Prognostic serum miRNA biomarkers associated with Alzheimer's disease shows concordance with neuropsychological and neuroimaging assessment.

There is no consensus for a blood-based test for the early diagnosis of Alzheimer's disease (AD). Expression profiling of small non-coding RNA's, microRNA (miRNA), has revealed diagnostic potential in human diseases. Circulating miRNA are found in small vesicles known as exosomes within biological fluids such as human serum. The aim of this work was to determine a set of differential exosomal miRNA biomarkers between healthy and AD patients, which may aid in diagnosis. Using next-generation deep sequencing, we profiled exosomal miRNA from serum (N=49) collected from the Australian Imaging, Biomarkers and Lifestyle Flagship Study (AIBL). Sequencing results were validated using quantitative reverse transcription PCR (qRT-PCR; N=60), with predictions performed using the Random Forest method. Additional risk factors collected during the 4.5-year AIBL Study including clinical, medical and cognitive assessments, and amyloid neuroimaging with positron emission tomography were assessed. An AD-specific 16-miRNA signature was selected and adding established risk factors including age, sex and apolipoprotein ɛ4 (APOE ɛ4) allele status to the panel of deregulated miRNA resulted in a sensitivity and specificity of 87% and 77%, respectively, for predicting AD. Furthermore, amyloid neuroimaging information for those healthy control subjects incorrectly classified with AD-suggested progression in these participants towards AD. These data suggest that an exosomal miRNA signature may have potential to be developed as a suitable peripheral screening tool for AD.

Rescue of neurophysiological phenotype seen in PrP null mice by transgene encoding human prion protein.

The prion protein (PrP) is central to the aetiology of the prion diseases, transmissible neurodegenerative conditions of humans and animals. PrP null mice show abnormalities of synaptic neurophysiology, in particular weakened GABAA receptor-mediated fast inhibition and impaired long-term potentiation in the hippocampus. Here we demonstrate that this PrP null phenotype is rescued in mice with a high copy number of a transgene encoding human PrP but not in low copy number mice, confirming the specificity of the phenotype for loss of function of PrP. The ability of human PrP to compensate for loss of murine PrP will allow direct study of the functional consequences of the 18 human PrP mutations, which cause the inherited prion diseases; this phenotype can now form the basis of the first functional assay for PrP.

Strain-specific prion-protein conformation determined by metal ions.

In animals infected with a transmissible spongiform encephalopathy, or prion disease, conformational isomers (known as PrPSc proteins) of the wild-type, host-encoded cellular prion protein (PrPc) accumulate. The infectious agents, prions, are composed mainly of these conformational isomers, with distinct prion isolates or strains being associated with different PrPSc conformations and patterns of glycosylation. Here we show that two different human PrPSc types, seen in clinically distinct subtypes of classical Creutzfeldt-Jakob disease, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrPSc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals.

Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations.

Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly alpha-helical to beta-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native alpha conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in beta structure. The soluble beta form (beta-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to beta-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation.

Non-invasive screening of a microRNA-based dysregulation signature in oral cancer and oral potentially malignant disorders.

INTRODUCTION: We have previously shown that oral swirls are a robust source of microRNA protected by extracellular vesicles, potentially useful to detect oral squamous cell carcinoma (OSCC)-associated molecular aberration. OBJECTIVES: To study a developed dysregulation score and risk classification algorithm based upon a panel of OSCC-associated microRNA in oral swirls from individuals with OSCC and oral potentially malignant disorders (OPMDs). MATERIALS AND METHODS: An OSCC-associated panel of 5 microRNAs (miR-24; miR-21; miR-99a; let-7c; miR-100;) was quantified by qPCR in 190 individuals with and without mucosal abnormalities, including OSCC (n = 53) and OPMDs (n = 74). Each sample was analyzed using a developed dysregulation score (dSCORE) and risk classification algorithm, allocating a LOW- or HIGH-RISK score. The influence of demographic, systemic, oral health and mucosal disease factors on the developed test was analyzed. RESULTS: MicroRNA for analysis can be predictably isolated from oral swirls sourced from individuals with a range of demographic, systemic and oral health findings. Utilizing the presence of HIGH-RISK identified OSCC patients with 86.8% sensitivity and 81.5% specificity. Older age and female gender were associated with higher dSCOREs and higher proportions of HIGH-RISK classification amongst individuals with no mucosal abnormalities. The dSCOREs for all subgroups of OPMDs were significantly different from the OSCC group. CONCLUSION: This is the first comparison of microRNA sourced from oral swirls from individuals with OPMDs with individuals with and without OSCC. A HIGH-RISK dysregulation signature was found to be accurate in indicating the presence of OSCC and exampled to parallel malignant transformation.

Molecular biology of prion propagation.

The occurrence of new variant Creutzfeldt-Jakob disease and the experimental confirmation that it is caused by the same prion strain as BSE has dramatically highlighted the need for a precise understanding of the molecular basis of prion propagation. The molecular basis of prion-strain diversity, previously a major challenge to the protein-only model, is now becoming clearer. The conformational change thought to be central to prion propagation, from a predominantly alpha-helical fold to one predominantly comprising beta-structure, can now be reproduced in vitro, and the ability of beta-PrP to form fibrillar aggregates provides a plausible molecular mechanism for prion propagation. These and other advances in the fundamental biology of prion propagation are leading to prion diseases becoming arguably the best understood of the neurodegenerative conditions and strategies for the development of rational therapeutics are becoming clearer.

Is covalently crosslinked Abeta responsible for synaptotoxicity in Alzheimer's disease?

Alzheimer's disease (AD) is the most common form of dementia in the elderly, and is characterized by the deposition of extracellular amyloid plaques primarily composed of the beta-amyloid peptide (Abeta). While these plaques define the pathology of AD, disease progression has been shown to correlate more closely with the level of synaptotoxicity induced by soluble Abeta oligomers. Recent evidence suggests that these oligomers are covalently crosslinked, possibly due to the interaction of Abeta with redox-active metal ions. These findings offer new avenues for the treatment and prevention of disease, by modulating metal binding or preventing the formation of neurotoxic Abeta oligomers. An understanding of the chemical nature of Abeta is also required to elucidate the synaptotoxic process or processes in AD, which have so far resisted explanation.

Multiple folding pathways for heterologously expressed human prion protein.

Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC. Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1. The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate. This equilibrium intermediate has a reduced helical content and aggregates over several hours. When the disulphide bond is reduced the protein adopts different conformations depending upon pH. At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure. In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo. Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.

Disruption of prion protein-HOP engagement impairs glioblastoma growth and cognitive decline and improves overall survival.

Glioblastomas (GBMs) are resistant to current therapy protocols and identification of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)-Hsp90-organizing protein (HOP) with cellular prion protein (PrP(C)) triggers a large number of trophic effects in the nervous system. We found that both PrP(C) and HOP are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grades I-III. High levels of PrP(C) and HOP were associated with greater GBM proliferation and lower patient survival. HOP-PrP(C) binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrP(C) binding site (HOP230-245) abrogates this effect. PrP(C) knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230-245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230-245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrP(C)-HOP engagement is a promising approach for GBM therapy.

Molecular screening of sheep for bovine spongiform encephalopathy.

Bovine spongiform encephalopathy (BSE) may have transmitted to sheep through feed and pose a risk to human health. Sheep BSE cannot be clinically distinguished from scrapie, and conventional strain typing would be impractical on a significant scale. As human prion strains can be distinguished by differences in prion protein (PrPsc) conformation and glycosylation we have applied PrP(Sc) typing to sheep. We found multiple Western blot patterns of PrP(Sc) in scrapie, consistent with the known scrapie strain diversity in sheep. Sheep passaged BSE showed a PrP(Sc) banding pattern similar to BSE passaged in other species [Collinge, J., Sidle, K.C.L., Meads, J., Ironside, J. and Hill, A.F., Nature, 383 (1996) 685-690], both in terms of fragment size following proteinase K cleavage and abundance of diglycosylated PrP. However, none of the historical or contemporary scrapie cases studied had a PrP(Sc) type identical to sheep BSE. While more extensive studies, including sheep of all PrP genotypes, will be required to fully evaluate these findings, these results suggest that large scale screening of sheep for BSE may be possible.

Generating recombinant C-terminal prion protein fragments of exact native sequence.

Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aß1-40 versus Aß1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or ß-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (∼10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations.

A cell line infectible by prion strains from different species.

It has been shown previously that ovine prion protein (PrP(C)) renders rabbit epithelial RK13 cells permissive to the multiplication of ovine prions, thus providing evidence that species barriers can be crossed in cultured cells through the expression of a relevant PrP(C). The present study significantly extended this observation by showing that mouse and bank vole prions can be propagated in RK13 cells that express the corresponding PrP(C). Importantly, the respective molecular patterns of abnormal PrP (PrP(res)) and, where examined, the neuropathological features of the infecting strains appeared to be maintained during the propagation in cell culture. These findings indicate that RK13 cells can be genetically engineered to replicate prion strains faithfully from different species. Such an approach may facilitate investigations of the molecular basis of strain identity and prion diversity.

Packaging of prions into exosomes is associated with a novel pathway of PrP processing.

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.

Australian sporadic CJD analysis supports endogenous determinants of molecular-clinical profiles.

OBJECTIVE: To define the protease-resistant prion protein (PrPres) types and associated clinical profiles in Australian patients with sporadic Creutzfeldt-Jakob disease (CJD) to allow comparison with those reported from other continents and concomitantly reaffirm absence of variant CJD (vCJD). METHODS: Reassessment of available clinical and neuropathologic data on patients referred to the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR) who died between January 1, 1992, and June 30, 2003, was conducted. Molecular classification of PrPres was determined by immunoblot analysis of available frozen brain tissue. Brain homogenate pH and codon 129 genotype on the prion protein gene (PRNP) were established. RESULTS: PrPres patterns in 35 of 37 patients with sporadic CJD conformed to one of three common reported types. Of a range of clinical features assessed, illness duration was the only clinical feature significantly associated with PrPres type. Two patients displayed coexistence of more than one PrP type, with one displaying a novel pattern of three PrPres types in a single brain region. The absence of vCJD was reconfirmed, supported by the lack of the typical PrPres glycoform pattern. CONCLUSIONS: Given Australia's geographic isolation and environmental uniqueness, the general congruity of these results with those reported from other continents suggests that endogenous factors predominantly determine sporadic Creutzfeldt-Jakob disease (CJD) phenotypic subtypes or "strains." These results support a clinicopathologic classification system whereby both PrPres type and codon 129 genotype are utilized to most accurately depict phenotypic subtypes or strains of sporadic CJD.

Protease-resistant prion protein produced in vitro lacks detectable infectivity.

The 'protein-only' hypothesis of prion propagation argues that infectious prions consist of PrP(Sc), a conformational isomer of host-derived prion protein (PrP(C)), which can be distinguished from PrP(C) by its partial resistance to proteases. While protease-resistant PrP has been produced by mixing PrP(Sc) and recombinant-derived PrP(C) in vitro, bioassay of any new infectivity has been precluded by the need to use a large molar excess of same species PrP(Sc). Transgenic mice expressing a chimaeric hamster-mouse PrPC (MH2M PrP(C)) are, unlike conventional mice, highly susceptible to Sc237 hamster scrapie. In addition, they produce MH2M PrP(Sc) and infectivity which is pathogenic for conventional mice. We have therefore attempted to produce MH2M PrP(Sc) in vitro as any infectivity produced could be distinguished from the hamster PrP(Sc) used to promote the conversion by bioassay in conventional mice. Although protease-resistant MH2M PrP was produced, no infectivity was detected on bioassay. These results argue that acquisition of protease resistance by PrP(C) is not sufficient for the propagation of infectivity.

Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD.

Strains of transmissible spongiform encephalopathies are distinguished by differing physicochemical properties of PrPSc, the disease-related isoform of prion protein, which can be maintained on transmission to transgenic mice. 'New variant' Creutzfeldt-Jakob disease (CJD) has strain characteristics distinct from other types of CJD and which resemble those of BSE transmitted to mice, domestic cat and macaque, consistent with BSE being the source of this new disease. Strain characteristics revealed here suggest that the prion protein may itself encode disease phenotype.

Species-barrier-independent prion replication in apparently resistant species.

Transmission of prions between mammalian species is thought to be limited by a "species barrier," which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.