Inability to form new memories is an early clinical sign of Alzheimer's disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ 1-42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ 1-42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrP C ) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ 1-42 oligomers interact with PrP C , with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ 1-42 oligomers are responsible for cognitive impairment in AD and that PrP C is not required.Alzheimer | neurotoxicity | object recognition test | surface plasmon resonance | protein aggregation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.

Background: The cellular prion protein (PrPC) could be a toxicity-transducing receptor for amyloid-β (Aβ) oligomers.Results: N1, a naturally occurring fragment of PrPC, binds Aβ oligomers, inhibits their polymerization into fibrils, and suppresses their neurotoxic effects in vitro and in vivo.Conclusion: N1 binds tightly to Aβ oligomers and blocks their neurotoxicity.Significance: Administration of exogenous N1 or related peptides may represent an effective therapy for Alzheimer disease.

The presence of the cellular prion protein (PrPC) on the cell surface is critical for the neurotoxicity of prions. Although a number of biological activities have been attributed to PrPC, a definitive demonstration of its physiological function remains elusive. In this review, we will discuss some of the proposed functions of PrPC, focusing on recently suggested roles in cell adhesion, regulation of ionic currents at the cell membrane, and neuroprotection. We will also discuss recent evidence supporting the idea that PrPC may function as a receptor for soluble oligomers of the amyloid β peptide and possibly other toxic protein aggregates. These data suggest surprising new connections between the physiological function of PrPC and its role in neurodegenerative diseases beyond those caused by prions.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrP Sc ) of the cellular prion protein (PrP C ), caused epidemic and epizootic episodes [ 1 ]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS) [ 2 ] or β-solenoid conformations [ 3 ]. Similar folding models have also been proposed for PrP Sc , although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrP Sc is structured as a 4-rung β-solenoid (4RβS) [ 4 , 5 ]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrP Sc , based on the 4RβS architecture. The stability of this new PrP Sc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrP C conversion into PrP Sc . This study provides the most updated, experimentally-driven and physically-coherent model of PrP Sc , together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.

There is evidence that alterations in the normal physiological activity of PrP C contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP C has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein.We report that the toxic activity of ⌬105-125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP Sc formation, suggesting that common structural features may govern both the functional activity of PrP C and its conversion to PrP Sc .Prion diseases or transmissible spongiform encephalopathies comprise a group of fatal neurodegenerative disorders in humans and animals that can be sporadic, infectious, or genetic in origin (1, 2). The prion protein (PrP C ) is a membrane-anchored glycoprotein with no widely agreed-upon physiological function, although its ability to convert into a self-propagating isoform (PrP Sc ) is associated with development of prion diseases. PrP C , thus, plays a crucial role in prion pathogenesis as a substrate for generation of PrP Sc , a conclusion that has been demonstrated by the resistance of PrP-null mice to prion infection (3). In addition, however, there is evidence that PrP C is required for delivery of a toxic signal during prion propagation. This is demonstrated by the fact that brain tissue from PrP-null mice grafted into wild-type animals remains healthy despite the presence of copious amounts of PrP Sc from the surrounding host brain (4). Moreover, conditional genetic ablation of neuronal PrP C allows pathological and clinical recovery of prioninfected mice (5). Therefore, prion neurotoxicity is likely due to subversion of normal PrP C function rather than loss of PrP C or gain of PrP Sc activity (6). However, progress in investigating this mechanism has been hampered by a lack of understanding of the physiological role of PrP C and the absence of assays to measure the functional activity of the protein. To address this issue, our laboratory has recently developed two assays that measure toxicity of PrP mutants expressed in cultured cells. The first assay is a drugbased cellular assay (DBCA) 3 that measures cell death resulting from increased accumulation of two classes of drugs that are normally used to select transfected cell lines (aminoglycosides and bleomycin analogues) (7, 8). The second assay utilizes whole-cell patch clamping to measure the la...

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

In prion diseases, the infectious isoform of the prion protein (PrP Sc ) may subvert a normal, physiological activity of the cellular isoform (PrP C ). A deletion mutant of the prion protein (⌬105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing ⌬105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of ⌬105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing ⌬105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.