (Rabbit polyclonal anti-Glyoxalase I antibody – FL-184, sc67351, Santa Cruz Biotechnology), and β-actin (Mouse Monoclonal anti-β-actin antibody in a dilution of 1:5000 in blocking solution, Ambion, Thermo Fisher Scientific; Waltham, MA, USA).

Techniques: Transgenic Assay, Injection, SDS Page, Dot Blot, Standard Deviation, Two Tailed Test