[show abstract][hide abstract] ABSTRACT: One of the main features of Alzheimer's disease (AD) is the presence of Abeta deposits, which accumulate in the brain years before the onset of symptoms. We and others have demonstrated that cerebral Abeta-amyloidosis can be induced in vivo by administration of AD-brain extracts into transgenic mice. However, it is currently unknown whether amyloid formation can be induced using extracts from individuals harboring Abeta deposits, but not clinical disease.
In this study we analyzed the amyloid-inducing capability of samples from individuals affected by mild cognitive impairment (MCI) and Non-Demented persons with Alzheimer's disease Neuropathology (NDAN). Our results show that inoculation of transgenic mice with MCI and NDAN brain samples accelerated Abeta pathology in a similar way as extracts from confirmed AD.
This data demonstrate that the sole presence of Abeta aggregates in a given sample, regardless of the clinical condition, is capable to accelerate Abeta deposition in vivo. These findings indicate that the amyloid-inducing activity may be present in the brain of people during pre-symptomatic or a-symptomatic stages of AD.
[show abstract][hide abstract] ABSTRACT: Prion diseases are a group of neurodegenerative disorders affecting humans as well as captive and wild animals. The mechanisms and routes governing the natural spread of prions are not completely understood and several hypotheses have been proposed. In this study, we analyzed the effect of gender in prion incubation period, as well as the possibility of prion transmission by sexual and parental contact using 263K infected hamsters as a model. Our results show that males have significantly longer incubation periods compared with females when exposed to the same quantity of infectious material. Importantly, no evidence of sexual or parental prion transmission was found, even 500 d after sexual contact or birth, respectively. Western blotting and PMCA were unable to detect sub-clinical levels of PrP(Sc) in experimental subjects, suggesting a complete absence of prion transmission by these routes. Our results show that sexual and parental transmission of prions does not occur in this model. It remains to be studied whether this conclusion is valid also for other prion strains and species.
[show abstract][hide abstract] ABSTRACT: Several epidemiological studies have shown that cigarette smoking might alter the incidence of Alzheimer's disease. However, inconsistent results have been reported regarding the risk of Alzheimer's disease among smokers. Previous studies in experimental animal models have reported that administration of some cigarette components (for example, nicotine) alters amyloid-β aggregation, providing a possible link. However, extrapolation of these findings towards the in vivo scenario is not straightforward as smoke inhalation involves a number of other components. Here, we analysed the effect of smoking under more relevant conditions. We exposed transgenic mouse models of Alzheimer's disease to cigarette smoke and analysed the neuropathological alterations in comparison with animals not subjected to smoke inhalation. Our results showed that smoking increases the severity of some abnormalities typical of Alzheimer's disease, including amyloidogenesis, neuroinflammation and tau phosphorylation. Our findings suggest that cigarette smoking may increase Alzheimer's disease onset and exacerbate its features and thus, may constitute an important environmental risk factor for Alzheimer's disease.
[show abstract][hide abstract] ABSTRACT: The once heretical concept that a misfolded protein is the infectious agent responsible for prion diseases is now widely accepted. Recent exciting research has led not only to the end of the skepticism that proteins can transmit disease, but also to expanding the concept that transmissible proteins might be at the root of some of the most prevalent human illnesses. At the same time, the idea that biological information can be transmitted by propagation of protein (mis)folding raises the possibility that heritable protein agents may be operating as epigenetic factors in normal biological functions and participating in evolutionary adaptation.
[show abstract][hide abstract] ABSTRACT: Neurodegenerative diseases (NDs) are some of the most debilitating human illnesses. Research over the past 10 years has provided evidence for a common mechanism of neurodegeneration in which the critical event is the brain accumulation of misfolded protein aggregates. Although it is well established that misfolded proteins play an important role in these diseases, the mechanisms by which they cause cellular and tissue dysfunction are still unknown. To understand the molecular basis of NDs and to develop therapeutic strategies against them, numerous transgenic rodent models have been produced, which reproduce some (but not all) of the features of these diseases. Importantly, some NDs are not exclusive to human beings, such as transmissible spongiform encephalopathies. Moreover, other diseases which are associated to aging (e.g. Alzheimer's disease) could be studied in aged mammals, which could reproduce the human disease in a more natural way. Although the usefulness of transgenic mice is unquestionable, the information obtained from natural non-transgenic models could be very valuable to fully understand the pathogenesis of these devastating diseases.
Current pharmaceutical design 01/2012; 18(8):1148-58. · 4.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrP(Sc)), which is able to template the conversion of the host's natively folded form of the protein (PrP(C)). The detailed mechanism of prion replication and the high-resolution structure of PrP(Sc) are unknown. The currently available information on PrP(Sc) structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrP(Sc) structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrP(Sc), arguably the major pending milestone to reach in understanding prions.
[show abstract][hide abstract] ABSTRACT: 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 (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). 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 PrP(C) 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 PrP(Sc) 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.
[show abstract][hide abstract] ABSTRACT: Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP), is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP) into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc)-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts) submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(-) or SO(4)(-2). The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc). The aggregates also exhibited PrP(Sc)-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc) biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.
PLoS ONE 01/2012; 7(2):e31678. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein misfolding cyclic amplification (PMCA) is a technique that takes advantage of the nucleation-dependent prion replication process to accelerate the conversion of PrP(C) into PrP(Sc) in the test tube. PMCA uses ultrasound waves to fragment the PrP(Sc) polymers, increasing the amount of seeds present in the infected sample without affecting their ability to act as conversion nuclei. Over the past 5 years, PMCA has become an invaluable technique to study diverse aspects of prions. The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the cellular factors involved in prion propagation, the intriguing phenomena of prion strains and species barriers, to detect PrP(Sc) in tissues and biological fluids, and to screen for inhibitors against prion replication. In this chapter, we describe a detailed protocol of the PMCA technique, highlighting some of the important technical aspects to obtain a successful and reproducible application of the technology.
Methods in molecular biology (Clifton, N.J.) 01/2012; 849:199-212.
[show abstract][hide abstract] ABSTRACT: Alzheimer's 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.Molecular Psychiatry advance online publication, 4 October 2011; doi:10.1038/mp.2011.120.
[show abstract][hide abstract] ABSTRACT: Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrP(Sc)), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrP(Sc) in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrP(Sc), the protein was eliminated rapidly from the serum (half-life of 3.24 h), mostly through tissue uptake. The quantity of intact PrP(Sc) reaching the brain was ∼ 0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (∼ 20% ID/g), spleen (∼ 13% ID/g), and kidney (∼ 7.4% ID/g). Cell surface PrP(C) does not appear to play a role in PrP(Sc) pharmacokinetics, since the infectious protein distributed similarly in wild-type and PrP-null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrP(Sc). Our results provide a fundamental pharmacokinetic characterization of PrP(Sc) in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.
The FASEB Journal 05/2011; 25(8):2792-803. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Some of the most prevalent human degenerative diseases appear as a result of the misfolding and aggregation of proteins. Compelling evidence suggest that misfolded protein aggregates play an important role in cell dysfunction and tissue damage, leading to the disease. Prion protein (Prion diseases), amyloid-beta (Alzheimer's disease), alpha-synuclein (Parkinson's disease), Huntingtin (Huntington's disease), serum amyloid A (AA amyloidosis) and islet amyloid polypeptide (type 2 diabetes) are some of the proteins that trigger disease when they get misfolded. The recent understanding of the crucial role of misfolded proteins as well as the structural requirements and mechanism of protein misfolding have raised the possibility that these diseases may be transmissible by self-propagation of the protein misfolding process in a similar way as the infamous prions transmit prion diseases. Future research in this field should aim to clarify this possibility and translate the knowledge of the basic disease mechanisms into development of novel strategies for early diagnosis and efficient treatment.
Seminars in Cell and Developmental Biology 05/2011; 22(5):482-7. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: A hallmark event in neurodegenerative diseases is the accumulation of misfolded aggregated proteins in the brain leading to neuronal dysfunction and disease. Compelling evidence suggests that misfolded proteins damage cells by inducing endoplasmic reticulum (ER) stress and alterations in calcium homeostasis. Changes in cytoplasmic calcium concentration lead to unbalances on several signaling pathways. Recent data suggest that calcium-mediated hyperactivation of calcineurin (CaN), a key phosphatase in the brain, triggers synaptic dysfunction and neuronal death, the two central events responsible for brain degeneration in neurodegenerative diseases. Therefore, blocking CaN hyper-activation might be a promising therapeutic strategy to prevent brain damage in neurodegenerative diseases.
Current opinion in cell biology 02/2011; 23(2):223-30. · 14.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prion diseases are infectious neurodegenerative disorders that affect humans and animals and that result from the conversion of normal prion protein (PrP(C)) into the misfolded prion protein (PrP(Sc)). Chronic wasting disease (CWD) is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. Determining the risk of transmission of CWD to humans is of utmost importance, considering that people can be infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrP(C) can be converted into the misfolded form by CWD PrP(Sc), we performed experiments using the protein misfolding cyclic amplification technique, which mimics in vitro the process of prion replication. Our results show that cervid PrP(Sc) can induce the conversion of human PrP(C) but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, the newly generated human PrP(Sc) exhibits a distinct biochemical pattern that differs from that of any of the currently known forms of human PrP(Sc). Our results also have profound implications for understanding the mechanisms of the prion species barrier and indicate that the transmission barrier is a dynamic process that depends on the strain and moreover the degree of adaptation of the strain. If our findings are corroborated by infectivity assays, they will imply that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading.
Journal of Biological Chemistry 01/2011; 286(9):7490-5. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Forty-three years have passed since it was first proposed that a protein could be the sole component of the infectious agent responsible for the enigmatic prion diseases. Many discoveries have strongly supported the prion hypothesis, but only recently has this once heretical hypothesis been widely accepted by the scientific community. In the past 3 years, researchers have achieved the 'Holy Grail' demonstration that infectious material can be generated in vitro using completely defined components. These breakthroughs have proven that a misfolded protein is the active component of the infectious agent, and that propagation of the disease and its unique features depend on the self-replication of the infectious folding of the prion protein. In spite of these important discoveries, it remains unclear whether another molecule besides the misfolded prion protein might be an essential element of the infectious agent. Future research promises to reveal many more intriguing features about the rogue prions.
Trends in Biochemical Sciences 12/2010; 36(3):151-8. · 13.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prion diseases are a group of infectious neurodegenerative diseases with an entirely novel mechanism of transmission, involving a protein-only infectious agent that propagates the disease by transmitting protein conformational changes. The disease results from extensive and progressive brain degeneration. The molecular mechanisms involved in neurodegeneration are not entirely known but involve multiple processes operating simultaneously and synergistically in the brain, including spongiform degeneration, synaptic alterations, brain inflammation, neuronal death and the accumulation of protein aggregates. Here, we review the pathways implicated in prion-induced brain damage and put the pieces together into a possible model of neurodegeneration in prion disorders. A more comprehensive understanding of the molecular basis of brain degeneration is essential to develop a much needed therapy for these devastating diseases.
Trends in Molecular Medicine 10/2010; · 9.57 Impact Factor