Clusterin protects neurons against intracellular proteotoxicity.

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.

Moving pathogen genomics out of the lab and into the clinic: what will it take?

Pathogen genomic analysis is a potentially transformative new approach to the clinical and public-health management of infectious diseases. Health systems investing in this technology will need to build infrastructure and develop policies that ensure genomic information can be generated, shared and acted upon in a timely manner.

Direct observations of amyloid ß self-assembly in live cells provide insights into differences in the kinetics of Aß(1-40) and Aß(1-42) aggregation.

Insight into how amyloid ß (Aß) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aß(1-40) and Aß(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aß(1-42) aggregation are considerably faster than those of Aß(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aß aggregation may occur within neurons.

Oligomer-targeting with a conformational antibody fragment promotes toxicity in Aß-expressing flies.

INTRODUCTION: The self-assembly of Aß peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer's disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments. RESULTS: We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aß-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aß(1-40) and not with those expressing Aß(1-42) or the arctic variant of Aß(1-42) This finding is consistent with the binding preference of KW1 for Aß(1-40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation. CONCLUSIONS: While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Single point mutations induce a switch in the molecular mechanism of the aggregation of the Alzheimer's disease associated Aß42 peptide.

Single point mutations in the Alzheimer's disease associated Aß42 peptide are found to alter significantly its neurotoxic properties in vivo and have been associated with early onset forms of this devastating condition. We show that such mutations can induce structural changes in Aß42 fibrils and are associated with a dramatic switch in the fibril-dependent mechanism by which Aß42 aggregates. These observations reveal how subtle perturbations to the physicochemical properties of the Aß peptide, and the structural properties of fibrils that it forms, can have profound effects on the mechanism of its aggregation and pathogenicity.

Proliferation of amyloid-ß42 aggregates occurs through a secondary nucleation mechanism.

The generation of toxic oligomers during the aggregation of the amyloid-ß (Aß) peptide Aß42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results reveal that the aggregation of Aß42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the Aß aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aß42 oligomers.

Selenium-enhanced electron microscopic imaging of different aggregate forms of a segment of the amyloid ß peptide in cells.

The aggregation of misfolded proteins is a common feature underlying a wide range of age-related degenerative disorders, including Alzheimer's and Parkinson's diseases. A key aspect of understanding the molecular origins of these conditions is to define the manner in which specific types of protein aggregates influence disease pathogenesis through their interactions with cells. We demonstrate how selenium-enhanced electron microscopy (SE-EM), combined with tomographic reconstruction methods, can be used to image, here at a resolution of 5-10 nm, the interaction with human macrophage cells of amyloid aggregates formed from Aß(25-36), a fragment of the Aß peptide whose self-assembly is associated with Alzheimer's disease. We find that prefibrillar aggregates and mature fibrils are distributed into distinct subcellular compartments and undergo varying degrees of morphological change over time, observations that shed new light on the origins of their differential toxicity and the mechanisms of their clearance. In addition, the results show that SE-EM provides a powerful and potentially widely applicable means to define the nature and location of protein assemblies in situ and to provide detailed and specific information about their partitioning and processing.

The aggregation and neurotoxicity of TDP-43 and its ALS-associated 25 kDa fragment are differentially affected by molecular chaperones in Drosophila.

Almost all cases of sporadic amyotrophic lateral sclerosis (ALS), and some cases of the familial form, are characterised by the deposition of TDP-43, a member of a family of heteronuclear ribonucleoproteins (hnRNP). Although protein misfolding and deposition is thought to be a causative feature of many of the most prevalent neurodegenerative diseases, a link between TDP-43 aggregation and the dysfunction of motor neurons has yet to be established, despite many correlative neuropathological studies. We have investigated this relationship in the present study by probing the effect of altering TDP-43 aggregation behaviour in vivo by modulating the levels of molecular chaperones in a Drosophila model. More specifically, we quantify the effect of either pharmacological upregulation of the heat shock response or specific genetic upregulation of a small heat shock protein, CG14207, on the neurotoxicity of both TDP-43 and of its disease associated 25 kDa fragment (TDP-25) in a Drosophila model. Inhibition of the aggregation of TDP-43 by either method results in a partial reduction of its neurotoxic effects on both photoreceptor and motor neurons, whereas inhibition of the aggregation of TDP-25 results not only in a complete suppression of its toxicity but also its clearance from the brain in both neuronal subtypes studied. The results demonstrate, therefore, that aggregation plays a crucial role in mediating the neurotoxic effects of both full length and truncated TDP-43, and furthermore reveal that the in vivo propensity of these two proteins to aggregate and their susceptibility to molecular chaperone mediated clearance are quite distinct.

Expression in drosophila of tandem amyloid ß peptides provides insights into links between aggregation and neurotoxicity.

The generation and subsequent aggregation of amyloid ß (Aß) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aß(40)) or 42 residues (Aß(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aß peptides to probe the manner in which changes in the aggregation kinetics of Aß affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aß(40) and Aß(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aß(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aß(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aß(40) rather than simply to its higher rate of aggregation.

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering.

Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.

ANS binding reveals common features of cytotoxic amyloid species.

Oligomeric assemblies formed from a variety of disease-associated peptides and proteins have been strongly associated with toxicity in many neurodegenerative conditions, such as Alzheimer's disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Abeta(1-42) peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Abeta(1-42), the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.

Intrinsic determinants of neurotoxic aggregate formation by the amyloid beta peptide.

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid beta peptide (Abeta) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Abeta40 peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Abeta42 peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Abeta peptide.

Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

Methods and models in neurodegenerative and systemic protein aggregation diseases.

Protein misfolding and aggregation are implicated in a wide range of increasingly prevalent human diseases ranging from dementia to diabetes. In this review we discuss the current experimental strategies that are being employed in the investigation of the pathogenesis of three important protein misfolding disorders. The first, Alzheimer's disease (AD), is the most prevalent neurodegenerative disease and is thought to be initiated by the aggregation of a natively unstructured peptide called amyloid beta (Abeta). We discuss methods for the characterization of the aggregation properties of Abeta in vitro and how the results of such experiments can be correlated with data from animal models of disease. We then consider another form of amyloidosis, where a systemic distribution of amyloid deposit is caused by aggregation and deposition of mutational variants of lysozyme. We describe how experiments in vitro, and more recently in vivo, have provided insights into the origins of this disease. Finally we outline the varied paradigms that have been employed in the study of the serpinopathies, and in particular, a dementia caused by neuroserpin polymerization.

Bridging the gap: from protein misfolding to protein misfolding diseases.

Protein misfolding and aggregation are pathognomic for a number of the most common age-related degenerative diseases. Great progress has been made in studying protein aggregation in the test tube and also in replicating protein aggregation in vertebrate animal models of these diseases. However, we argue here that the development and effective integration of emerging techniques such as the methods of nanoscience and the use of invertebrate models are now providing powerful new opportunities to advance our current understanding of the fundamental origins of these disorders.

Protein misfolding and disease: from the test tube to the organism.

Protein misfolding is the underlying cause of many highly debilitating disorders ranging from Alzheimer's Disease to Cystic Fibrosis. Great strides have been made recently in understanding what causes proteins to misfold, primarily through the use of biophysical and computational techniques that enable systematic and quantitative analysis of the effects of a range of different perturbations in proteins. Correlation of the results of such analyses with observations made in animal models of disease has however been limited by their seemingly irreconcilable differences in methodology and scope. Several recent studies have however begun to overcome this limitation by combining the two approaches. This strategy has made it possible to investigate many of the consequences of protein misfolding in vivo, ranging from disease pathogenesis to epigenetic regulation, in the context of the fundamental physico-chemical principles derived from extensive and highly detailed studies undertaken in vitro.

Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity.

Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Abeta42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Abeta42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Abeta and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism.

Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils.

Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of beta sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.