The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS‐expressed Doppel or internally deleted PrP (‘ΔPrP’). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC‐like protein, ‘π’. Shadoo (Sho) is a hypothetical GPI‐anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N‐terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC‐deficient dendritic processes. In Prnp 0/0 CGNs, Sho transgenes were PrPC‐like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion‐infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC‐like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP ^C ). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP ^C -like function to maintain the viability of Prnp ^0/0 mice lacking the PrP ^C 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 Mtg 1 gene transcription unit that overlaps the Sprn 3′ UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn ^0/0 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 Prnp ^0/0 embryos using lentiviruses targeted against the Sprn 3′ UTR, we established that double-knockout mice deficient in both Sho and PrP ^C 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 PrP ^C and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP ^C , we infer that Sho’s activity will prove germane to the maintenance of neuronal viability in postnatal life.

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrP^Sc), which is derived from its cellular precursor (PrP^C), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrP^C and Sho, we inferred that PrP^C levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrP^C glycosylation and proteolytic processing, net reductions in PrP^C occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrP^C results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrP^Sc in vitro. While PrP^C downregulation is not discernible in animals with unusually short incubation periods and high PrP^C expression, slowly evolving prion infections exhibit downregulation of the PrP^C substrate required for new PrP^Sc synthesis and as a receptor for pathogenic signaling. Our data reveal PrP^C downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains.

Rapid replication in vitro implicates distinct populations of misfolded hippocampal 4R TAU protein in accelerated progression of Alzheimer’s disease.

Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer’s disease (AD) pathology. Amyloid beta (Aβ) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aβ activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors. The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aβ1–42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain. Acute applications of human amylin or Aβ1–42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1β, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1β. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls. Microglial amylin receptors mediate Aβ-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Cellular prion protein (PrP^C) misfolds to form infectivity‐associated scrapie prion protein and generates C‐terminal fragments C1 and C2 in healthy and prion‐infected animals. C1 cleavage occurs N‐terminally of PrP^C's hydrophobic domain, whereas the larger C2 fragment is generated by cleavage at the end of the octarepeat region. As the PrP‐like proteins Doppel and Shadoo (Sho) have been reported to inhabit similar membrane environments as PrP^C, we investigated endoproteolysis by using a panel of mutant alleles. Doppel undergoes efficient in vivo cleavage at a C1 site mapped to the start of the globular domain, which is a structurally similar cleavage site to that in PrP^C. Sho is processed to C1 and C2 fragments, and proved refractory to mutagenesis to inactivate C1 cleavage. As a reciprocal product of C1 cleavage, Sho also engenders a metabolically stable N1 fragment with a C‐terminus after its hydrophobic domain, an observation that may account for N1's association with membrane and/or cellular fractions in vitro and in vivo. Our data indicate that glycosylation status and yet to be identified proteases modulate internal C1 and C2 proteolysis events within the mammalian prion protein family.