Proteomics applications in prion biology and structure

Prion diseases are a heterogeneous class of fatal neurodegenerative disorders associated with misfolding of host cellular prion protein (PrP^C) into a pathological isoform, termed PrP^Sc. Prion diseases affect various mammals, including humans, and effective treatments are not available. Prion diseases are distinguished from other protein misfolding disorders – such as Alzheimer’s or Parkinson’s disease – in that they are infectious. Prion diseases occur sporadically without any known exposure to infected material, and hereditary cases resulting from rare mutations in the prion protein have also been documented. The mechanistic underpinnings of prion and other neurodegenerative disorders remain poorly understood. Various proteomics techniques have been instrumental in early PrP^Sc detection, biomarker discovery, elucidation of PrP^Sc structure and mapping of biochemical pathways affected by pathogenesis. Moving forward, proteomics approaches will likely become more integrated into the clinical and research settings for the rapid diagnosis and characterization of prion pathogenesis.     

Is loss of function of the prion protein the cause of prion disorders?

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.     

Neurodegenerative amyloidoses: Yeast model

More than 20 human diseases are associated with protein misfolding, which results in the appearance of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are termed amyloid diseases, or amyloidoses. Of these, only prion diseases are transmissible. Amyloids of the prion type are known for lower eukaryotes. While mammalian prions cause neurodegenerative diseases, prions of lower eukaryotes are associated with some nonchromosomally inherited phenotypic traits. The review summarizes the results of studying the prions of yeast Saccharomyces cerevisiae and data obtained using S. cerevisiae as a model to investigate some human amyloidoses such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases.     

Molecular pathogenesis of sporadic prion diseases in man

The yeast, fungal and mammalian prions determine heritable and infectious traits that are encoded in alternative conformations of proteins. They cause lethal sporadic, familial and infectious neurodegenerative conditions in man, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, sporadic fatal insomnia (SFI) and likely variable protease-sensitive prionopathy (VPSPr). The most prevalent of human prion diseases is sporadic (s)CJD. Recent advances in amplification and detection of prions led to considerable optimism that early and possibly preclinical diagnosis and therapy might become a reality. Although several drugs have already been tested in small numbers of sCJD patients, there is no clear evidence of any agent’s efficacy. Therefore, it remains crucial to determine the full spectrum of sCJD prion strains and the conformational features in the pathogenic human prion protein governing replication of sCJD prions. Research in this direction is essential for the rational development of diagnostic as well as therapeutic strategies. Moreover, there is growing recognition that fundamental processes involved in human prion propagation – intercellular induction of protein misfolding and seeded aggregation of misfolded host proteins – are of far wider significance. This insight leads to new avenues of research in the ever-widening spectrum of age-related human neurodegenerative diseases that are caused by protein misfolding and that pose a major challenge for healthcare.     

Yeast-based screening of natural product extracts results in the identification of prion inhibitors from a marine sponge

ABSTRACT

One of the major medical challenges of the twenty-first century is the treatment of incurable and fatal neurodegenerative disorders caused by misfolded prion proteins. Since the discovery of these diseases a number of studies have been conducted to identify small molecules for their treatment, however to date no curative treatment is available. These studies can be highly expensive and time consuming, but more recent experimental approaches indicate a significant application for yeast prions in these studies. We therefore used yeast prions to optimize previous high-throughput methods for the cheaper, easier and more rapid screening of natural extracts. Through this approach we aimed to identify natural yeast-prion inhibitors that could be useful in the development of novel treatment strategies for neurodegenerative disorders. We screened 500 marine invertebrate extracts from temperate waters in Australia allowing the identification of yeast-prion inhibiting extracts. Through the bioassay-driven chemical investigation of an active Suberites sponge extract, a group of bromotyrosine derivatives were identified as potent yeast-prion inhibitors. This study outlines the importance of natural products and yeast prions as a first-stage screen for the identification of new chemically diverse and bioactive compounds.     

Prion protein function and the disturbance of early embryonic development in zebrafish

Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist’s view of PrP functions might be clearer at a greater phylogenetic distance.     

Prion Pathogenesis is Independent of Caspase-12

The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer's disease and many others. The ER stress response and up-regulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.     

Prion Pathogenesis is Independent of Caspase-12

The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer''s disease and many others. The ER stress response and upregulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.     

Prion Domain of Yeast Ure2 Protein Adopts a Completely Disordered Structure: A Solid-Support EPR Study

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer’s, Parkinson’s, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer’s amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.     

The neuroprotective potential of carotenoids in vitro and in vivo

BackgroundDespite advances in research on neurodegenerative diseases, the pathogenesis and treatment response of neurodegenerative diseases remain unclear. Recent studies revealed a significant role of carotenoids to treat neurodegenerative diseases. The aim of this study was to systematically review the neuroprotective potential of carotenoids in vivo and in vitro and the molecular mechanisms and pathological factors contributing to major neurodegenerative diseases (Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and stroke).HypothesisCarotenoids as therapeutic molecules to target neurodegenerative diseases.ResultsAggregation of toxic proteins, mitochondrial dysfunction, oxidative stress, the excitotoxic pathway, and neuroinflammation were the major pathological factors contributing to the progression of neurodegenerative diseases. Furthermore, in vitro and in vivo studies supported the beneficiary role of carotenoids, namely lycopene, β-carotene, crocin, crocetin, lutein, fucoxanthin and astaxanthin in alleviating disease progression. These carotenoids provide neuroprotection by inhibition of neuro-inflammation, microglial activation, excitotoxic pathway, modulation of autophagy, attenuation of oxidative damage and activation of defensive antioxidant enzymes. Additionally, studies conducted on humans also demonstrated that dietary intake of carotenoids lowers the risk of neurodegenerative diseases.ConclusionCarotenoids may be used as drugs to prevent and treat neurodegenerative diseases. Although, the in vitro and in vivo results are encouraging, further well conducted clinical studies on humans are required to conclude about the full potential of neurodegenerative diseases.     

Genetic and epigenetic control of the efficiency and fidelity of cross‐species prion transmission

Self‐perpetuating amyloid‐based protein isoforms (prions) transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Although mechanisms that control species specificity of prion transmission are poorly understood, studies of closely related orthologues of yeast prion protein Sup35 demonstrate that cross‐species prion transmission is modulated by both genetic (specific sequence elements) and epigenetic (prion variants, or ‘strains’) factors. Depending on the prion variant, the species barrier could be controlled at the level of either heterologous co‐aggregation or conversion of the aggregate‐associated heterologous protein into a prion polymer. Sequence divergence influences cross‐species transmission of different prion variants in opposing ways. The ability of a heterologous prion domain to either faithfully reproduce or irreversibly switch the variant‐specific prion patterns depends on both sequence divergence and the prion variant. Sequence variations within different modules of prion domains contribute to transmission barriers in different cross‐species combinations. Individual amino acid substitutions within short amyloidogenic stretches drastically alter patterns of cross‐species prion conversion, implicating these stretches as major determinants of species specificity.     

Harnessing the power of yeast to unravel the molecular basis of neurodegeneration

Several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration has been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well‐characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders.     

Cellular cholesterol enrichment prevents prion peptide-induced neuron cell damages

The prion diseases are neurodegenerative disorders characterized by the conversion of the PrPc (normal cellular prion) to the PrPsc (misfolded isoform). The accumulation of PrPsc within the central nervous system (CNS) leads to neurocytotoxicity by increasing oxidative stress. In addition, many neurodegenerative disorders including prion, Parkinson’s and Alzheimer’s diseases may be regulated by cholesterol homeostasis. The effects of cholesterol balance on prion protein-mediated neurotoxicity and ROS (reactive oxygen species) generation were the focus of this study. Cholesterol treatment inhibited PrP (106-126)-induced neuronal cell death and ROS generation in SH-SY5Y neuroblastoma cells. In addition, the PrP (106-126)-mediated increase of p53, p-p38, p-ERK and the decrease of Bcl-2 were blocked by cholesterol treatment. These results indicated that cellular cholesterol enrichment is a key regulator of PrP-106-126-mediated oxidative stress and neurotoxicity. Taken together, the results of this study suggest that modulation of cellular cholesterol appears to prevent the neuronal cell death caused by prion peptides.     

Delineating common molecular mechanisms in Alzheimer's and prion diseases

The structure of the infectious agent responsible for prion diseases has not been fully characterized, but evidence points to a beta-rich conformer of the host-encoded prion protein. Amyloid-beta peptide (Abeta), a proteolytic fragment generated from the amyloid precursor protein, has been implicated as the toxic molecule involved in the pathogenesis of Alzheimer's disease. The mechanism of Abeta toxicity might be mediated through the coordination of redox-active transition-metal ions such as copper leading to the generation of reactive oxygen species, coupled with the propensity to interact with lipid bilayers. Key sequence and chemical similarities between prion protein (PrP) and Abeta indicate that similar therapeutic strategies might be applicable for the treatment of Alzheimer's and prion diseases.     

Molecular pathogenesis of sporadic prion diseases in man

The yeast, fungal and mammalian prions determine heritable and infectious traits that are encoded in alternative conformations of proteins. They cause lethal sporadic, familial and infectious neurodegenerative conditions in man, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, sporadic fatal insomnia (SFI) and likely variable protease-sensitive prionopathy (VPSPr). The most prevalent of human prion diseases is sporadic (s)CJD. Recent advances in amplification and detection of prions led to considerable optimism that early and possibly preclinical diagnosis and therapy might become a reality. Although several drugs have already been tested in small numbers of sCJD patients, there is no clear evidence of any agent’s efficacy. Therefore, it remains crucial to determine the full spectrum of sCJD prion strains and the conformational features in the pathogenic human prion protein governing replication of sCJD prions. Research in this direction is essential for the rational development of diagnostic as well as therapeutic strategies. Moreover, there is growing recognition that fundamental processes involved in human prion propagation – intercellular induction of protein misfolding and seeded aggregation of misfolded host proteins – are of far wider significance. This insight leads to new avenues of research in the ever-widening spectrum of age-related human neurodegenerative diseases that are caused by protein misfolding and that pose a major challenge for healthcare.     

Prion protein function and the disturbance of early embryonic development in zebrafish

Transmissible Spongiform Encephal-opathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist''s view of PrP functions might be clearer at a greater phylogenetic distance.Key words: prion protein, zebrafish, gene expression, embryo development, neurogenesis     

Suppression of polyglutamine toxicity by the yeast Sup35 prion domain in Drosophila

The propensity of proteins to form beta-sheet-rich amyloid fibrils is related to a variety of biological phenomena, including a number of human neurodegenerative diseases and prions. A subset of amyloidogenic proteins forms amyloid fibrils through glutamine/asparagine (Q/N)-rich domains, such as pathogenic polyglutamine (poly(Q)) proteins involved in neurodegenerative disease, as well as yeast prions. In the former, the propensity of an expanded poly(Q) tract to abnormally fold confers toxicity on the respective protein, leading to neuronal dysfunction. In the latter, Q/N-rich prion domains mediate protein aggregation important for epigenetic regulation. Here, we investigated the relationship between the pathogenic ataxin-3 protein of the human disease spinocerebellar ataxia type 3 (SCA3) and the yeast prion Sup35, using Drosophila as a model system. We found that the capacity of the Sup35 prion domain to mediate protein aggregation is conserved in Drosophila. Although select yeast prions enhance poly(Q) toxicity in yeast, the Sup35N prion domain suppressed poly(Q) toxicity in the fly. Suppression required the oligopeptide repeat of the Sup35N prion domain, which is critical for prion properties in yeast. These results suggest a trans effect of prion domains on pathogenic poly(Q) disease proteins in a multicellular environment and raise the possibility that Drosophila may allow studies of prion mechanisms.     

Understanding and exploiting interactions between cellular proteostasis pathways and infectious prion proteins for therapeutic benefit

Several neurodegenerative diseases of humans and animals are caused by the misfolded prion protein (PrP^Sc), a self-propagating protein infectious agent that aggregates into oligomeric, fibrillar structures and leads to cell death by incompletely understood mechanisms. Work in multiple biological model systems, from simple baker''s yeast to transgenic mouse lines, as well as in vitro studies, has illuminated molecular and cellular modifiers of prion disease. In this review, we focus on intersections between PrP and the proteostasis network, including unfolded protein stress response pathways and roles played by the powerful regulators of protein folding known as protein chaperones. We close with analysis of promising therapeutic avenues for treatment enabled by these studies.     

In vitro self-propagation of recombinant PrPSc-like conformation generated in the yeast cytoplasm

Self-propagation is characteristic property for a prion conformation. Previous studies revealed that prion protein expressed in the cytoplasm gained a PrP(Sc)-like conformation. However, it remains unclear whether the PrP(Sc)-like conformation has the self-propagating property. We found that PrP partially purified from yeast cytoplasm formed amyloid fiber like structures, and we found that the PrP(Sc)-like conformation is able to convert normal PrP(C) in the brain homogenate to a proteinase K-resistant conformation. These results suggest that yeast cytoplasm expressed recombinant PrP(Sc)-like conformation has the characteristic self-propagating property of a prion, which may have implications in the pathogenesis of sporadic and inherited prion diseases.     

Diagnostic function of the neuroinflammatory biomarker YKL-40 in Alzheimer’s disease and other neurodegenerative diseases

Introduction: Neuroinflammation is a crucial mechanism in the pathophysiology of neurodegenerative diseases pathophysiology. Cerebrospinal fluid (CSF) YKL-40 – an indicator of microglial activation ? has recently been identified by proteomic studies as a candidate biomarker for Alzheimer’s disease (AD).

Areas covered: We review the impact of CSF YKL-40 as a pathophysiological biomarker for AD and other neurodegenerative diseases. CSF YKL-40 concentrations have been shown to predict progression from prodromal mild cognitive impairment to AD dementia. Moreover, a positive association between CSF YKL-40 and other biomarkers of neurodegeneration – particularly total tau protein ? has been reported during the asymptomatic preclinical stage of AD and other neurodegenerative diseases. Albeit preliminary, current data do not support an association between APOE-ε4 status and CSF YKL-40 concentrations. When interpreting the diagnostic/prognostic significance of CSF YKL-40 concentrations in neurodegenerative diseases, potential confounders – including age, metabolic and cardiovascular risk factors, diagnostic criteria for selecting cases/controls – need to be considered.

Expert opinion/commentary: CSF YKL-40 represents a pathophysiological biomarker reflecting immune/inflammatory mechanisms in neurodegenerative diseases, associated with tau protein pathology. Besides being associated with tau pathology, CSF YKL-40 adds to the growing array of biomarkers reflecting distinct molecular brain mechanisms potentially useful for stratifying individuals for biomarker-guided, targeted anti-inflammatory therapies emerging from precision medicine.