Neuroprotective properties of the PrP-like Shadoo glycoprotein assessed in the middle cerebral artery occlusion model of ischemia

<p>Biochemical similarities have been noted between the natively unstructured region of the cellular prion protein, PrP^C, and a GPI-linked glycoprotein called Shadoo (Sho); these proteins are encoded by the <i>Prnp</i> and <i>Sprn</i> genes, respectively. Both proteins are expressed in the adult central nervous system and they share overlapping partners, including each other, in interactome studies. As prior studies have ascribed neuroprotective properties to the N-terminal region of PrP^C, specifically the octarepeat region, we investigated Sho's neuroprotective properties. To this end we assessed Sho-null (<i>Sprn</i>^0/0) and hemizygous (<i>Sprn</i>^{<i><i>0</i>/+</i>}) mice in the middle cerebral artery occlusion (MCAO) model versus wild type mice and also vs. transgene-rescued <i>Sprn</i>^0/0-Tg<i>Sprn</i> mice. <i>Sprn</i>^0/0 mice had a tendency to greater fragility in reaching endpoint and deficits in parameters including infarct volume and neurogenesis, with a reciprocal trend noted in transgene-rescued mice; however these effects did not reach significance. Loss of both PrP^C and Sho immunostaining occurred in parallel to neuronal loss on the ipsilateral side of MCAO-lesioned animals; while focal elevations in immunostaining in the penumbra region were sometimes evident for PrP^C, they were not noted for Sho. Our studies argue against discernible neuroprotective action of Sho in the genetic backgrounds used for this MCAO paradigm. Whether or not the positively charged N-terminal regions in Sho and PrP^C fulfil different roles <i>in vivo</i> remains to be determined.</p

Apoptosis is synergistically increased when Appa and Prp1 levels are reduced. A–D.

<p>Zebrafish injected with a control morpholino (MO), low dose (sub-effective) <i>prp1</i> MO, low dose (sub-effective) <i>appa</i> MO, or a combination of sub-effective <i>appa</i> and <i>prp1</i> MOs (A–D, respectively) showed increased abundance of activated-caspase 3-positive cells (A′–D′, respectively). Higher doses of MOs used in this same assay showed individual MOs can also produce this effect (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s007" target="_blank">Fig. S7</a>). <b>E.</b> Activated caspase 3-positive cells were slightly increased when low doses of <i>prp1</i> or <i>appa</i> MOs were injected alone and synergistically increased when they were combined in one injection solution. N = 5. ** = P<0.01, * = P<0.05.</p

APP interactions with PrP are conserved from fish to mammals. A. Mouse <i>Prnp</i> can replace zebrafish <i>prp1</i> in the context of its genetic interaction with <i>appa</i>.

<p>Co-injecting zebrafish <i>prp1</i> mRNA, in concert with the Appa+Prp1 co-knockdown, rescues the observed phenotypes (first two sets of bars). <i>prp1</i>’s paralog, zebrafish <i>prp2</i>, does <u>not</u> rescue this co-knockdown, nor does another prion family member from zebrafish, <i>shadoo1</i>. In contrast, mouse <i>Prnp</i> mRNA (<i>moPrP</i>) can partially alleviate the Appa & Prp1 co-knockdown. Thus mouse PrP can replace Prp1 in the context of its interaction with App, indeed with greater efficacy than zebrafish orthologs. * p<0.05. **p<0.01. <b>B. Human </b><b><i>APP</i></b><b> can replace zebrafish </b><b><i>appa</i></b><b> in the context of its genetic interaction with </b><b><i>prp1</i></b><b>.</b> We established above that <i>appa</i> mRNA from zebrafish can rescue the co-knockdown of Appa+Prp1; Here we use <i>APPb</i> as a negative control comparator mRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone-0051305-g003" target="_blank">Fig. 3K</a>). Human <i>APP₆₉₅</i> mRNA (huAPPwt) was effective in replacing zebrafish APPa in the context of Prp1 knockdown. <b>C. Co-immunoprecipitation demonstrates an interaction between human PrP and human APP in N2a cells.</b> Left: Inputs as whole cell lysate showing expression of human PrP using the human PrP specific antibody 3F4 in N2a cells (wild type and stably transfected with human APP) transiently transfected with pcDNA3-human PrP construct but not with empty vector (“EV”). Expression of human APP is only observed in N2a cells with human APP using 6E10 antibody, specific for human APP. Input represented 7% of whole cell lysate used for co-immunoprecipitation. Right: whole cell lysates were co-immunoprecipitated using a human specific anti-APP antibody followed by immunoblotting with a human PrP specific antibody. Detection of human APP bound human PrP was observed only in N2a cells stably transfected with human APP and transiently transfected with human PrP construct. A no lysate immunoprecipitation experiment was included as an additional negative control.</p

Summary of phenotypes attained and number of trials per treatment applied.

1<p>Treatments were combinations of morpholino (MO, designed to block normal splicing) gene knockdown and/or mRNA gene expression reagents.</p>2<p>A translation blocking MO (TB-MO) was used as an independent knockdown reagent to validate some results.</p

Appa and Appb can replace each other and thus are redundant in early zebrafish development. A–D

<p>. Embryos were injected with sub-effective doses of <i>appa</i> and/or <i>appb</i> morpholino (MO). These doses produced no phenotype in the fish compared to control MO (A–C, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone-0051305-g001" target="_blank">Figure 1</a>). <b>D.</b> When sub-effective doses of <i>appa</i> and <i>appb</i> MO were combined and injected, a strong phenotype emerged consisting of morphological malformations and death of tissues within the CNS (*). <b>E.</b> Quantifying this effect, the co-injection of sub-effective doses of both MOs produced a significant decrease in the number of normal fish (green bars) and a significant increase in number of fish displaying CNS cell death (mild in light orange bars; severe in dark orange bars). ** = P<0.01. <b>F.</b> Fish injected with <i>appa</i> MO can be rescued by co-injection with <i>appa</i> mRNA * = P<0.05. A similar result was attained for <i>appb</i> MO and its cognate mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s002" target="_blank">Fig. S2</a>). <b>G, H.</b> Fish were injected with an effective dose of one MO along with cognate mRNA from the other paralog to see if rescue of the phenotype occurred. <i>appb</i> mRNA was able to effectively alleviate the phenotype caused by injection of the <i>appa</i> MO (G) and vice versa (H). ** = P<0.01.</p

<i>appa</i> interacts with <i>prp1,</i> but <i>appb</i> does not.

<p>Panels <b>A–E</b>: Sub-effective doses of <i>appa</i> and <i>prp1</i> gene knockdown synergize to produce an overt phenotype in the fish. Fish injected with a control morpholino (MO) (<b>A</b>), a sub-effective dose of <i>appa</i> (<b>B</b>) or <i>prp1</i> (<b>C</b>) MO fail to display any signs of CNS cell death or disruptions in development, i.e. no severe phenotypes. <b>D.</b> When sub-effective doses of <i>appa</i> and <i>prp1</i> are combined a severe phenotype emerges comprised of prominent morphological disruptions and an overt appearance of cell death within the CNS. <b>E.</b> The abundance of fish with normal morphology observed is significantly reduced, and the percentage of fish displaying cell death within the CNS is significantly increased when sub-effective doses of <i>appa</i> and <i>prp1</i> MOs are combined. ** = P<0.01. Panels <b>F–J</b> present a similar experimental design to panels A–E, but represent <i>appb</i> knockdown instead of <i>appa</i>. When a sub-effective doses of <i>appb</i> and <i>prp1</i> MOs are combined there is no significant increase in the number of fish showing developmental abnormalities or cell death within the CNS. <b>K</b>. Despite Appa and Appb being largely redundant during normal development (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone-0051305-g002" target="_blank">Fig. 2</a>), they cannot replace each other when PrP1 abundance is reduced. <i>appa</i> mRNA is able to alleviate the phenotype caused by co-injection of sub-effective doses of <i>appa</i> and <i>prp1</i> MOs. <i>appa</i> mRNA significantly reduced the percentage of fish displaying a severe phenotype. <i>appb</i> mRNA at an equivalent dose failed to reduce the percentage of fish displaying a phenotype. ** = P<0.01. <b>L. </b><i>app</i> mRNAs with stop codon mutations are not able to rescue the <i>app</i> or <i>appa</i>+<i>prp1</i> knockdown phenotypes. Data from the mutations S3X;E5X and 14_15 insT are shown (WT = wild type). Further analysis of these mRNAs and similar ones for <i>appb</i> was carried out in other knockdown backgrounds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s005" target="_blank">Fig. S5</a>).</p

Knockdown of APP and PrP synergize to reduce cell aggregation.

<p>Low doses of <i>prp1</i> and <i>appa</i> knockdown reagents (morpholinos, MO) are used here to show that their effects on cell adhesion synergize; higher doses of MOs used in this same assay showed individual MOs can also produce this effect. <b>A–D.</b> Zebrafish embryos injected with fluorescent dyes along with control MO (A), low dose of <i>prp1</i> MO (B), low dose of <i>appa</i> MO (C), or a combination of the two sub-effective MOs (D), were dissociated to single cells and subjected to an aggregation assay. Insets show clumped cells (or lack thereof) at higher magnification. <b>E, F.</b> Aliquots of dissociated cells taken prior to aggregation confirmed that dissociation was successful. <b>G.</b> The ability of these cells to form aggregates (10 or more cells in direct physical contact) rather than stay alone in solution was quantified. Cells with slightly reduced App or slightly reduced Prp had only marginal decreases in aggregation ability, whereas cells with both proteins reduced were significantly reduced in their aggregation ability. N = 3. * = P<0.05.</p

Primers used for gene cloning, checking morpholino efficacy and site-directed mutagenesis.

<p>Primers used for gene cloning, checking morpholino efficacy and site-directed mutagenesis.</p

Knockdown of Appa, Appb, or Prp1 results in impaired development and death of head region. A–L.

<p>Morpholino (MO) was delivered to disrupt translation of endogenous amyloid β precursor protein (APP) and prion protein (PrP) paralogs in zebrafish: <i>appa</i>, <i>appb</i>, or <i>prp1</i> (top-bottom rows, respectively). Standard control MO at levels equivalent to our effective dose fail to induce any CNS cell death or disruptions in morphology of the fish (left column). Low doses of <i>appa, appb, or prp1</i> MOs (0.5, 1.0, 0.5 ng respectively) were empirically determined to be sub-effective (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s001" target="_blank">Fig. S1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s002" target="_blank">S2</a>), leading to mild changes, but no death of CNS tissues (2^nd column). Effective doses (1.0, 2.5, 1.0 ng, respectively) lead to severe alterations in CNS morphology (*) and death of CNS tissues (3^rd column). Specificity of the MOs was demonstrated by rescuing the injection of an effective dose of <i>appa, appb, or prp1</i> MO by co-injection of the cognate mRNA (200 pg, 200 pg or 100 pg, respectively; Right column). These data are quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone-0051305-g002" target="_blank">Figs. 2F</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s001" target="_blank">S1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s002" target="_blank">S2</a>. <b>M.</b> Western blots of zebrafish lysates reveal efficacy of our MO knockdown reagents (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s003" target="_blank">Fig. S3</a>). The <i>appa</i> and <i>appb</i> splice blocking (SB) MOs used above (A–H) significantly decreases detection of protein species by the antibody 22C11 (top row). Bottom row is a β-actin loading control. An additional, independent MO that acts as a translation block of <i>appa</i> (<i>appa</i> TB) confirms this protein knockdown and produces similar phenotypes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s001" target="_blank">Fig. S1</a>). The <i>prp1</i> MO reagents used here were previously shown to be effective in knocking down protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305-MalagaTrillo2" target="_blank">[27]</a>. <b>N.</b> Quantification of western blots from three biological replicates (three independent injection trials on three separate days) demonstrate a significant decrease (*p<0.05, **p<0.01) of the APP immunoreactivity compared to β-actin with all three MO reagents at their effective doses.</p

A Common Phenotype Polymorphism in Mammalian Brains Defined by Concomitant Production of Prolactin and Growth Hormone

<div><p>Pituitary Prolactin (PRL) and Growth Hormone (GH) are separately controlled and sub-serve different purposes. Surprisingly, we demonstrate that extra-pituitary expression in the adult mammalian central nervous system (CNS) is coordinated at mRNA and protein levels. However this was not a uniform effect within populations, such that wide inter-individual variation was superimposed on coordinate PRL/GH expression. Up to 44% of individuals in healthy cohorts of mice and rats showed protein levels above the norm and coordinated expression of PRL and GH transcripts above baseline occurred in the amygdala, frontal lobe and hippocampus of 10% of human subjects. High levels of PRL and GH present in <i>post mortem</i> tissue were often presaged by altered responses in fear conditioning and stress induced hyperthermia behavioral tests. Our data define a common phenotype polymorphism in healthy mammalian brains, and, given the pleiotropic effects known for circulating PRL and GH, further consequences of coordinated CNS over-expression may await discovery.</p></div