Susceptibility of Beavers to Chronic Wasting Disease

Chronic wasting disease (CWD) is a contagious, fatal, neurodegenerative prion disease of cervids. The expanding geographical range and rising prevalence of CWD are increasing the risk of pathogen transfer and spillover of CWD to non-cervid sympatric species. As beavers have close contact with environmental and food sources of CWD infectivity, we hypothesized that they may be susceptible to CWD prions. We evaluated the susceptibility of beavers to prion diseases by challenging transgenic mice expressing beaver prion protein (tgBeaver) with five strains of CWD, four isolates of rodent-adapted prions and one strain of Creutzfeldt–Jakob disease. All CWD strains transmitted to the tgBeaver mice, with attack rates highest from moose CWD and the 116AG and H95+ strains of deer CWD. Mouse-, rat-, and especially hamster-adapted prions were also transmitted with complete attack rates and short incubation periods. We conclude that the beaver prion protein is an excellent substrate for sustaining prion replication and that beavers are at risk for CWD pathogen transfer and spillover. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP

BACKGROUND: The cellular prion protein PrP(C) is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP(Sc) isoform in prion diseases. The prototype prion disease is scrapie in sheep, and whereas Prnp exhibits common missense polymorphisms for V136A, R154H and Q171R in ovine populations, genetic variation in mouse Prnp is limited. Recently the CNS glycoprotein Shadoo (Sho) has been shown to resemble PrP(C) both in a central hydrophobic domain and in activity in a toxicity assay performed in cerebellar neurons. Sho protein levels are reduced in prion infections in rodents. Prompted by these properties of the Sho protein we investigated the extent of natural variation in SPRN. PRINCIPAL FINDINGS: Paralleling the case for ovine versus human and murine PRNP, we failed to detect significant coding polymorphisms that alter the mature Sho protein in a sample of neurologically normal humans, or in diverse strains of mice. However, ovine SPRN exhibited 4 missense mutations and expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats R1-R5 encoding Sho's hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)(3)Gly70 to 67(Ala)(5)Gly72 reached frequencies of 20%, with other alleles including Delta67-70 and a 67(Ala)(6)Gly73 expansion. Sheep V71, A71, Delta67-70 and 67(Ala)(6)Gly73 SPRN alleles encoded proteins with similar stability and posttranslational processing in transfected neuroblastoma cells. SIGNIFICANCE: Frequent coding polymorphisms are a hallmark of the sheep PRNP gene and our data indicate a similar situation applies to ovine SPRN. Whether a common selection pressure balances diversity at both loci remains to be established

<i>Still Heart</i> Encodes a Structural HMT, SMYD1b, with Chaperone-Like Function during Fast Muscle Sarcomere Assembly

<div><p>The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The <i>still heart</i> mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be <i>smyd1b</i> and the <i>still heart</i> mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors <i>gata4</i>, <i>gata5</i> and <i>hand2</i>. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.</p></div

SMYD1b is co-regulated with other myosin chaperones HSP90a1 and UNC45b.

<p>In-situ hybridization staining of wild type and <i>still heart</i> embryos shows that <i>hsp90a1</i> expression is normal in <i>still heart</i> mutants, when compared to wild type embryos at 19hpf (A&B). However, the expression of <i>hsp90a1</i> and <i>unc45b</i> dramatically increases in <i>still heart</i> mutants at 24hpf (C-F & K) and 48hpf throughout the somites (G-J & M). Additionally, <i>smyd1b</i> expression increases significantly when UNC45b is absent in <i>steif</i> mutants (N), supporting co-regulation of these three genes. (O) A time course of <i>smyd1b</i> expression during muscle formation reveals that <i>smyd1b</i> is expressed early at 10hpf when <i>unc45b</i> is expressed and increases in its expression as muscle development progresses. Due to the rapid development of <i>unc45b</i> staining in the somites of the embryo in panel J, the head while present, has no background staining and is not clear in this focal plane. (qPCR: n = 3, 30 embryos each time/phenotype. Error bars are standard deviation.).</p

SMYD1b is required for fast myosin incorporation during sarcomere assembly.

<p>At 48hpf, fast myosin (F310—green) and actin (phalloidin—red) staining is visible in the premyofibrils in wt zebrafish tails (A&B), while absent from the premyofibrils in <i>sth</i> fast muscle tissue (C&D). Slow muscle (F59) develops normally in both wild type and <i>still heart</i> zebrafish at 48hpf (E&F, G&H). At 19hpf, in wild type embryos, fast myosin (F310) is beginning to be incorporated into the maturing myofibril (I) and overlaps (white arrowheads) with the developing actin (phalloidin) (J) fibers in trunk muscle (K, merge, K’ inset, white arrowhead). Fast myosin is not incorporated into the maturing premyofibril (L), although actin fibers are still present (M&N, N’ inset).</p

<i>Still heart</i>, a <i>smyd1b</i> mutant, has defects in heart and fast skeletal muscle tissue.

<p>A lateral view of 48hpf wild type (A) and <i>still heart</i> (<i>sth</i>) mutants (B), which have pericardial edema, small eyes, malformed head and reduced motility. Black arrowheads highlight the pericardial edema in sth mutants and the absence of edema in wild type. White arrowheads indicate blood pooling in the mutant and the absence of pooling in wild type. (C&D) <i>Sth</i> mutant hearts are underdeveloped and do not beat. (E-G) Examination of lateral myofibers at 5dpf under DIC microscopy revealed striations, indicative of fully formed sarcomeres, are visible in the myofibers of wild type muscle (E, black arrowheads), while absent in the fast muscle of <i>still heart</i> mutants (F); striations are present in <i>sth</i> slow muscle (G, black arrowhead) but are disturbed by nuclei and fluid-filled spaces (G, white arrowhead). Sequencing of <i>smyd1b</i> cDNA from wild type embryos (H) and <i>sth</i> mutant embryos (I) revealed a 9 nucleotide insertion between exon 1 and 2 in the <i>smyd1b</i> mRNA, creating an in-frame stop codon (I, underlined sequence). The insertion is the first 9 nucleotides of intron one as sequenced from wild type smyd1b genomic sequence (J). This is a result of a transition mutation in the splice donor site of intron 1 (I, green letter in sequence, J, outlined letter in sequence). This results in a premature truncation of the SMYD1b protein after exon 1 (K).</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

<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

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

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