Assessment of the Efficacy of Solutes from Extremophiles on Protein Aggregation in Cell Models of Huntington’s and Parkinson’s Diseases

Protein misfolding and deposition in the brain are implicated in the etiology of numerous neurodegenerative disorders. Here, organic solutes characteristic of microorganisms adapted to hot environments, were tested on experimental cell models of Huntington’s and Parkinson’s diseases. Diglycerol phosphate, di-myo-inositol phosphate, mannosylglycerate, and mannosylglyceramide were not toxic to the cells, at 10 mM concentration, but caused a decrease in cell density, which suggested an effect on proliferation. In contrast, mannosyl-lactate, an artificial analogue of mannosylglycerate, had a negative impact on cell viability. Concerning protein aggregation, inclusions of mutant huntingtin were reduced in the presence of diglycerol phosphate and di-myo-inositol phosphate, increased with mannosylglycerate, while mannosyl-lactate and mannosylglyceramide had no significant effect. α-Synuclein aggregation was not affected by the solutes tested, except for di-myo-inositol phosphate that led to a slight increased percentage of cells displaying visible aggregates. These solutes might be useful in the development of therapies for protein misfolding diseases.     

Pathological implications of nucleic acid interactions with proteins associated with neurodegenerative diseases

Protein misfolding disorders (PMDs) refer to a group of diseases related to the misfolding of particular proteins that aggregate and deposit in the cells and tissues of humans and other mammals. The mechanisms that trigger protein misfolding and aggregation are still not fully understood. Increasing experimental evidence indicates that abnormal interactions between PMD-related proteins and nucleic acids (NAs) can induce conformational changes. Here, we discuss these protein–NA interactions and address the role of deoxyribonucleic (DNA) and ribonucleic (RNA) acid molecules in the conformational conversion of different proteins that aggregate in PMDs, such as Alzheimer’s, Parkinson’s, and prion diseases. Studies on the affinity, stability, and specificity of proteins involved in neurodegenerative diseases and NAs are specifically addressed. A landscape of reciprocal effects resulting from the binding of prion proteins, amyloid-β peptides, tau proteins, huntingtin, and α-synuclein are presented here to clarify the possible role of NAs, not only as encoders of genetic information but also in triggering PMDs.     

Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes

The protection of mannosylglycerate, at 0.5 M concentration, against heat inactivation of the model enzyme lactate dehydrogenase (LDH) was compared to that exerted by other compatible solutes, namely, trehalose, ectoine, hydroxyectoine, di- myo-inositol phosphate, diglycerol phosphate, and mannosylglyceramide. Mannosylglycerate and hydroxyectoine were the best stabilizers of the enzyme and showed comparable protective effects. Diglycerol phosphate, trehalose, and mannosylglyceramide protected the enzyme to a lower extent. Ectoine conferred no protection, and di- myo-inositol phosphate had a strong destabilizing effect. The superior ability of mannosylglycerate to prevent LDH inactivation was accompanied by a higher efficiency in preventing LDH aggregation induced by heat stress. Moreover, mannosylglycerate induced an increase of 4.5 degrees C in the melting temperature of LDH, whereas the same molar concentration of trehalose caused an increase of only 2.2 degrees C. The effectiveness of mannosylglycerate in protecting LDH was also compared to that of other chemically related compounds: mannose, methyl-mannoside, potassium glycerate, glucosylglycerol, glycerol, and glucose. Mannosylglycerate conferred the highest protection, but glucosylglycerol and potassium glycerate were very efficient; glucose exerted a low degree of protection, glycerol and methyl-mannoside had no significant effect, and mannose caused destabilization. Mannosylglycerate was also a good thermoprotectant of glucose oxidase from Aspergillus niger, an enzyme with a net charge opposite to that of LDH under the working conditions. Given the superior performance of mannosylglycerate as a thermoprotectant of enzyme activity in vitro, it is conceivable that it also fulfills a protein thermoprotective function in vivo.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

The expanding realm of prion phenomena in neurodegenerative disease

The aggregation of a soluble protein into insoluble, β-sheet rich amyloid fibrils is a defining characteristic of many neurodegenerative diseases, including prion disorders. The prion protein has so far been considered unique because of its infectious nature. Recent investigations, however, suggest that other amyloid-forming proteins associated with much more common diseases, such as tau, α-synuclein, amyloid β, and polyglutamine proteins, while not infectious in the classical sense, share certain essential properties with prions that may explain phenotypic diversity, and patterns of spread within the nervous system. We suggest a common mechanism of pathogenesis of myriad sporadic and inherited neurodegenerative diseases based on templated conformational change.     

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.     

The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach

Protein glycation is initiated by a nucleophilic addition reaction between the free amino group from a protein, lipid or nucleic acid and the carbonyl group of a reducing sugar. This reaction forms a reversible Schiff base, which rearranges over a period of days to produce ketoamine or Amadori products. The Amadori products undergo dehydration and rearrangements and develop a cross-link between adjacent proteins, giving rise to protein aggregation or advanced glycation end products (AGEs). A number of studies have shown that glycation induces the formation of the β-sheet structure in β-amyloid protein, α-synuclein, transthyretin (TTR), copper-zinc superoxide dismutase 1 (Cu, Zn-SOD-1), and prion protein. Aggregation of the β-sheet structure in each case creates fibrillar structures, respectively causing Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, and prion disease. It has been suggested that oligomeric species of glycated α-synuclein and prion are more toxic than fibrils. This review focuses on the pathway of AGE formation, the synthesis of different types of AGE, and the molecular mechanisms by which glycation causes various types of neurodegenerative disease. It discusses several new therapeutic approaches that have been applied to treat these devastating disorders, including the use of various synthetic and naturally occurring inhibitors. Modulation of the AGE-RAGE axis is now considered promising in the prevention of neurodegenerative diseases. Additionally, the review covers several defense enzymes and proteins in the human body that are important anti-glycating systems acting to prevent the development of neurodegenerative diseases.     

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.     

Interaction between pathogenic proteins in neurodegenerative disorders

The misfolding and progressive aggregation of specific proteins in selective regions of the nervous system is a seminal occurrence in many neurodegenerative disorders, and the interaction between pathological/toxic proteins to cause neurodegeneration is a hot topic of current neuroscience research. Despite clinical, genetic and experimental differences, increasing evidence indicates considerable overlap between synucleinopathies, tauopathies and other protein-misfolding diseases. Inclusions, often characteristic hallmarks of these disorders, suggest interactions of pathological proteins enganging common downstream pathways. Novel findings that have shifted our understanding in the role of pathologic proteins in the pathogenesis of Alzheimer, Parkinson, Huntington and prion diseases, have confirmed correlations/overlaps between these and other neurodegenerative disorders. Emerging evidence, in addition to synergistic effects of tau protein, amyloid-β, α-synuclein and other pathologic proteins, suggests that prion-like induction and spreading, involving secreted proteins, are major pathogenic mechanisms in various neurodegenerative diseases, depending on genetic backgrounds and environmental factors. The elucidation of the basic molecular mechanisms underlying the interaction and spreading of pathogenic proteins, suggesting a dualism or triad of neurodegeneration in protein-misfolding disorders, is a major challenge for modern neuroscience, to provide a deeper insight into their pathogenesis as a basis of effective diagnosis and treatment.     

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

Taking advantage of physiological proteolytic processing of the prion protein for a therapeutic perspective in prion and Alzheimer diseases

Prion and Alzheimer diseases are fatal neurodegenerative diseases caused by misfolding and aggregation of the cellular prion protein (PrP^C) and the β-amyloid peptide, respectively. Soluble oligomeric species rather than large aggregates are now believed to be neurotoxic. PrP^C undergoes three proteolytic cleavages as part of its natural life cycle, α-cleavage, β-cleavage, and ectodomain shedding. Recent evidences demonstrate that the resulting secreted PrP^C molecules might represent natural inhibitors against soluble toxic species. In this mini-review, we summarize recent observations suggesting the potential benefit of using PrP^C-derived molecules as therapeutic agents in prion and Alzheimer diseases.     

The expanding realm of prion phenomena in neurodegenerative disease

The aggregation of a soluble protein into insoluble, β-sheet rich amyloid fibrils is a defining characteristic of many neurodegenerative diseases, including prion disorders. The prion protein has so far been considered unique because of its infectious nature. Recent investigations, however, suggest that other amyloidforming proteins associated with much more common diseases, such as tau, α-synuclein, amyloid β and polyglutamine proteins, while not infectious in the classical sense, share certain essential properties with prions that may explain phenotypic diversity, and patterns of spread within the nervous system. We suggest a common mechanism of pathogenesis of myriad sporadic and inherited neurodegenerative diseases based on templated conformational change.Key words: tau, prion, amyloid beta, α-synuclein, polyglutamine, neurodegeneration, fibril, propagation     

α-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells

α-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimer’s disease patients. Moreover, a proteolytic fragment of α-synuclein, the so-called non-amyloid component of Alzheimer’s disease amyloid, was found to be an integral part of Alzheimer’s dementia related plaques. However, the role of α-synuclein in pathomechanism of Alzheimer’s disease remains elusive. In particular, the relationship between α-synuclein and amyloid beta is unknown. In the present study we showed the involvement of α-synuclein in amyloid beta secretion and in the mechanism of amyloid beta evoked mitochondria dysfunction and cell death. Rat pheochromocytoma PC12 cells transfected with amyloid beta precursor protein bearing Swedish double mutation (APPsw) and control PC12 cells transfected with empty vector were used in this study. α-Synuclein (10 μM) was found to increase by twofold amyloid beta secretion from control and APPsw PC12 cells. Moreover, α-synuclein decreased the viability of PC12 cells by about 50% and potentiated amyloid beta toxicity leading to mitochondrial dysfunction and caspase-dependent programmed cell death. Inhibitor of caspase-3 (Z-DEVD-FMK, 100 μM), and a mitochondrial permeability transition pore blocker, cyclosporine A (2 μM) protected PC12 cells against α-synuclein or amyloid beta evoked cell death. In contrast Z-DEVD-FMK and cyclosporine A were ineffective in APPsw cells containing elevated amount of amyloid beta treated with α-synuclein. It was found that the inhibition of neuronal and inducible nitric oxide synthase reversed the toxic effect of α-synuclein in control but not in APPsw cells. Our results indicate that α-synuclein enhances the release and toxicity of amyloid beta leading to nitric oxide mediated irreversible mitochondria dysfunction and caspase-dependent programmed cell death.     

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.     

A standard model of Alzheimer’s disease?

ABSTRACT

The recent Research Framework proposed by the US National Institute on Aging and the Alzheimer’s Association (NIA-AA) recommends that Alzheimer’s disease be defined by its specific biology rather than by non-specific neurodegenerative and syndromal features. By affirming markers of abnormal Aβ and tau proteins as the essential pathobiological signature of Alzheimer’s disease, the Framework tacitly reinforces the amyloid (Aβ) cascade as the leading theory of Alzheimer pathogenesis. In light of recent evidence that the cascade is driven by the misfolding and templated aggregation of Aβ and tau, we believe that an empirically grounded Standard Model of Alzheimer’s pathogenesis is within reach. A Standard Model can clarify and consolidate existing information, contextualize risk factors and the complex disease phenotype, identify testable hypotheses for future research, and pave the most direct path to effective prevention and treatment.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Protein Misfolding and Aggregation in Ageing and Disease

Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.