Rodrigo Morales

Molecular cross talk between misfolded proteins in animal models of alzheimer's and prion diseases

The central event in protein misfolding disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated with different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimers and prion diseases. For this purpose, we inoculated prions in an Alzheimers transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion-infected mice compared with their noninoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimers and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs.

De novo induction of amyloid-β deposition in vivo

Alzheimers disease (AD), the most common type of senile dementia, is associated to the build-up of misfolded amyloid-β (Aβ) in the brain. Although compelling evidences indicate that the misfolding and oligomerization of Aβ is the triggering event in AD, the mechanisms responsible for the initiation of Aβ accumulation are unknown. In this study, we show that Aβ deposition can be induced by injection of AD brain extracts into animals, which, without exposure to this material, will never develop these alterations. The accumulation of Aβ deposits increased progressively with the time after inoculation, and the Aβ lesions were observed in brain areas far from the injection site. Our results suggest that some of the typical brain abnormalities associated with AD can be induced by a prion-like mechanism of disease transmission through propagation of protein misfolding. These findings may have broad implications for understanding the molecular mechanisms responsible for the initiation of AD, and may contribute to the development of new strategies for disease prevention and intervention.

Protein misfolding cyclic amplification for diagnosis and prion propagation studies

Diverse human disorders are thought to arise from the misfolding and aggregation of an underlying protein. Among them, prion diseases are some of the most intriguing disorders that can be transmitted by an unprecedented infectious agent, termed prion, composed mainly (if not exclusively) of the misfolded prion protein. The hallmark event in the disease is the conversion of the native prion protein into the disease-associated misfolded protein. We have recently described a novel technology to mimic the prion conversion process in vitro. This procedure, named protein misfolding cyclic amplification (PMCA), conceptually analogous to DNA amplification by polymerase chain reaction (PCR), has important applications for research and diagnosis. In this chapter we describe the rational behind PMCA and some of the many potential applications of this novel technology. We also describe in detail the technical and methodological aspects of PMCA, as well as its application in automatic and serial modes that have been developed with a view to improving disease diagnosis.

Estimating prion concentration in fluids and tissues by quantitative PMCA

Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrPSc) in brain, spleen, blood and urine of scrapie-affected hamsters.

Cross-seeding of misfolded proteins: implications for etiology and pathogenesis of protein misfolding diseases.

Accumulation of misfolded protein aggregates is a hallmark event in diverse diseases. These structures are able to seed their own polymerization by acting as aggregation nuclei both in vitro and in vivo. Recent studies in animal models suggest that misfolded proteins associated with different diseases can synergize in a heterologous fashion, potentiating pathological mechanisms and accelerating disease progression. The coexistence of misfolded protein aggregates has been described in patients affected by several protein misfolding disorders, suggesting a possible molecular cross-talk between pathological processes associated with different diseases. One putative mechanism for this cross-talk is a direct interaction between misfolded proteins, leading to cross-seeding of protein aggregation. This article summarizes the evidence for the cross-seeding phenomenon recently obtained in studies performed in vitro, in animal models, and in human patients, as well as the potential contribution of this mechanism to our understanding of the still elusive etiology and progression of maladies such as Alzheimers disease, where no effective diagnostic or therapeutic strategies exist.

Protein misfolding cyclic amplification of infectious prions

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrPC) is converted into the abnormal isoform (PrPSc). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrPC and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrPSc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.

Cross currents in protein misfolding disorders: interactions and therapy.

Protein Misfolding Disorders (PMDs) are a group of diseases characterized by the accumulation of abnormally folded proteins. Despite the wide range of proteins and tissues involved, PMDs share similar molecular and pathogenic mechanisms. Several epidemiological, clinical and experimental reports have described the co-existence of PMDs, suggesting a possible cross-talk between them. A better knowledge of the molecular basis of PMDs could have important implications for understanding the mechanism by which these diseases appear and progress and ultimately to develop novel strategies for treatment. Due to their similar molecular mechanisms, common therapeutic strategies could be applied for the diseases in this group.

Prion Protein Glycosylation Is Not Required for Strain-Specific Neurotropism

ABSTRACT In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrPSc) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrPC) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrPSc molecules. Both RML- and 301C-derived prions containing unglycosylated PrPSc molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrPSc deposition and neuronal vacuolation. These results show that PrPSc glycosylation is not necessary for strain-dependent prion neurotropism.

Grass Plants Bind, Retain, Uptake, and Transport Infectious Prions

Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrP(Sc)) to plants. Small quantities of PrP(Sc) contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-plant interaction occurs with prions from diverse origins, including chronic wasting disease. Furthermore, leaves contaminated by spraying with a prion-containing preparation retained PrP(Sc) for several weeks in the living plant. Finally, plants can uptake prions from contaminated soil and transport them to aerial parts of the plant (stem and leaves). These findings demonstrate that plants can efficiently bind infectious prions and act as carriers of infectivity, suggesting a possible role of environmental prion contamination in the horizontal transmission of the disease.

Prion-like features of misfolded Aβ and tau aggregates

Recent findings have shown that several misfolded proteins can transmit disease pathogenesis in a prion-like manner by transferring their conformational properties to normally folded units. However, the extent by which these molecule-to-molecule or cell-to-cell spreading processes reflect the entire prion behavior is now subject of controversy, especially due to the lack of epidemiological data supporting inter-individual transmission of non-prion protein misfolding diseases. Nevertheless, extensive research has shown that several of the typical characteristics of prions can be observed for Aβ and tau aggregates when administered in animal models. In this article we review recent studies describing the prion-like features of both proteins, highlighting the similarities with bona fide prions in terms of inter-individual transmission, their strain-like conformational diversity, and the transmission of misfolded aggregates by different routes of administration.