QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 19 February 2008
doi: 10.1242/jcs.021816

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parkyn, C. J. Articles by Morris, R. J. Search for Related Content PubMed PubMed Citation Articles by Parkyn, C. J. Articles by Morris, R. J. Social Bookmarking                         What's this?

LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein

Celia J. Parkyn¹, Esmeralda G. M. Vermeulen¹, Roy C. Mootoosamy¹, Claire Sunyach^1,*, Christian Jacobsen², Claus Oxvig², Søren Moestrup², Qiang Liu³, Guojun Bu³, Angela Jen¹ and Roger J. Morris^1,

¹ King's College London, Wolfson Centre for Age Related Disease, Guy's Campus, London SE1 1UL, UK
² Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
³ Department of Pediatrics, Washington University School of Medicine, St Louis Children's Hospital, St Louis MO 63110, USA

View larger version (16K): [in this window] [in a new window]   Fig. 1. Schematic view of the domain structure of LRP1 [adapted from Li et al. (Li et al., 2000) and Springer (Springer, 1998)] and PrPC, for which the flexible N- and structured C-terminal domains, and GPI anchor, are indicated; grey lines represent the surface membrane. LRP1 domains and the site of furin cleavage are indicated; Roman numerals indicate the four repeats of the ligand-binding complement-like domains; endocytic motifs are indicated in the cytoplasmic domain. LRP1, with >4500 amino acids, is more than 20 times larger than PrPC (208 amino acids).

View larger version (71K): [in this window] [in a new window]   Fig. 2. Immunohistochemical localisation of PrPC and LRP1 on sensory neurons. (A,B) Immunolabelling for cell-surface PrPC with 2S Alexa-Fluor-594–Fab (red), 3 µm (A) and 9 µm (B) above the plane of the laminin substrate. PrPC-expressing neurons (1-3; arrowheads point to their surface labelling), plus axons (arrows), and PrPC-negative substrate cells (visible by their blue DAPI-stained chromatin, asterisks) can be seen in (A); only their cell bodies are visible in B. These images are from a series (supplementary material Fig. S1), collected every 100 nm in vertical steps, which were deconvolved and assembled into ascending stacks of ten sections (i.e. 1 µm optical sections). Neuronal cell bodies shown subsequently in this paper are combined from stacks of 10-30 images taken at a level corresponding to B, with DAPI-stained chromatin. (C,D) Surface immunolabelling of PrPC (2S Alexa-Fluor-594–Fab, red) and green Alexa-Fluor-488-labelled anti-LRP1 (C) or Ctx (D) on sensory neurons fixed at 37°C before immunolabelling. On the cells shown, 37% and 77% of PrP colocalised with LRP1 and Ctx (data are in supplementary material Tables S1a and S1b). (E) Neuron with surface PrPC prelabelled at 10°C with Alexa-Fluor-594–Fab (red), then allowed to endocytose at 37°C for 1 minute, then fixed, permeabilised and immunolabelled for LRP1. It had endocytosed 82% of its labelled PrPC, most to perinuclear tubular structures (yellow owing to colocalised LRP1); arrow points to labelled PrPC still on the surface. (F) Transmission EM showing labelling within a coated pit on a neuron that has endocytosed (1.5 minutes at 37°C) its prelabelled 5 nm gold-Fab to PrPC, and 10 nm gold-2M*. Bars, 5 µm (A,C-E), 100 nm (F).

View larger version (42K): [in this window] [in a new window]   Fig. 3. Inhibition of endocytosis of PrPC on sensory neurons by RAP and siRNALRP1. (A,B) 1 µM RAP (B; A is vehicle control) inhibits the endocytosis of neuronal surface-labelled PrPC (red) but not Tf (green) after 2 minutes at 37°C. (C,D) Neurons treated for 90 minutes with 250 nM siRNACon (C) or siRNALRP1.1 (D) before endocytosing surface-labelled PrPC (red) and Tf (green) for 2 minutes at 37°C. (E,F) Immunocytochemical labelling of surface PrPC (red) and total LRP1 (green) in neurons preincubated for 90 minutes with 250 nM siRNACon (E) or siRNALRP1 (F), used to assess the effect of LRP1 knockdown; data in supplementary material Tables S2c and S2d. Bars, 5 µm. (G,H) Reduction of LRP1 protein shown by immunoblot (G; the 515-kDa band is shown) and quantitated in H (mean band intensity normalised to actin, ±s.d., n=4 independent knockdown experiments) after 250 nM penetratin-siRNA addition for the times shown (plus a 4-hour point showing the effect of adding additional 250 nM siRNA at 2 hours). The samples shown in G were from the 4-hour time point with two additions of siRNA.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Effect of 4 hours of treatment with siRNALRP1 upon PrPC distribution within neurons, and upon LRP1-dependent endocytosis. (A-D) Neurons pretreated with 250 nM siRNACon (A), or with siRNALRP1.1 (B), siRNALRP1.2 (C) or siRNALRP1.3 (D), were surface labelled with Alexa-Fluor-488–2S Fab anti-PrP (green), then fixed and permeabilised for labelling internal PrPC with Alexa-Fluor-594–SAF83 (red). Quantitation is given in supplementary material Table S4a. (E,F) Neurons treated as above with siRNACon (E) or siRNALRP1.1 (F), fixed, permeabilised and labelled (both surface and internal) with Alexa-Fluor-488 anti-Thy-1 antibody (green). Quantitation is given in supplementary material Table S3b. (G,H) Neurons treated with siRNACon (G) or siRNALRP1.1 (H) were prelabelled with Alexa-Fluor-488 2M* (green) and Texas Red-Tf and then placed at 37°C for 2 minutes. (I,J) Neurons treated with siRNACon (I) or siRNALRP1.1 (J) were prelabelled for PrPC with Alexa-Fluor-594–Fab (red), and the transferrin receptor with Alexa-Fluor-488–Tf (green), and placed at 37°C for 2 minutes. Bars, 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Effect of prolonged downregulation of LRP1 on the neuronal proteins Thy-1 and APP. Immunoblots, with the neurofilament light chain used as a loading control, of cells treated for 5 hours with two additions of 250 nM siRNA, control (left) and LRP1.1 (right).

View larger version (84K): [in this window] [in a new window]   Fig. 6. Effect of lowering LRP1 levels upon internal PrPC in sensory neurons. (A-F) Neurons treated for 4 hours with 250 nM siRNACon (A,C,E) or siRNALRP1.1 (B,D,F) were fixed, permeabilised and immunolabelled for PrPC (green) and (in red) BiP (A,B), -mannosidase II (C,D) or syntaxin 6 (E,F). Bars, 5 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 7. Effect of overexpression of PrPC upon its endocytic and biosynthetic trafficking. (A) The percentage of surface-labelled PrPC on WT (left panel) and Tg20 neurons (right panel) that has been endocytosed is plotted as a function of their relative level of surface PrPC (measured as the fluorescence intensity of Alexa-Fluor-594–2S Fab bound per µm3). The correlation coefficient and its significance (p) are shown. To avoid saturation of the camera by the 14-fold brighter fluorescence of Tg20 neurons, excitation intensity was turned down to give an overall fluorescence measured that was approximately comparable for the two sets of neurons. (B,C) tg20 neurons, treated for 90 minutes with siRNACon (B) or siRNALRP1.1 (C), labelled with 2S Alexa-Fluor-594–Fab (red) and Alexa-Fluor-488–Tf (green) were placed at 37°C for 6 minutes. In the control cell shown, 50% of the labelled PrPC has been internalised, and 35% in the siRNALRP1.1-treated cell. (D,E) Examples of immunolabelling of WT (D) and Tg20 (E) neurons for cell-surface PrP (Alexa-Fluor-488–2S fab; green) and internal PrPC (Alexa-Fluor-594–SAF83, red). Bars, 5 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 8. Biochemical analysis of interaction between PrPC and LRP1 on sensory neurons. (A) Immunoblot for PrP and Thy-1 of sequential immunoprecipitates (IP) of surface then internal antigen using antibody against PrPC or LRP1. The large arrow denotes the major 37-kDa di-glycosylated surface form of PrPC, the mid-size arrow shows monoglycosylated PrPC, and the small arrow shows non-glycosylated 23-kDa PrPC. PrP immunoblots are from the same gel, with duplicate samples run separately for Thy-1. (B) Immunoprecipitates for internal PrP or LRP1 (from part A) were diluted in endo H digestion buffer and either digested or not with the glycosidase, as indicated, before being immunoblotted for PrP. Arrows denote the relevant PrPC glycoforms as in A; the asterisk denotes the (slanted) band of endo H enzyme that binds antibodies nonspecifically.

View larger version (15K): [in this window] [in a new window]   Fig. 9. Surface plasmon resonance (SPR) analysis of the binding of GST-PrP23-107 to human LRP1. (A) Binding of 350 nM GST-PrP23-107 compared with GST; dissociation started at 600 seconds. (B) Binding of 1 µM RAP, with a second addition of 1 µM RAP without (solid upper line) or with 100 nM GST-PrP23-107 (dashed line); binding of 100 nM GST-PrP23-107 alone is shown in the lower solid trace; dissociation started at 1150 seconds.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter