Nathalie Daude

Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrPC) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrPC interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrPC paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrPC and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrPC with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrPSc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrPC organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrPC-deficiency

The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrPC). Sho has been considered a candidate for the hypothetical π protein that supplies a PrPC-like function to maintain the viability of Prnp0/0 mice lacking the PrPC protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a “C1” C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, “N1,” which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg1 gene transcription unit that overlaps the Sprn 3′ UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn0/0 mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp0/0 embryos using lentiviruses targeted against the Sprn 3′ UTR, we established that double-knockout mice deficient in both Sho and PrPC are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrPC and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrPC, we infer that Sho’s activity will prove germane to the maintenance of neuronal viability in postnatal life.

PrPc Does Not Mediate Internalization of PrPSc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells

ABSTRACT We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface.

Amyloid Beta Precursor Protein and Prion Protein Have a Conserved Interaction Affecting Cell Adhesion and CNS Development

Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of APP and/or PRNP homologs in the zebrafish (appa, appb, prp1, and prp2) produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrPC proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.

Down-Regulation of Shadoo in Prion Infections Traces a Pre-Clinical Event Inversely Related to PrPSc Accumulation

During prion infections of the central nervous system (CNS) the cellular prion protein, PrPC, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp a and Prnp b mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.

Regulation of amyloid β oligomer binding to neurons and neurotoxicity by the prion protein-mGluR5 complex

The prion protein (PrPC) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrPC, laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrPC and mGluR5 are co-receptors also for β-amyloid oligomers (AβOs) and have been shown to modulate toxicity and neuronal death in Alzheimers disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrPC-mGluR5 or AβO-PrPC-mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrPC-mGluR5 has an important role in AβO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrPC (Ln-γ1) binds to PrPC and induces intracellular Ca2+ increase in neurons via the complex PrPC-mGluR5. Ln-γ1 promotes internalization of PrPC and mGluR5 and transiently decreases AβO biding to neurons; however, the peptide does not impact AβO toxicity. Given that mGluR5 is critical for toxic signaling by AβOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrPC-mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrPC-mGluR5 may modulate signaling intensity by different PrPC ligands.

Wild-type Shadoo proteins convert to amyloid-like forms under native conditions

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06575.x

Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains

ABSTRACT Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrPC). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrPC modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95+) with distinct biological properties. Transmission of first-passage tg60CWD-H95+ isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrPC primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrPC molecules results in the generation of novel CWD strains. IMPORTANCE CWD prions are contagious among wild and captive cervids in North America and in South Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrPC) to the emergence of a novel CWD strain (H95+). We show that, upon infection, deer expressing H95-PrPC molecules accumulated a mixture of CWD strains that selectively propagated depending on the PRNP genotype of the host in which they were passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95+ CWD prions. The potential for the generation of novel strains raises the possibility of an expanded host range for CWD.

Biological properties of the PrP-like Shadoo protein.

The SPRN gene encodes the Shadoo glycoprotein (Sho), a central nervous system-expressed member of the prion protein superfamily. Sho has similarity to two features within PrPCs natively unstructured N-terminus, a hydrophobic domain and tandem repeats with positively charged residues. Indeed, scrutiny of Shos biochemical properties in uninfected cells has revealed overlaps with the properties of PrPC, these including shared protein binding partners. SPRN is conserved in mammals, as is the prion gene PRNP, but in sheep SPRN and PRNP are both marked by polymorphic variation, suggestive of a shared selection pressure within these scrapie disease-prone livestock animals. In rodent models of prion disease there are reduced levels of Sho in infected tissues, defining a form of cross-regulation between full-length Sho holoprotein and PrPSc. In human prion disease an SPRN signal peptide polymorphism is associated with risk for sporadic Creutzfeldt-Jakob Disease (CJD), while two patients with early-onset variant CJD carried putatively inactive SPRN alleles. Further investigation of Sho as a novel tracer or modifier for the accumulation of pathologic forms of PrP may prove advantageous.

Frequent missense and insertion/deletion polymorphisms in the ovine Shadoo gene parallel species-specific variation in PrP.

Background The cellular prion protein PrPC is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrPSc 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 PrPC 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 Shos hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)3Gly70 to 67(Ala)5Gly72 reached frequencies of 20%, with other alleles including Δ67–70 and a 67(Ala)6Gly73 expansion. Sheep V71, A71, Δ67–70 and 67(Ala)6Gly73 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.