The typical abnormalities observed in the brain of Alzheimer's disease (AD) patients include synaptic alterations, neuronal death, brain inflammation, and the accumulation of protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Despite the development of many animal and in vitro models for AD, there is a lack of an experimental approach that fully recapitulates essential aspects of the disease in human cells. Here, we report the generation of a new model to study AD, consisting of cerebral organoids (COs) produced from human-induced pluripotent stem cells (iPSCs). Under our experimental conditions, COs grow to form three-dimensional (3D) structures containing neural areas with cortical-like organization. Analysis of COs by histological and biochemical methods revealed that organoids produced from iPSCs derived from patients affected by familial AD or Down syndrome (DS) spontaneously develop over time pathological features of AD, including accumulation of structures highly reminiscent to amyloid plaques and neurofibrillary tangles. These pathological abnormalities were not observed in COs generated from various controls, including human iPSCs from healthy individuals, human iPSCs from patients affected by Creutzfeldt-Jakob disease, mouse embryonic stem cells (ESCs), or mouse iPSCs. These findings enable modeling genetic AD in a human cellular context in a 3D cortical-like tissue developed in vitro from patient-specific stem cells. This system provides a more relevant disease model compared to pre-existing methods and offers a new platform for discovery of novel targets and screening of drugs for therapeutic intervention.

Protein misfolding and aggregation into fibrillar deposits is a common feature of a large group of degenerative diseases affecting the central nervous system or peripheral organs, termed protein misfolding disorders (PMDs). Despite their established toxic nature, clinical trials aiming to reduce misfolded aggregates have been unsuccessful in treating or curing PMDs. An interesting possibility for disease intervention is the regular intake of natural food or herbal extracts, which contain active molecules that inhibit aggregation or induce the disassembly of misfolded aggregates. Among natural compounds, phenolic molecules are of particular interest, since most have dual activity as amyloid aggregation inhibitors and antioxidants. In this article, we review many phenolic natural compounds which have been reported in diverse model systems to have the potential to delay or prevent the development of various PMDs, including Alzheimer's and Parkinson's diseases, prion diseases, amyotrophic lateral sclerosis, systemic amyloidosis, and type 2 diabetes. The lower toxicity of natural compounds compared to synthetic chemical molecules suggest that they could serve as a good starting point to discover protein misfolding inhibitors that might be useful for the treatment of various incurable diseases.

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.

The cellular prion protein (PrPC) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrPSc in Creutzfeldt-Jakob disease. PrPC β-endoproteolysis to the C2 fragment allows PrPSc formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, ‘S1’ and ‘S3’, locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrPSc and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrPSc and infectivity can either uncouple or engage to drive the onset of clinical disease.

A tag-recapture study of the Ozark Hellbender salamander, Cryptobranc/ws a. bishopi, was made on the North Fork of the White River, Ozark Co., Missouri. During the summers of 1969 and 1970, animals were tagged along a 2.67-km stretch of stream bed. Population estimates were 428 with 95% C.L. of 341-573 hellbenders/km of stream bed. Biomass estimates were 156 kg/km with 95% C.L. of 124.5-210 kg/km of stream bed. Density estimate in "prime habitat" was one/8-10 m2 with 95% C.L. of one/6-7 mz_one/13-16 m 2 . Recaptures indicated little movement. Ozark Hellbenders are one of the dominant organisms in this stream system.

During prion infections of the central nervous system (CNS) the cellular prion protein, PrPC, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp a and Prnp b mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.

In lethal prion neurodegenerative diseases, misfolded prion proteins (PrP Sc ) replicate by redirecting the folding of the cellular prion glycoprotein (PrP C ). Infections of different durations can have a subclinical phase with constant levels of infectious particles, but the mechanisms underlying this plateau and a subsequent exit to overt clinical disease are unknown. Using tandem biophysical techniques, we show that attenuated accumulation of infectious particles in presymptomatic disease is preceded by a progressive fall in PrP C level, which constricts replication rate and thereby causes the plateau effect. Furthermore, disease symptoms occurred at the threshold associated with increasing levels of small, relatively less protease-resistant oligomeric prion particles (oPrP Sc ). Although a hypothetical lethal isoform of PrP cannot be excluded, our data argue that diminishing residual PrP C levels and continuously increasing levels of oPrP Sc are crucial determinants in the transition from presymptomatic to symptomatic prion disease. IMPORTANCEPrions are infectious agents that cause lethal brain diseases; they arise from misfolding of a cell surface protein, PrP C to a form called PrP Sc . Prion infections can have long latencies even though there is no protective immune response. Accumulation of infectious prion particles has been suggested to always reach the same plateau in the brain during latent periods, with clinical disease only occurring when hypothetical toxic forms (called PrP L or TPrP) begin to accumulate. We show here that infectivity plateaus arise because PrP C precursor levels become downregulated and that the duration of latent periods can be accounted for by the level of residual PrP C , which transduces a toxic effect, along with the amount of oligomeric forms of PrP Sc . Prions are proteinaceous, infectious particles responsible for a group of incurable neurodegenerative diseases in humans and animals. A posttranslationally misfolded version of the cellular prion protein (PrP C ), known as PrP Sc , is the primary component of a prion and propagates by acting as a template for the conformational conversion of PrP C substrate (1). Analysis of brain material by prion bioassays has shown that infectivity plateaus can exist early during disease, suggesting that infections can be divided into an infectivity phase and a toxicity phase (2-4). The accumulation of a hypothetical toxic PrP form (PrP L , "L" for lethal), distinct from PrP Sc , has been proposed to explain the transition from a subclinical phase to the appearance of clinical signs and progression to end-stage disease at the time when the prion levels plateau. It has been further suggested that prions are infectious, but nontoxic, entities that act as a catalyst for the generation of toxic PrP L at a rate with direct proportionality to PrP C expression levels in the animal models used for these experiments (2). However, this hypothetical protein has yet to be isolated.In other studies, based on falling levels of the PrP-like Sh...

Poly-L-lysine (PLL), a homopolymer of amino acid L-lysine (LL), has been frequently used for drug delivery. Here, we report that PLL is an effective agent to inhibit propagation of prions that cause fatal and incurable neurologic disorders in humans and animals, termed prion diseases. In our recent investigation on prion propagation facilitated by conversion of the cellular prion protein (PrP) to the misfolded, disease-associated PrP (PrP Sc ), we demonstrated that plasminogen stimulates PrP conversion as a cellular cofactor. In the current study, we targeted plasminogen using PLL and assessed its anti-prion efficacy. The results showed that PLL strongly inhibited PrP Sc propagation in the cell-free, cell culture, and mouse models of prion disease. These results confirm the role of plasminogen in PrP Sc propagation, validates plasminogen as a therapeutic target to combat prion disease, and suggests PLL as a potential anti-prion agent. Therefore, our study represents a proof-of-concept that targeting plasminogen, a cofactor for PrP conversion, using PLL results in suppression of prion propagation, which represents a successful translation of our understanding on details of prion propagation into a potential therapeutic strategy for prion diseases.