A nine amino acid domain Is essential for mutant prion protein toxicity

Transgenic mice expressing PrP molecules with several different internal deletions display spontaneous neurodegenerative phenotypes that can be dose-dependently suppressed by co-expression of wild-type PrP. Each of these deletions, including the largest one (Δ32–134), retains nine amino acids immediately following the signal peptide cleavage site (residues 23–31; KKRPKPGGW). These residues have been implicated in several biological functions of PrP, including endocytic trafficking and binding of glycosaminoglycans. We report here on our experiments to test the role of this domain in the toxicity of deleted forms of PrP. We find that transgenic mice expressing Δ23–134 PrP display no clinical symptoms or neuropathology, in contrast to mice expressing Δ32–134 PrP, suggesting that residues 23–31 are essential for the toxic phenotype. Using a newly developed cell culture assay, we narrow the essential region to amino acids 23–26, and we show that mutant PrP toxicity is not related to the role of the N-terminal residues in endocytosis or binding to endogenous glycosaminoglycans. However, we find that mutant PrP toxicity is potently inhibited by application of exogenous glycosaminoglycans, suggesting that the latter molecules block an essential interaction between the N-terminus of PrP and a membrane-associated target site. Our results demonstrate that a short segment containing positively charged amino acids at the N-terminus of PrP plays an essential role in mediating PrP-related neurotoxicity. This finding identifies a protein domain that may serve as a drug target for amelioration of prion neurotoxicity

The Toxicity of a Mutant Prion Protein Is Cell-Autonomous, and Can Be Suppressed by Wild-Type Prion Protein on Adjacent Cells

Insight into the normal function of PrPC, and how it can be subverted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants, Δ105–125 (called ΔCR), produces a spontaneous neurodegenerative illness when expressed in transgenic mice, and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous, or can be exerted on neighboring cells, is unknown. To investigate this question, we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice, which are marked by the presence or absence of GFP, are differentiated together to yield neurons, astrocytes, and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity, we assayed sensitivity of the cells to the cationic antibiotic, Zeocin. In a previous study, we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics, an effect that is suppressed by co-expression of wild type PrP, similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system, we find that while ΔCR-dependent toxicity is cell-autonomous, the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrPC, and the cellular mechanisms underlying the rescuing process

The N-Terminal, Polybasic Region Is Critical for Prion Protein Neuroprotective Activity

Several lines of evidence suggest that the normal form of the prion protein, PrPC, exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrPC to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32–134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23–31, Δ23–111, and Δ23–134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23–31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrPC neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases

Meta-Analysis Reveals Reproducible Gut Microbiome Alterations in Response to a High-Fat Diet

Multiple research groups have shown that diet impacts the gut microbiome; however, variability in experimental design and quantitative assessment have made it challenging to assess the degree to which similar diets have reproducible effects across studies. Through an unbiased subject-level meta-analysis framework, we re-analyzed 27 dietary studies including 1,101 samples from rodents and humans. We demonstrate that a high-fat diet (HFD) reproducibly changes gut microbial community structure. Finer taxonomic analysis revealed that the most reproducible signals of a HFD are Lactococcus species, which we experimentally demonstrate to be common dietary contaminants. Additionally, a machine-learning approach defined a signature that predicts the dietary intake of mice and demonstrated that phylogenetic and gene-centric transformations of this model can be translated to humans. Together, these results demonstrate the utility of microbiome meta-analyses in identifying robust and reproducible features for mechanistic studies in preclinical models

F35 mice co-expressing N-terminal deletion mutants are normal at 3 weeks.

<p>Animals of the indicated genotypes were sacrificed at 3 weeks and brain sections were stained with hematoxylin and eosin. Images show the whole cerebellum (A–F), the granule cell layer of the second cerebellar lobe (G–L), and the cerebellar white matter (M–R). Scale bars = 1 mm (A–F) and 100 µm (G–R).</p

F35 does not interact with either WT PrP or Δ23–31 PrP in co-immunoprecipitation experiments.

<p>Brain lysates from mice expressing F35 PrP in the presence or absence of either WT or Δ23–31 PrP were immunoprecipitated with Dynabeads coupled to anti-PrP antibody 6D11, or with naked beads as a control. Immunopreciptated proteins were then analyzed by Western blotting with anti-PrP antibody 6H4. The arrowhead indicates the position of F35 PrP. The faint band appearing in lane 6 between 15 and 20 kDa is distinguishable from the F35 protein, and could represent a C-terminal fragment of Δ23–31 PrP.</p

Expression of transgenes.

<p>(<b>A</b>) Brain lysates from a non-transgenic WT mouse (expressing 1× PrP), and from Tg mice expressing F35 PrP (2×), Δ23–31 PrP (1× and 6×), Δ23–111 PrP (7×), and Δ23–134 PrP (1×) were Western blotted and probed with anti-PrP antibody 6H4. (<b>B</b>) Lysates from the brains of 10 week old mice were treated with PNGase F to removed N-linked oligosaccharides. Digestion products were subjected to Western blotting using antibody 6H4 to detect PrP. Single and double asterisks mark the positions of the endogenous C1 and C2 cleavage fragments, respectively.</p

Δ23–31, Δ23–111, Δ23–134, and Δ32–134 (F35) PrP are GPI-anchored and have a cellular localization pattern similar to WT PrP.

<p>(<b>A–L</b>) The indicated constructs were transiently expressed in BHK cells along with dsRedER. Cells were incubated in the absence (A–F) or presence (G–L) of PIPLC, then surface stained for PrP (A–C, G–I: 6D11 or D–F, J–L: 6H4) on ice prior to incubating with secondary antibody (dsRedER signal in red, PrP in green). DAPI staining is shown in blue for panels D–F, J–L. Like WT PrP (H), the mutant PrP molecules are released from the plasma membrane by PIPLC treatment (I–L). (<b>M–R</b>) BHK cells transfected with the indicated constructs were permeabilized and stained with anti-PrP antibody [M–O: 6D11, P–R: 6H4 (green)], anti-giantin antibody (red), and DAPI (blue). Like WT PrP, each mutant is present both at the cell surface and intracellularly, where it colocalizes with the Golgi marker, giantin. [Scale bar in A (applicable to panels A–L, P–R) = 25 µm. Scale bar in M–O = 15 µm.].</p

N-terminally deleted forms of PrP are impaired in their ability to suppress the neurodegenerative phenotype of Tg(F35) mice.

<p>The genotype, number of mice, age at death, and relative expression levels PrP are shown for each transgenic line. While 0.5× expression of WT PrP greatly prolongs the lifespan of Tg(F35) mice, the N-terminal mutants have only a modest effect on lifespan, even at elevated expression levels. Asterisks indicate statistically significant differences in age at death compared to Tg(F35)/<i>Prn-p</i>^−/− mice (<b>**</b> p<0.001, <b>*</b> p<0.01 by Kruskal-Wallis with Dunn's secondary test).</p

Survival of mice co-expressing N-terminal deletion mutants.

<p>Each point represents the percentage of animals alive at the indicated age. Statistical analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025675#pone-0025675-t001" target="_blank">Table 1</a>.</p