The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide—either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome. They form an important line of defense against misfolded proteins and are part of the cellular quality control system. The heat shock protein (Hsp) family, particularly Hsp70 and Hsp90, plays a major part in this process and it is well-known to regulate protein misfolding in a variety of diseases, including tau levels and toxicity in AD. However, the role of Hsp90 in regulating protein misfolding is not yet fully understood. For example, knockdown of Hsp90 and its co-chaperones in a Caenorhabditis elegans model of Aβ misfolding leads to increased toxicity. On the other hand, the use of Hsp90 inhibitors in AD mouse models reduces Aβ toxicity, and normalizes synaptic function. Stress-inducible phosphoprotein 1 (STI1), an intracellular co-chaperone, mediates the transfer of clients from Hsp70 to Hsp90. Importantly, STI1 has been shown to regulate aggregation of amyloid-like proteins in yeast. In addition to its intracellular function, STI1 can be secreted by diverse cell types, including astrocytes and microglia and function as a neurotrophic ligand by triggering signaling via the cellular prion protein (PrPC). Extracellular STI1 can prevent Aβ toxic signaling by (i) interfering with Aβ binding to PrPC and (ii) triggering pro-survival signaling cascades. Interestingly, decreased levels of STI1 in C. elegans can also increase toxicity in an amyloid model. In this review, we will discuss the role of intracellular and extracellular STI1 and the Hsp70/Hsp90 chaperone network in mechanisms underlying protein misfolding in neurodegenerative diseases, with particular focus on AD.

We report the results of solid state nuclear magnetic (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23-231, Syrian hamster sequence). Measurements of intermolecular 13 C-13 C dipole-dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer's disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13 C-13 C and 15 N-13 C solid state NMR spectra of a uniformly 15 N, 13 C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals can not be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173-224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90-231 of the human PrP sequence, formed under somewhat different conditions, suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.

The transmissible agent of prion disease consists of prion protein in β-sheet rich state (PrPSc), which can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrPSc template. Here three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrPSc, whereas an alternative amyloid state, with a folding pattern different from that of PrPSc induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrPSc emerged. The dynamics between the two forms suggest that atypical PrPres gave rise to PrPSc. While no PrPSc was found in preparations of fibrils using Protein Misfolding Cyclic Amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb suggesting that atypical PrPres was the first product of PrPC misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrPSc-templated or spontaneous conversion of PrPC into PrPSc exists. This study provides compelling evidence that non-infectious amyloids with a structure different from that of PrPSc could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

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Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro.