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

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 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

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

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

Additional file 1: Table S1. of A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

Densitometry analysis of monomers in the pellet (P) and in the supernatant (S) fractions to determine ratios (P/S). (PDF 54 kb

Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

<div><p>Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, <i>PrP1</i> and <i>PrP2</i>, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in <i>PrP2</i> morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in <i>PrP2</i> morphants as well as in <i>PrP2^−/−</i> mutants. Rosette formation defects were observed in <i>PrP2</i> morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.</p></div

Loss of PLL nerve fasciculation and associated myelination in absence of PrP2.

<p><b>A–C</b>. In control larvae at 5 dpf, <i>sox10-GFP</i> cells are tightly organized along the PLL nerve (<i>nbt-dsred</i>). <b>D–F</b>. Double morphant <i>PrP2/p53</i> displayed an enlarged PLL nerve (with defasciculated axons, arrows) and rounded Schwann cells (arrowheads). <b>G–G</b>′. In 5 dpf larvae MBP labeling is observed in close apposition to <i>sox10-GFP</i> cells and formed a homogeneous line. <b>H–H′</b>. MBP labeling is altered and partially missing while Schwann cells are disorganized. <b>I–I′</b>. At 7 dpf, control larvae display tightly and regularly organized <i>sox10-GFP</i> positive cells. <b>J–K′</b>. In <i>PrP2/p53</i> morphants (J–J″), loosened Schwann cell processes are observed (arrows) as well as rounded cells (arrowheads) in <i>PrP2</i> morphants (K–K′). Results obtained from five independent experiments (n = 160 embryos).</p

PrP2 decrease expression results in abnormal PLL development.

<p><b>A</b>. In control embryos at 48 hours post fertilization (hpf) from <i>nbt-dsred</i> line that labels neurons and axons, the PLL nerve develops from the PLL ganglia until the tip of the tail (arrow). <b>B–C</b>. In <i>PrP2</i>-MO embryos, a range of defects for the PLL nerve is observed with premature arrest (B, C). <b>D</b>. Control PLL nerve fibers are tightly while <i>PrP2</i>-MO fibers exhibit abnormal branches were observed in severe cases (<b>E</b>). <b>F</b>. In control <i>claudinB-GFP</i> embryos at 48 hpf all derivatives issuing from the primordium express GFP and appear normal. Five regularly spaced neuromasts are present, with 3 terminal neuromasts at the tip of the tail. Due to embryo transparency, the neuromasts on the other side of the embryo are also visible. In <i>PrP2</i>-MO (<b>G</b>) and <i>PrP2</i>-MO/<i>p53</i>-MO (<b>H</b>) injected embryos (in order to avoid off-target defects), neuromast numbers are reduced, and often irregularly spaced. <b>I–J</b>. Western blot analysis of PrP2 expression using SAF84 antibody shows a decrease of the PrP2 protein after morpholino injections (n = 3 independent experiments).</p

PrP2 is involved in PLL development.

<p><b>A, C.</b> Alkaline phosphatase staining of the trunk neuromasts (arrows) in wild type embryos at 72 hpf. <b>C</b>. High magnification of boxed area in A. <b>B, D.</b> In <i>Prp2^−/−</i> mutant, less neuromasts are visible with the first neuromast displaced anteriorly (<b>B</b>, white arrow) <b>D</b>. High magnification of boxed area in B. <b>E–G.</b> In Control <b>(E)</b>, <i>PrP2</i>-MO/<i>p53</i>-MO (<b>F</b>) and <i>PrP2</i>-MO (<b>G</b>), the total neuromast number is decreased in morphants. <b>H.</b> Quantification of total neuromast number shows a significant decrease in morphants and mutants compared to control. <b>I.</b> Neuromast position along the somite axis, scheme adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331-Matsuda1" target="_blank">[28]</a>. In <i>Prp2^−/−</i> mutant, L1 is retrieved anteriorly to the normal L1 position while in morphants, the present neuromasts are displaced posteriorly. *: p<0.05, ***: p<0.001.</p