Efficient <i>in vitro</i> ubiquitination of alpha-synuclein filaments by Nedd4.

<p>Ubiquitination assays of Nedd4 with alpha-synuclein (antibody Syn-1). (A) Coomassie-stained gels of purified wild-type (WT) and A53T mutant alpha-synuclein. (B) Time course of polymerization of WT and mutant alpha-synuclein assayed by thioflavin dye fluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#sec007" target="_blank">Methods</a>). (C) Coomassie-stained gel of purified Nedd4 ligase. (D) <i>In vitro</i> ubiquitination assays using Nedd4 [200nM] with alpha-synuclein [200nM] showing efficient ubiquitination of filamentous (fil.) wild-type alpha-synuclein with strongly reduced modification of the filamentous A53T mutant and hardly any detectable ubiquitination of the monomeric forms (mo.). (E) Ubiquitination assays of Nedd4 [150nM] with alpha-synuclein filaments [600nM] in the presence of ubiquitin chain-type specific deubiquitinases as indicated [UbiCREST, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#pone.0200763.ref014" target="_blank">14</a>]] demonstrating mainly K63, but also some K29 and K33, linkages.</p

Ligase-specific recognition of alpha-synuclein filaments.

<p>(A) Cartoon of the modular architecture of Nedd4-type E3 ligases and overview of the constructs used in this study. (B) Western blots of <i>in vitro</i> ubiquitination assays comparing different Itch versions (full-length, ΔC2, WW mutants [175nM, left panel]) and full-length or HECT-only versions of Nedd4, Nedd4L and Smurf2 [100nM, right panel] in their ability to ubiquitinate alpha-synuclein [600nM, upper panels]. The lower panels (anti-ubiquitin blot) show overall ubiquitination, demonstrating the activity of the E3 ligase constructs by their ability to auto-ubiquitinate. Note that full-length Smurf2 is auto-inhibited in the presence of monomeric alpha-synuclein. (C) Activation of autoinhibited Smurf2 by alpha-synuclein filaments. (D) Western blots of <i>in vitro</i> ubiquitination assays comparing full-length, ΔC2 or HECT-only versions of Nedd4, Itch and Smurf2 [100nM] in their ability to ubiquitinate alpha-synuclein [400nM], indicating ligase-specific recognition of filaments (antibody Syn-1). Three sections of the same exposure of the same gel are shown.</p

Clustering of alpha-synuclein does not result in efficient ubiquitination.

<p>(A) Schematic of the polymerization domains (DIX and TPR) fused to a soluble substrate, either the cytoplasmic domain of Ndfip2 containing three PY motifs or full-length alpha-synuclein with mutants that were used in this study. The location of the A53T mutation is indicated, but mutant protein was not used in Fig 4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#pone.0200763.g005" target="_blank">Fig 5A</a>). (B) <i>In vitro</i> ubiquitination assays using Smurf2 with purified recombinant proteins as indicated above the panels, showing efficient Smurf2 autoubiquitination when incubated with polymerization-competent DIX-TPR-Ndfip2 with intact PY motifs (D-T-N2), but not when all three PY motifs were mutated (D-T-PPAG). No Smurf2 autoubiquitination was observed with DIX-TPR-alpha-synuclein (D-T-Syn) and only modest effects were observed with an introduced weak PY motif (D-T-Syn PY1). (C) Western blots of His pull downs from HEK293T cells co-transfected with His-Ub, HA-tagged Nedd4, WWP2 and the GFP-tagged highly efficient polymerizing DIX-TPR fusions as indicated. N2-Pym, Ndfip2 with all three PY motifs mutated; N2-1xPY, Ndfip2 with one remaining LPxY motif. (D) Live-cell images showing single confocal sections of representative HeLa cells transfected with DIX-TPR-alpha-synuclein-EGFP either with polymerization defective M4 mutant (top left), wild-type alpha-synuclein (middle left) or introduced weak PY motif (bottom left panel) and co-transfected with mCherry-Nedd4 (center panels). Recruitment of Nedd4 into larger punctate aggregates was observed only in the presence of the weak PY motif (see arrows). Insets show enlarged versions of the outlined areas.</p

<i>In vitro</i> ubiquitination of alpha-synuclein by different Nedd4-type ligases.

<p>Western blots of <i>in vitro</i> ubiquitination assays using different members of the Nedd4 family [250nM] with alpha-synuclein [600nM] (antibody Syn-1). (A) Comparing the ability of the Nedd4-type E3 ligases to ubiquitinate monomeric (mo.) or filamentous (fil.) alpha-synuclein indicates a general preference for filaments. (B) Ubiquitination assays of different Nedd4 family members with filamentous alpha-synuclein with or without the ubiquitin chain-type specific deubiquitinase Trabid as indicated above the panel. In each case the ubiquitin chains were sensitive to Trabid, implying similar linkages.</p

Expression of human tau in the optic nerve of mice transgenic for human mutant P301S tau.

<p>(A–C), Staining for human tau (A, green) and βIII tubulin (B, red) showed co-localisation in the axons of the optic nerve (C, overlay image of A and B; example from 1 month old mouse). (D–F), Staining for tau phosphorylated at S202/T205 (D, green) and βIII tubulin (E, red) showed co-localisation in the axons of the optic nerve (F, overlay image of D and E; example from 5 month old mouse). Arrows indicate examples of co-localisation. Scale bar, 20 µm.</p

Quantification Methodology.

<p>(A), Retinal ganglion cell (RGC) survival following excitotoxic injury <i>in vivo</i> was quantified using retinal flat mounts immunohistochemically labelled for NeuN. Twelve images (3 per quadrant at central, medial and peripheral locations; approximate positions defined by boxes) were captured per retina using a 40× objective; NeuN-positive nuclei were counted in each image and their average number calculated for each retina. RGC loss was calculated compared to NeuN counts from the uninjured contralateral eye. (B), Axonal transport of fluorescent cholera toxin B (CTB) in the optic nerve was quantified by measuring average fluorescence intensity across the width of the optic nerve at 100 µm intervals along the full length of each nerve. A representative image is shown, with the white lines indicating example regions where average fluorescence intensity was measured. Scale bar, 100 µm.</p

Binding of cholera toxin B (CTB) protein to GM1 receptor in P301S tissue.

<p>Fixed retinal (A–B) and brain (C–F) tissue was exposed to fluorescently tagged CTB (red) in order to visualise GM1 receptor binding in both C57/Bl6 control tissue (A, C–D) and P301S tissue (B, E–F). CTB binding was observed in RGCs within the retina (A–B), counterstained for the marker βIII tubulin (green; arrows indicate co-localisation), and punctate staining within the inner retina was also seen. Nuclei were counterstained with DAPI (blue). No difference in the pattern of CTB binding to the GM1 receptor in the retina was found between control (A) and P301S (B) tissue. Furthermore, no difference in the pattern of CTB binding to the GM1 receptor in the superior colliculus (outlined) of the brain was observed between control (D) and P301S (F) tissue. The RGC axon terminals in the superior colliculus were counterstained for the marker vGluT2 (vesicular glutamate transporter 2; green) in both control (C) and P301S (E) tissue. Scale bar, 25 µm A–B, 500 µm C–F.</p

Sequence Determinants for Amyloid Fibrillogenesis of Human a-Synuclein

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the presence of filamentous inclusions in nerve cells. These filaments are amyloid fibrils that are made of the protein a-synuclein, which is genetically linked to rare cases of PD and DLB. ß-Synuclein, which shares 60% identity with a-synuclein, is not found in the inclusions. Furthermore, while recombinant a-synuclein readily assembles into amyloid fibrils, ß-synuclein fails to do so. It has been suggested that this may be due to the lack in ß-synuclein of a hydrophobic region that spans residues 73¿83 of a-synuclein. Here, fibril assembly of recombinant human a-synuclein, a-synuclein deletion mutants, ß-synuclein and ß/a-synuclein chimeras was assayed quantitatively by thioflavin T fluorescence and semi-quantitatively by transmission electron microscopy. Deletion of residues 73¿83 from a-synuclein did not abolish filament formation. Furthermore, a chimera of ß-synuclein with a-synuclein(73¿83) inserted was significantly less fibrillogenic than wild-type a-synuclein. These findings, together with results obtained using a number of recombinant synucleins, showed a correlation between fibrillogenesis and mean ß-strand propensity, hydrophilicity and charge of the amino acid sequences. The combination of these simple physicochemical properties with a previously described calculation of ß-strand contiguity allowed us to design mutations that changed the fibrillogenic propensity of a-synuclein in predictable way

Retrograde axonal transport is reduced in optic nerve of mice transgenic for human mutant P301S tau.

<p>Fluorescent cholera toxin B was injected bilaterally into the superior colliculus and the amount transported measured in 5-month-old (A) and 3-month-old (C) P301S tau transgenic and C57/Bl6 control mice. Fluorescence intensity was lower along the length of the optic nerve in transgenic mice at all ages, compared to C57/Bl6 controls. Statistical analysis of the area under the fluorescence intensity curve for each individual showed a significant reduction in retrograde axonal transport in optic nerves from P301S tau transgenic mice at 5 months (B) and at 3 months (D), compared to controls. Data are presented as mean±SEM.</p

Reduced axonal transport in optic nerve of mice transgenic for human mutant P301S tau increases neuronal susceptibility to injury.

<p>Retinal ganglion cell (RGC) survival was quantified following a mild unilateral excitotoxic injury of the left eye (LE) by counting NeuN-positive nuclei in the RGC layer of the whole-mounted retina of 1, 3 and 5 month old mice (A–D). Percentage RGC loss was calculated by comparing the number of surviving RGCs in injured retinas to that of the uninjured right eye (RE). Representative images of NeuN-positive nuclei in injured P301S tau transgenic (A) and C57/Bl6 (C) retinas from 5 month old animals, compared to uninjured contralateral retinas (B and D), are shown as an example. Statistical analysis revealed a significant increase in RGC death following mild excitotoxic injury in P301S tau transgenic retinas, compared to C57/Bl6 control retinas (E), at both 3 months and 5 months of age; however no difference in RGC excitotoxic death was found at 1 month of age between control and transgenic retinas. N-methyl-D-aspartic acid (NMDA) was used as the excitotoxin. Scale bar, 100 µm.</p