oligomeric α syn  (Exosome Diagnostics)

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: Commercial recombinant proteins, including human Tau (R&D Systems, SP-495), α-Syn (Proteos, cat# RP-003), oligomeric α-Syn (StressMarq, cat# SPR-484), α-Syn preformed fibrils (PFFs) (StressMarq, cat# SPR-322-C), α-Syn N (1–60) fragment (rPeptide, cat# S-1011–1), and α-Syn ΔN (61–140) fragment (rPeptide, cat# S-1013–1) were labeled with Alexa Fluor® 488 ester (Life Technologies, cat# A10235) according to the manufacturer’s instructions.

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: Commercial recombinant proteins, including human Tau (R&D Systems, SP-495), α-Syn (Proteos, cat# RP-003), oligomeric α-Syn (StressMarq, cat# SPR-484), α-Syn preformed fibrils (PFFs) (StressMarq, cat# SPR-322-C), α-Syn N (1–60) fragment (rPeptide, cat# S-1011–1), and α-Syn ΔN (61–140) fragment (rPeptide, cat# S-1013–1) were labeled with Alexa Fluor® 488 ester (Life Technologies, cat# A10235) according to the manufacturer’s instructions.

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: Commercial recombinant proteins, including human Tau (R&D Systems, SP-495), α-Syn (Proteos, cat# RP-003), oligomeric α-Syn (StressMarq, cat# SPR-484), α-Syn preformed fibrils (PFFs) (StressMarq, cat# SPR-322-C), α-Syn N (1–60) fragment (rPeptide, cat# S-1011–1), and α-Syn ΔN (61–140) fragment (rPeptide, cat# S-1013–1) were labeled with Alexa Fluor® 488 ester (Life Technologies, cat# A10235) according to the manufacturer’s instructions.

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