Tunneling nanotubes spread fibrillar α‐synuclein by intercellular trafficking of lysosomes

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α‐synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α‐synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co‐cultured neurons, here we show that α‐synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α‐synuclein fibrils are able to seed soluble α‐synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α‐synuclein aggregates in lysosomes dispose of this material by hijacking TNT‐mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies.     

Monoclonal antibodies selective for α‐synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α‐synuclein transgenic mice with the disease‐causing A30P mutation

Inclusions of intraneuronal alpha‐synuclein (α‐synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α‐synuclein is a central feature of the disease pathogenesis. Among the different α‐synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α‐synuclein oligomers were generated. These antibodies, which do not bind amyloid‐beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α‐synuclein transgenic mice than linear epitope antibodies. An oligomer‐selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α‐synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α‐synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer‐selective α‐synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.     

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/α‐synuclein complex by Hip

The ATP‐dependent protein chaperone heat‐shock protein 70 (Hsp70) displays broad anti‐aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α‐synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation‐prone polypeptides. We show a nucleotide‐dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co‐aggregation phenomenon can be prevented in vitro by the co‐chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.     

Denaturation induced aggregation in α‐crystallin: differential action of chaotropes

α‐Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α‐crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α‐crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α‐crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α‐crystallin at 216 nm, suggesting gain in β‐sheet structure in the presence of GdnHCl (0.5–1 M) followed by unfolding at higher concentration (2–6 M). However, only minor changes in the secondary structure of α‐crystallin were observed in the presence of urea. Moreover, 8‐anilinonaphthalene‐1‐sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α‐crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α‐crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α‐crystallin in the presence of GdnHCl, whereas only aggregate‐like structures were observed in α‐crystallin treated with urea. Our results suggest that α‐crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

STITCHER: Dynamic assembly of likely amyloid and prion β‐structures from secondary structure predictions

The supersecondary structure of amyloids and prions, proteins of intense clinical and biological interest, are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Previous work has demonstrated that probability‐based prediction of discrete β‐strand pairs can offer insight into these structures. Here, we devise a system of energetic rules that can be used to dynamically assemble these discrete β‐strand pairs into complete amyloid β‐structures. The STITCHER algorithm progressively ‘stitches’ strand‐pairs into full β‐sheets based on a novel free‐energy model, incorporating experimentally observed amino‐acid side‐chain stacking contributions, entropic estimates, and steric restrictions for amyloidal parallel β‐sheet construction. A dynamic program computes the top 50 structures and returns both the highest scoring structure and a consensus structure taken by polling this list for common discrete elements. Putative structural heterogeneity can be inferred from sequence regions that compose poorly. Predictions show agreement with experimental models of Alzheimer's amyloid beta peptide and the Podospora anserina Het‐s prion. Predictions of the HET‐s homolog HET‐S also reflect experimental observations of poor amyloid formation. We put forward predicted structures for the yeast prion Sup35, suggesting N‐terminal structural stability enabled by tyrosine ladders, and C‐terminal heterogeneity. Predictions for the Rnq1 prion and alpha‐synuclein are also given, identifying a similar mix of homogenous and heterogeneous secondary structure elements. STITCHER provides novel insight into the energetic basis of amyloid structure, provides accurate structure predictions, and can help guide future experimental studies. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

α‐Synuclein sequesters arachidonic acid to modulate SNARE‐mediated exocytosis

α‐Synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. α‐Synuclein has been proposed to regulate soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, α‐synuclein fails to interact with SNARE proteins in conventional protein‐binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both α‐synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by α‐synuclein, both in vitro and in vivo. α‐Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of α‐synuclein in the modulation of neurotransmission.     

The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein

In a group of neurodegenerative diseases, collectively termed transmissible spongiform encephalopathies, the prion protein aggregates into β‐sheet rich amyloid‐like deposits. Because amyloid structure has been connected to different prion strains and cellular toxicity, it is important to obtain insight into the structural properties of prion fibrils. Using a combination of solution NMR spectroscopy, thioflavin‐T fluorescence and electron microscopy we here show that within amyloid fibrils of a peptide containing residues 108–143 of the human prion protein [humPrP (108–143)]—the evolutionary most conserved part of the prion protein ‐ residue H111 and S135 are in close spatial proximity and their interaction is critical for fibrillization. We further show that residues H111 and H140 share the same microenvironment in the unfolded, monomeric state of the peptide, but not in the fibrillar form. While protonation of H140 has little influence on fibrillization of humPrP (108–143), a positive charge at position 111 blocks the conformational change, which is necessary for amyloid formation of humPrP (108–143). Our study thus highlights the importance of protonation of histidine residues for protein aggregation and suggests point mutations to probe the structure of infectious prion particles.     

Amyloids, prions and the inherent infectious nature of misfolded protein aggregates

Misfolded aggregates present in amyloid fibrils are associated with various diseases known as "protein misfolding" disorders. Among them, prion diseases are unique in that the pathology can be transmitted by an infectious process involving an unprecedented agent known as a "prion". Prions are infectious proteins that can transmit biological information by propagating protein misfolding and aggregation. The molecular mechanism of prion conversion has a striking resemblance to the process of amyloid formation, suggesting that misfolded aggregates have an inherent ability to be transmissible. Intriguing recent data suggest that other protein misfolding disorders might also be transmitted by a prion-like infectious process.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of intrinsically disordered proteins with electrospray ionization mass spectrometry: Conformational heterogeneity of α‐synuclein

Conformational heterogeneity of α‐synuclein was studied with electrospray ionization mass spectrometry by analyzing protein ion charge state distributions, where the extent of multiple charging reflects compactness of the protein conformations in solution. Although α‐synuclein lacks a single well‐defined structure under physiological conditions, it was found to sample four distinct conformational states, ranging from a highly structured one to a random coil. The compact highly structured state of α‐synuclein is present across the entire range of conditions tested (pH ranging from 2.5 to 10, alcohol content from 0% to 60%), but is particularly abundant in acidic solutions. The only other protein state populated in acidic solutions is a partially folded intermediate state lacking stable tertiary structure. Another, more compact intermediate state is induced by significant amounts of ethanol used as a co‐solvent and appears to represent a partially folded conformation with high β‐sheet content. Protein dimerization is observed throughout the entire range of conditions tested, although only acidic solutions favor formation of highly structured dimers of α‐synuclein. These dimers are likely to present the earliest stages in protein aggregation leading to globular oligomers and, subsequently, protofibrils. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Conformational transitions induced by in vitro macromolecular crowding lead to the amyloidogenesis of buffalo heart cystatin

The biological cells and extracellular matrix exhibit a highly crowded environment, called as macromolecular crowding. Crowding significantly influences protein structure and may lead to its aggregation. In the present study, buffalo heart cystatin (BHC), after purification from buffalo heart tissue, has been used as a model protein for studying effect of macromolecular crowding in the presence of high concentrations of bovine serum albumin (BSA), poly‐ethylene glycol‐1000 (PEG‐1000), and poly‐ethylene glycol‐4000 (PEG‐4000). Cystatins are thiol protease inhibitors and found to be involved in various important physiological processes. Functional inactivation of BHC was observed upon crowding, which varied as a function of concentration and molecular weight of crowding agents as well as incubation time. Structural changes of BHC at tertiary and secondary level were detected with the help of fluorescence and CD spectroscopy. CD analysis showed changes of α‐helix to β‐sheet, which could be due to aggregation. The ANS‐fluorescence study suggested the unfolding and presence of some partially folded intermediates. Increase in ThT‐fluorescence and absorption of Congo red spectra with red shift, confirmed the amyloid type aggregation of BHC in the presence of various crowding agents. Finally, electron microscopy provided the physical evidence about the formation of amyloid fibrils. Results suggested that among the various crowding agents used, amyloidogenesis of BHC was maximal in case of BSA followed by PEG‐4000 and least for PEG‐1000. The present work makes an important contribution in crowding mediated protein aggregation, which can have implications of potential interest. Copyright © 2015 John Wiley & Sons, Ltd.     

Amyloid formation modulates the biological activity of a bacterial protein

The aggregation of proteins into amyloid fibrils is the hallmark feature of a group of late-onset degenerative diseases including Alzheimer, Parkinson, and prion diseases. We report here that microcin E492, a peptide naturally produced by Klebsiella pneumoniae that kills bacteria by forming pores in the cytoplasmic membrane, assembles in vitro into amyloid-like fibrils. The fibrils have the same structural, morphological, tinctorial, and biochemical properties as the aggregates observed in the disease conditions. In addition, we found that amyloid formation also occurs in vivo where it is associated with a loss of toxicity of the protein. The finding that microcin E492 naturally exists both as functional toxic pores and as harmless fibrils suggests that protein aggregation into amyloid fibrils is an evolutionarily conserved property of proteins that can be successfully employed by bacteria to fulfill specific physiological needs.     

Defining the pathway of worm-like amyloid fibril formation by the mouse prion protein by delineation of the productive and unproductive oligomerization reactions

Aggregation reactions of proteins leading to amyloid fibril formation are often characterized by early transient accumulation of a heterogeneous population of soluble oligomers differing in size and structure. Delineating the kinetic roles of the different oligomeric forms in fibril formation has been a major challenge. The aggregation of the mouse prion protein to form worm-like amyloid fibrils at low pH is known to proceed via a β-rich oligomer ensemble, which is shown here to be comprised of two subpopulations of oligomers that differ in size and internal structure. The relative populations of the two oligomers can be tuned by varying the concentration of NaCl present. By demonstrating that the apparent rate constant for the formation of fibrils is dependent linearly on the concentration of the larger oligomer and is independent of the concentration of the smaller oligomer, we show that the larger oligomer is a productive intermediate that accumulates on the direct pathway of aggregation from monomer to worm-like fibrils. The smaller oligomer is shown to be populated off the pathway of the larger oligomer and, hence, is not directly productive for fibril formation. The relative populations of the two oligomers can also be tuned by single-amino acid residue changes in the sequence of the protein. The different protein variants yield worm-like fibrils of different lengths, and the apparent rate of formation of the fibrils by the mutant variants is also shown to be dependent on the concentration of the larger but not of the smaller oligomer formed.     

Long‐lasting pathological consequences of overexpression‐induced α‐synuclein spreading in the rat brain

Increased expression of α‐synuclein can initiate its long‐distance brain transfer, representing a potential mechanism for pathology spreading in age‐related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpression‐induced α‐synuclein transfer were assessed over a 1‐year period after injection of viral vectors carrying human α‐synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long‐lasting pathological changes. Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression‐induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by α‐synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpression‐induced spreading, even if temporary, causes long‐lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder α‐synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation.     

A dimer model of human calcitonin13‐32 forms an α‐helical structure and robustly aggregates in 50% aqueous 2,2,2‐trifluoroethanol solution

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13‐32 aggregated to a greater degree than native hCT under aqueous 2,2,2‐trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine‐T binding assays and atomic force microscopy suggest that the α‐helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β‐sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α‐helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of prion protein structure by pressure and temperature

High pressure and temperature have been used efficiently to shed light on prion protein structure and folding. These physical parameters induce different conformational states of the prion protein, suggesting that prion structural changes occur within a complex energy landscape. Pressure has been used to prevent and even reverse prion protein aggregation. Alternatively, depending on experimental conditions, pressure also promotes prion protein aggregation leading to the formation of amorphous aggregates and amyloid fibrils. The latter ones show all characteristics of the pathogenic scrapie form. Furthermore, the pressure effects on prion protein structure appear to be strongly dependent on the integrity of the disulfide bond. In this paper, we discuss the mechanism and the origin of these opposing effects of pressure, taking the truncated form of hamster prion protein (SHaPrP(90-231)) as a model.