Infectious prions and prion-like proteins.

<p>Infectious prions and prion-like proteins.</p

Electron cryomicroscopy analysis of infectious prion protein amyloid fibrils.

<p><b>(A)</b> Section of a cryo electron micrograph showing prion fibrils lacking the glycosylphosphatidylinositol (GPI) anchor. A single isolated and twisted fibril used for the 3-D reconstruction is enclosed by a black box. <b>(B)</b> Close-up view of the isolated prion fibril. <b>(C)</b> Reprojected image of the 3-D fibril map for comparison with the unprocessed image (B). <b>(D)</b> 3-D reconstruction of the GPI-anchorless prion fibril. <b>(E)</b> Cross section of the reconstructed fibril showing two distinct protofilaments. <b>(F)</b> Contoured density maps of the cross section with lines contoured at increasing levels of 0.125 σ. <b>(G)</b> Cartoon depicting the proposed configuration of the polypeptide chains in the prion fibril. Please note that this is not an atomistic model. <b>(H)</b> Close-up view of the possible ß-sheet stacking in a four-rung ß-solenoid architecture for illustration purposes only. Different colors represent different ß-solenoid rungs. Characteristic distances of the four-rung ß-solenoid architecture are labeled. Figure adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006229#ppat.1006229.ref010" target="_blank">10</a>].</p

Colloid Formation by Drugs in Simulated Intestinal Fluid

Many organic molecules form colloidal aggregates in aqueous solution at micromolar concentrations. These aggregates promiscuously inhibit soluble proteins and are a major source of false positives in high-throughput screening. Several drugs also form colloidal aggregates, and there has been speculation that this may affect the absorption and distribution of at least one drug in vivo. Here we investigate the ability of drugs to form aggregates in simulated intestinal fluid. Thirty-three Biopharmaceutics Classification System (BCS) class II and class IV drugs, spanning multiple pharmacological activities, were tested for promiscuous aggregation in biochemical buffers. The 22 that behaved as aggregators were then tested for colloid formation in simulated intestinal fluid, a buffer mimicking conditions in the small intestine. Six formed colloids at concentrations equal to or lower than the concentrations reached in the gut, suggesting that aggregation may have an effect on the absorption and distribution of these drugs, and potentially others, in vivo

A Neuronal Culture System to Detect Prion Synaptotoxicity

<div><p>Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrP^Sc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrP^C, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrP^C molecule. Both protease-resistant and protease-sensitive forms of PrP^Sc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrP^Sc and testing potential therapeutic agents.</p></div

The N-terminal domain of PrP^C is essential for PrP^Sc-induced dendritic spine loss.

<p>Hippocampal neurons from Tg(Δ23–111) mice (<b>A-D</b>) and Tg(Δ23–31) mice (<b>E-H</b>) (both on the <i>Prn-p</i>^0/0 background) were treated for 24 hr with 4.4 μg/ml of PrP^Sc purified without proteases (<b>B, F</b>), or with an equivalent amount of mock-purified material from uninfected brains (<b>A, E</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel F = 20 μm (applicable to panels A, B, E). Pooled measurements of spine number (<b>C, G</b>) and area (<b>D, H</b>) were collected from 20–24 cells from 4 independent experiments. N.S., not significantly different by Student’s t-test.</p

Purified PrP^Sc, prepared using pronase E, causes PrP^C-dependent spine loss.

<p>(<b>A</b>) Silver stain and Western blot analysis (using anti-PrP antibody IPC1) of PrP^Sc purified from scrapie-infected brains using pronase E, and mock-purified material from uninfected brains. Lane M, molecular size markers in kDa. Hippocampal neurons from wild-type (WT) mice (<b>B, C</b>) and PrP knockout (<i>Prn-p</i>^0/0) mice (<b>D, E</b>) were treated for 24 hr with 4.4 μg/ml of purified PrP^Sc (<b>C, E</b>), or with an equivalent amount of material mock-purified from uninfected brains (<b>B, D</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel E = 20 μm (applicable to panels B-D). Pooled measurements of spine number (<b>F</b>) and area (<b>G</b>) were collected from 16–18 cells from 3 independent experiments. ***p<0.001 or *p<0.05 by Student’s t-test; N.S., not significantly different.</p

PK-digested PrP^Sc causes dendritic spine loss.

<p>(<b>A</b>) Silver stain and Western blot (using anti-PrP antibody D18) of a PrP^Sc sample and a mock-purified control sample, after digestion with PK. Lane M, molecular size markers in kDa. Hippocampal neurons from wild-type (WT) mice (<b>B, C</b>) and PrP knockout (<i>Prn-p</i>^0/0) mice (<b>D, E</b>) were treated for 24 hr with 4.4 μg/ml of purified, PK-treated PrP^Sc (<b>C, E</b>), or with an equivalent amount of mock-purified sample (<b>B, D</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel E = 20 μm (applicable to panels B-D). Pooled measurements of spine number (<b>F</b>) and area (<b>G</b>) were collected from 20–24 cells from 3 independent experiments. ***p<0.001 by Student’s t-test; N.S., not significantly different.</p

Purified PrP^Sc, prepared without proteases, causes PrP^C-dependent spine loss.

<p>(<b>A</b>) Silver stain and Western blot analysis (using anti-PrP antibody D18) of PrP^Sc purified from scrapie-infected brains without proteases, and mock-purified material from uninfected brains. Lane M, molecular size markers in kDa. Hippocampal neurons from wild-type (WT) mice (<b>B, C</b>) and PrP knockout (<i>Prn-p</i>^0/0) mice (<b>D, E</b>) were treated for 24 hr with 4.4 μg/ml of purified PrP^Sc (<b>C, E</b>), or with an equivalent amount of material mock-purified from uninfected brains (<b>B, D</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel E = 20 μm (applicable to panels B-D). Pooled measurements of spine number (<b>F</b>) and area (<b>G</b>) were collected from 22–25 cells from 4 independent experiments. ***p<0.001 by Student’s t-test; N.S., not significantly different.</p

Molecular dynamics simulations.

<p>Comparison of wt and 116G PrP via MD simulations. (<b>a</b>) Cartoon representation of wild type PrP (residues 112 to 233): red spheres represent alanine in position 116. H1, H2 and H3 stand for helix one, two, and three, respectively. S1 and S2 stand for β-strands one and two. (<b>b</b>) Cartoon representation of 116G PrP: red spheres represent glycine in position 116. (<b>c</b>) RMSD plot for the folded domains of both wt (blue) and 116G polymorphism (red). (<b>d</b>) RMSF plot comparing wt (blue) and 116G polymorphism (red). The location of the polymorphism at residue 116 is shown with a black line and a label. The yellow and blue bars highlight the location of individual β-strands and α-helices, respectively. Both graphs show the average of three rounds (R1, R2, R3) of simulation. (<b>e)</b> Radius of gyration (Rg) values for both wt (blue) and the 116G polymorphism (red) as a function of simulation time. Both graphs are the average of three rounds (R1, R2, R3) of simulation. (<b>f</b>) Per-residue percentage of dominant secondary structure for the last 20 ns of production simulation. The blue and red graphs represent wt and the 116G polymorphism, respectively. The location of known secondary structure elements are shown using yellow and blue bars (top), on the crystal structure of deer prion protein (PDB ID: 4yxh [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006553#ppat.1006553.ref066" target="_blank">66</a>]. (<b>g</b>) Solvent accessible surface area for both 116G PrP and wt PrP. (<b>h</b>) Hydrophobic surface area for both 116G and wt. (<b>g</b>) and (<b>h</b>) both show the average of three rounds (R1, R2, R3) of simulation and the dashed lines represent the linear fit for the solvent accessible surface area and the hydrophobic surface area values, respectively.</p

RT-QuIC analysis of WTD prion seeding and amplification characteristics using deer and mouse rPrP substrates.

<p><b>(a)</b> The curves depict a representative RT-QuIC response of serially diluted (2x10^-2 to 2x10^-7) wt (left panels) and 116AG (right panels) brain homogenates using rPrP deer or mouse substrate. Fluorescence signals were measured every 15 min. The x-axis represents the reaction time (hours) and the y-axis represents the relative fluorescence units (RFUs), and each curve represents a different dilution. Mean values of four replicates were used for each dilution. The cut-off is indicated at app. 50,000 RFU based on the average fluorescence values of negative control +5SD. <b>(b, c)</b> The RT-QuIC responses of wt and 116AG were quantified by calculating different parameters: lag phase <b>(b)</b>, and log phase <b>(c)</b>. Mean values of 5 experiments with 4 replicates each were used and statistical analyses were evaluated using log-rank (Mantel-Cox) test for the lag phase (<b>b</b>) and unpaired t-test for the log phase (<b>c</b>). *<i>P</i> <0.05, **<i>P</i> <0.01 and ***<i>P</i> <0.005 refers to differences between WTD isolates (GraphPad Prism software).</p