Non-coding RNA as a trigger of neuropathologic disorder phenotypes in transgenic Drosophila.

At most, many protein-misfolding diseases develop as environmentally induced sporadic disorders. Recent studies indicate that the dynamic interplay between a wide repertoire of noncoding RNAs and the environment play an important role in brain development and pathogenesis of brain disorders. To elucidate this new issue, novel animal models which reproduce the most prominent disease manifestations are required. For this, transgenic Drosophila strains were constructed to express small highly structured, non-coding RNA under control of a heat shock promoter. Expression of the RNA induced formation of intracellular aggregates revealed by Thioflafin T in embryonic cell culture and Congo Red in the brain of transgenic flies. Also, this strongly perturbed the brain control of locomotion monitored by the parameters of sound production and memory retention of young 5-day-old males. This novel model demonstrates that expression of non-coding RNA alone is sufficient to trigger neuropathology.

Alpha2-macroglobulin is a potential facilitator of prion protein transformation.

Cellular prion protein changes conformation during transformation to an infectious scrapie isoform. One measure of transformation is the development of partial resistance to protease treatment. A fraction of human and bovine plasma was identified containing activity that facilitates transformation of cellular prion protein to a protease resistant isoform in the presence of RNA in the absence of seeded scrapie prion protein. Purification of proteins from this fraction led to the identification of alpha2-macroglobulin as an active component suggesting that it may facilitate conformational changes in prion protein in spontaneous forms of prion disease.

Pathogenic chaperone-like RNA induces congophilic aggregates and facilitates neurodegeneration in Drosophila.

Protein aggregation is a hallmark of many neurodegenerative diseases. RNA chaperones have been suggested to play a role in protein misfolding and aggregation. Noncoding, highly structured RNA recently has been demonstrated to facilitate transformation of recombinant and cellular prion protein into proteinase K-resistant, congophilic, insoluble aggregates and to generate cytotoxic oligomers in vitro. Transgenic Drosophila melanogaster strains were developed to express highly structured RNA under control of a heat shock promoter. Expression of a specific construct strongly perturbed fly behavior, caused significant decline in learning and memory retention of adult males, and was coincident with the formation of intracellular congophilic aggregates in the brain and other tissues of adult and larval stages. Additionally, neuronal cell pathology of adult flies was similar to that observed in human Parkinson's and Alzheimer's disease. This novel model demonstrates that expression of a specific highly structured RNA alone is sufficient to trigger neurodegeneration, possibly through chaperone-like facilitation of protein misfolding and aggregation.

Interaction of prion protein with small highly structured RNAs: detection and characterization of PrP-oligomers.

Conformational modification of normal prion protein (PrP(c)) to protease-resistant, beta-sheet rich, aggregates (PrP(sc)) is commonly accepted cause for prion diseases. On the other hand, several studies in recent years implicate soluble, protease-sensitive, oligomers of PrP(c) in neuronal damage. Previously, our group has shown that small, highly structured RNAs (shsRNAs), in conjunction with a serum factor, facilitated the conversion of hrPrP to a protease resistant, high molecular weight isoform. In the current study we demonstrate that shsRNAs, in the absence of the serum factor, generate soluble, protease-sensitive, and potentially toxic oligomers of ovrPrP. We have isolated a 500 kD oligomer by size exclusion chromatography of the reaction mixture and identified the accessible epitopes. The soluble PrP-oligomers were present in enhanced amounts in scrapie infected sheep brain and treating extracts of normal sheep brain with shsRNA resulted in oligomerization of endogenous PrP. Isolation, characterization of PrP-oligomers and their possible implication in prion diseases is discussed.

Concentration of prion protein from biological samples to increase the limits of detection by immunoassay.

An RNA-ligand-based adsorbent has been shown to concentrate prion protein (PrP) from solutions in a model system. The work presented here extends the utility of the RNA-based adsorbent to brain homogenates of cow, sheep, mule deer (Odocoileus hemionus) and elk (Cervus elaphus). Brain homogenates were diluted either in buffer, representing specimens used in post-mortem tests, or in serum, modelling specimens used in biological-fluid-based tests. The RNA adsorbent was effective in binding PrPC (cellular PrP,) and PrPres (proteinase K-resistant PrP) from the brain homogenates of all the species tested in both model systems. The three antibodies against PrP used in the experiments identified PrP in immunoblot analysis after concentrating PrP from brain homogenates with the adsorbent, indicating the general applicability of this technology for improving the detection of PrP in immunoassays. Utilization of RNA adsorbent increased the level of detection of PrPres by immunoblot over several-hundredfold. The results obtained suggest that this RNA adsorbent can be used to increase detection in current post-mortem immunoassays and for the development of a blood-based ante-mortem test.

Small, highly structured RNAs participate in the conversion of human recombinant PrP(Sen) to PrP(Res) in vitro.

We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP). We also demonstrate the ability of this shsRNA to form highly stable nucleoprotein complexes with hrPrP and cellular PrP (PrP(C)) from various cell extracts and mammalian brain homogenates. The apparent mass of the nucleoprotein complex is dependent on the molar ratio of hrPrP to RNA during complex formation. The hrPrP in these complexes acquires resistance to degradation by Proteinase K (PK). Other shsRNAs, however, under identical conditions, neither form stable complexes with hrPrP nor do they induce resistance to PK digestion. We also demonstrate that the RNAs in these nucleoprotein complexes become resistant to ribonuclease A hydrolysis. These interactions between shsRNAs and hrPrP suggest possible roles of RNAs in the modulation of PrP structure and perhaps disease development. ShsRNAs that bind to hrPrP with high affinity and induce resistance to PK digestion can be used to develop molecular biology assays for the screening of compounds associated with PrP structure transformation or for drugs that inhibit this process.

Prion protein interactions with nucleic acid: possible models for prion disease and prion function.

Several models for the transmission and progression of prion diseases have arisen, evolving with the acquisition of new experimental results. It is generally accepted that the PrP(Sc) protein is at least part of the infectious particle and the major protein component of the scrapie-associated fibrils (SAFs) that characterize the disease. An additional, unknown cofactor is most likely involved in transmission of the disease, perhaps by influencing the PrP(c) --> PrP(Sc) transition. This review relates experimental observations on the interactions of nucleic acids (NAs) and PrP with specific focus on alterations in structure. In particular, NAs appear to induce PrP(c) to acquire some of the structural and biochemical characteristics of PrP(Sc). An updated hypothesis is related wherein NAs, on the basis of their structure, act in the PrP(c) --> PrP(Sc) transformation by serving as catalysts and/or chaperones and not by encoding genetic information.

Concentration and removal of prion proteins from biological solutions.

The pathogenesis of prion diseases is characterized by the accumulation of amyloid-like rods or scrapie-associated fibrils. The major protein component of scrapie-associated fibrils is an abnormally folded isoform of the normal cellular prion protein (PrP(C)) that is resistant to digestion by proteinase K and is referred to as PrP(Sc). Purified human recombinant (hr PrP) was used to characterize the binding of a set of RNAs with affinity to PrP proteins. We report here that hr PrP has two RNA-binding activities at physiological pH. One activity is capable of binding all of the screened RNAs with high affinity, whereas the other activity can bind only to a subset of the RNAs with high affinity in the presence of non-specific competitor RNAs. A novel RNA belonging to the latter class, RQ11+12, bound to hr PrP with high affinity in the presence of vast molar excesses of competing RNAs. Beads impregnated with the RQ11+12 RNA were used to construct a filtration column. The column efficiently bound hr PrP and native PrP(C) from serum and urine. Importantly, the filtration device was also capable of binding proteinase K-treated PrP(Sc) from serum and urine. The level of sensitivity of detection of PrP by standard Western blotting was increased at least 1000-fold by first concentrating PrP from solution with the filtration column.