Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species.

The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-edged sword, as it eliminates Tau amyloids at the cost of generating new seeds.

Bin1 directly remodels actin dynamics through its BAR domain.

Endocytic processes are facilitated by both curvature-generating BAR-domain proteins and the coordinated polymerization of actin filaments. Under physiological conditions, the N-BAR protein Bin1 has been shown to sense and curve membranes in a variety of cellular processes. Recent studies have identified Bin1 as a risk factor for Alzheimer's disease, although its possible pathological function in neurodegeneration is currently unknown. Here, we report that Bin1 not only shapes membranes, but is also directly involved in actin binding through its BAR domain. We observed a moderate actin bundling activity by human Bin1 and describe its ability to stabilize actin filaments against depolymerization. Moreover, Bin1 is also involved in stabilizing tau-induced actin bundles, which are neuropathological hallmarks of Alzheimer's disease. We also provide evidence for this effect in vivo, where we observed that downregulation of Bin1 in a Drosophila model of tauopathy significantly reduces the appearance of tau-induced actin inclusions. Together, these findings reveal the ability of Bin1 to modify actin dynamics and provide a possible mechanistic connection between Bin1 and tau-induced pathobiological changes of the actin cytoskeleton.

Cellular and molecular modifier pathways in tauopathies: the big picture from screening invertebrate models.

Abnormal tau accumulations were observed and documented in post-mortem brains of patients affected by Alzheimer's disease (AD) long before the identification of mutations in the Microtubule-associated protein tau (MAPT) gene, encoding the tau protein, in a different neurodegenerative disease called Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). The discovery of mutations in the MAPT gene associated with FTDP-17 highlighted that dysfunctions in tau alone are sufficient to cause neurodegeneration. Invertebrate models have been diligently utilized in investigating tauopathies, contributing to the understanding of cellular and molecular pathways involved in disease etiology. An important discovery came with the demonstration that over-expression of human tau in Drosophila leads to premature mortality and neuronal dysfunction including neurodegeneration, recapitulating some key neuropathological features of the human disease. The simplicity of handling invertebrate models combined with the availability of a diverse range of experimental resources make these models, in particular Drosophila a powerful invertebrate screening tool. Consequently, several large-scale screens have been performed using Drosophila, to identify modifiers of tau toxicity. The screens have revealed not only common cellular and molecular pathways, but in some instances the same modifier has been independently identified in two or more screens suggesting a possible role for these modifiers in regulating tau toxicity. The purpose of this review is to discuss the genetic modifier screens on tauopathies performed in Drosophila and C. elegans models, and to highlight the common cellular and molecular pathways that have emerged from these studies. Here, we summarize results of tau toxicity screens providing mechanistic insights into pathological alterations in tauopathies. Key pathways or modifiers that have been identified are associated with a broad range of processes including, but not limited to, phosphorylation, cytoskeleton organization, axonal transport, regulation of cellular proteostasis, transcription, RNA metabolism, cell cycle regulation, and apoptosis. We discuss the utility and application of invertebrate models in elucidating the cellular and molecular functions of novel and uncharacterized disease modifiers identified in large-scale screens as well as for investigating the function of genes identified as risk factors in genome-wide association studies from human patients in the post-genomic era. In this review, we combined and summarized several large-scale modifier screens performed in invertebrate models to identify modifiers of tau toxicity. A summary of the screens show that diverse cellular processes are implicated in the modification of tau toxicity. Kinases and phosphatases are the most predominant class of modifiers followed by components required for cellular proteostasis and axonal transport and cytoskeleton elements.

Reticulon-like-1, the Drosophila orthologue of the hereditary spastic paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons.

Several causative genes for hereditary spastic paraplegia encode proteins with intramembrane hairpin loops that contribute to the curvature of the endoplasmic reticulum (ER), but the relevance of this function to axonal degeneration is not understood. One of these genes is reticulon2. In contrast to mammals, Drosophila has only one widely expressed reticulon orthologue, Rtnl1, and we therefore used Drosophila to test its importance for ER organization and axonal function. Rtnl1 distribution overlapped with that of the ER, but in contrast to the rough ER, was enriched in axons. The loss of Rtnl1 led to the expansion of the rough or sheet ER in larval epidermis and elevated levels of ER stress. It also caused abnormalities specifically within distal portions of longer motor axons and in their presynaptic terminals, including disruption of the smooth ER (SER), the microtubule cytoskeleton and mitochondria. In contrast, proximal axon portions appeared unaffected. Our results provide direct evidence for reticulon function in the organization of the SER in distal longer axons, and support a model in which spastic paraplegia can be caused by impairment of axonal the SER. Our data provide a route to further understanding of both the role of the SER in axons and the pathological consequences of the impairment of this compartment.

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.

Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival.

Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. In the present study we have generated Drosophila transgenic for ovine PrP (prion protein) to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ-line transformation under expression control by the bi-partite GAL4/UAS (upstream activating sequence) system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and the phenotype of the fly when expressed in the Drosophila nervous system. The Arg(154) ovine PrP variants showed higher levels of PrP expression in neuronal cell bodies and insoluble PrP conformer than did His(154) variants. High levels of ovine PrP expression in Drosophila were associated with phenotypic effects, including reduced locomotor activity and decreased survival. Significantly, the present study highlights a critical role for helix-1 in the formation of distinct conformers of ovine PrP, since expression of His(154) variants were associated with decreased survival in the absence of high levels of PrP accumulation. Collectively, the present study shows that variants of the ovine PrP are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.

Uncovering specificity of endogenous TAU aggregation in a human iPSC-neuron TAU seeding model.

Tau pathobiology has emerged as a key component underlying Alzheimer's disease (AD) progression; however, human neuronal in vitro models have struggled to recapitulate tau phenomena observed in vivo. Here, we aimed to define the minimal requirements to achieve endogenous tau aggregation in functional neurons utilizing human induced pluripotent stem cell (hiPSC) technology. Optimized hiPSC-derived cortical neurons seeded with AD brain-derived competent tau species or recombinant tau fibrils displayed increases in insoluble, endogenous tau aggregates. Importantly, MAPT-wild type and MAPT-mutant hiPSC-neurons exhibited unique propensities for aggregation dependent on the seed strain rather than the repeat domain identity, suggesting that successful templating of the recipient tau may be driven by the unique conformation of the seed. The in vitro model presented here represents the first successful demonstration of combining human neurons, endogenous tau expression, and AD brain-derived competent tau species, offering a more physiologically relevant platform to study tau pathobiology.

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate.

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.

Identification of cis-acting determinants mediating the unconventional secretion of tau.

The deposition of tau aggregates throughout the brain is a pathological characteristic within a group of neurodegenerative diseases collectively termed tauopathies, which includes Alzheimer's disease. While recent findings suggest the involvement of unconventional secretory pathways driving tau into the extracellular space and mediating the propagation of the disease-associated pathology, many of the mechanistic details governing this process remain elusive. In the current study, we provide an in-depth characterization of the unconventional secretory pathway of tau and identify novel molecular determinants that are required for this process. Here, using Drosophila models of tauopathy, we correlate the hyperphosphorylation and aggregation state of tau with the disease-related neurotoxicity. These newly established systems recapitulate all the previously identified hallmarks of tau secretion, including the contribution of tau hyperphosphorylation as well as the requirement for PI(4,5)P2 triggering the direct translocation of tau. Using a series of cellular assays, we demonstrate that both the sulfated proteoglycans on the cell surface and the correct orientation of the protein at the inner plasma membrane leaflet are critical determinants of this process. Finally, we identify two cysteine residues within the microtubule binding repeat domain as novel cis-elements that are important for both unconventional secretion and trans-cellular propagation of tau.

Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila.

We investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1ß, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects.Abbreviations: AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; H2O2: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; Pink1: PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.

HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein beta(2)-microglobulin upon release from the MHC-1.

The light chain of the major histocompatibility complex class 1 (MHC-1), the protein beta(2)-microglobulin (beta(2)m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of beta(2)m in its MHC-1 bound state compared with those of beta(2)m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound beta(2)m for the first time. The HDX-ESI-MS data show clearly that the H-->D exchange of MHC-1 bound beta(2)m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric beta(2)m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound beta(2)m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound beta(2)m are consistent with unfolding of its exchange-protected core with a t(1/2) of 68 min (pH 7, 37 degrees C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free beta(2)m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein.

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

Folding versus aggregation: polypeptide conformations on competing pathways.

Protein aggregation has now become recognised as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are caused by protein aggregation, the biophysical characterisation of misfolded states and their aggregation mechanisms has received increased attention. Utilising experimental techniques and computational approaches established for the analysis of protein folding reactions has ensured rapid advances in the study of pathways leading to amyloid fibrils and amyloid-related aggregates. Here we describe recent experimental and theoretical advances in the elucidation of the conformational properties of dynamic, heterogeneous and/or insoluble protein ensembles populated on complex, multidimensional protein energy landscapes. We discuss current understanding of aggregation mechanisms in this context and describe how the synergy between biochemical, biophysical and cell-biological experiments are beginning to provide detailed insights into the partitioning of non-native species between protein folding and aggregation pathways.

Changes in Glutathione Redox Potential Are Linked to Aß42-Induced Neurotoxicity.

Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aß) aggregation. We find that changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. EGSH changes and Aß42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aß42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.

Unconventional Secretion Mediates the Trans-cellular Spreading of Tau.

The progressive deposition of misfolded hyperphosphorylated tau is a pathological hallmark of tauopathies, including Alzheimer's disease. However, the underlying molecular mechanisms governing the intercellular spreading of tau species remain elusive. Here, we show that full-length soluble tau is unconventionally secreted by direct translocation across the plasma membrane. Increased secretion is favored by tau hyperphosphorylation, which provokes microtubule detachment and increases the availability of free protein inside cells. Using a series of binding assays, we show that free tau interacts with components enriched at the inner leaflet of the plasma membrane, finally leading to its translocation across the plasma membrane mediated by sulfated proteoglycans. We provide further evidence that secreted soluble tau species spread trans-cellularly and are sufficient for the induction of intracellular tau aggregation in adjacent cells. Our study demonstrates the mechanistic details of tau secretion and provides insights into the initiation and progression of tau pathology.

Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry.

Numerous debilitating human disorders result from protein misfolding and amyloid formation. Despite the grave nature of these maladies, our understanding of the structural mechanism of fibril assembly is limited. Of paramount importance is the need to identify and characterize oligomeric species formed early during fibril assembly, so that the nature of the initiating assembly mechanism can be revealed and species that may be toxic to cells identified. However, the transient nature of early oligomeric species, combined with their heterogeneity and instability, has precluded detailed analysis to date. Here, we have used electrospray ionisation mass spectrometry (ESI-MS), complemented by analytical ultracentrifugation (AUC) and measurements of thioflavin-T fluorescence, to monitor the early stages of assembly of amyloid-like fibrils formed from human beta-2-microglobulin (beta2m) in vitro. We show that worm-like fibrils that form with nucleation-independent kinetics assemble by a mechanism consistent with monomer addition, with species ranging from monomer to > or = 13-mer being identified directly and uniquely as transient assembly intermediates. By contrast, only monomers, dimers, trimers and tetramers are observed during nucleated growth, which leads to the formation of long straight fibrils. The results highlight the unique power of non-covalent ESI-MS to identify protein assembly intermediates in complex heterogeneous systems and demonstrate its great potential to identify and characterise individual species formed early during amyloid assembly.

Seed-induced acceleration of amyloid-ß mediated neurotoxicity in vivo.

Seeded propagation of amyloid-beta (Aß) pathology is suggested to contribute to the progression of Alzheimer's disease. Local overproduction of aggregation-prone Aß variants could explain the focal initiation of a seeding cascade that subsequently triggers widespread pathology. Several animal models support this seeding concept by demonstrating accelerated Aß deposition following inoculation with Aß-containing homogenates, however its role in progressive neurodegeneration remains unclear. Here, we present a non-invasive approach to study Aß seeding processes in vivo using Drosophila models. We show that small amounts of aggregation-competent Aß42 seeds, generated in selected neuronal clusters, can induce the deposition of the pan-neuronally expressed and otherwise soluble Aß40. Moreover, our models visualize the accelerated formation and propagation of amyloid pathology throughout the brain, which correlates with severe neurotoxicity. Taken together, these in vivo models provide mechanistic insights into disease-related processes and represent versatile genetic tools to determine novel modifiers of the Aß seeding cascade.Seeding of amyloid beta from one brain region to another is thought to contribute to the progression of Alzheimer's disease, although to date most studies have depended on inoculation of animals with exogenous amyloid. Here the authors describe a genetic seed and target system in Drosophila which may be useful for the mechanistic study of seeding of amyloid in vivo.

The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization.

The kinesin-3 family member KIF1A has been shown to be important for experience dependent neuroplasticity. In Drosophila, amorphic mutations in the KIF1A homolog unc-104 disrupt the formation of mature boutons. Disease associated KIF1A mutations have been associated with motor and sensory dysfunctions as well as non-syndromic intellectual disability in humans. A hypomorphic mutation in the forkhead-associated domain of Unc-104, unc-104bris, impairs active zone maturation resulting in an increased fraction of post-synaptic glutamate receptor fields that lack the active zone scaffolding protein Bruchpilot. Here, we show that the unc-104brismutation causes defects in synaptic transmission as manifested by reduced amplitude of both evoked and miniature excitatory junctional potentials. Structural defects observed in the postsynaptic compartment of mutant NMJs include reduced glutamate receptor field size, and altered glutamate receptor composition. In addition, we observed marked loss of postsynaptic scaffolding proteins and reduced complexity of the sub-synaptic reticulum, which could be rescued by pre- but not postsynaptic expression of unc-104. Our results highlight the importance of kinesin-3 based axonal transport in synaptic transmission and provide novel insights into the role of Unc-104 in synapse maturation.

The Drosophila KIF1A Homolog unc-104 Is Important for Site-Specific Synapse Maturation.

Mutations in the kinesin-3 family member KIF1A have been associated with hereditary spastic paraplegia (HSP), hereditary and sensory autonomic neuropathy type 2 (HSAN2) and non-syndromic intellectual disability (ID). Both autosomal recessive and autosomal dominant forms of inheritance have been reported. Loss of KIF1A or its homolog unc-104 causes early postnatal or embryonic lethality in mice and Drosophila, respectively. In this study, we use a previously described hypomorphic allele of unc-104, unc-104(bris) , to investigate the impact of partial loss-of-function of kinesin-3 on synapse maturation at the Drosophila neuromuscular junction (NMJ). Unc-104(bris) mutants exhibit structural defects where a subset of synapses at the NMJ lack all investigated active zone (AZ) proteins, suggesting a complete failure in the formation of the cytomatrix at the active zone (CAZ) at these sites. Modulating synaptic Bruchpilot (Brp) levels by ectopic overexpression or RNAi-mediated knockdown suggests that the loss of AZ components such as Ca(2+) channels and Liprin-α is caused by impaired kinesin-3 based transport rather than due to the absence of the key AZ organizer protein, Brp. In addition to defects in CAZ assembly, unc-104(bris) mutants display further defects such as depletion of dense core and synaptic vesicle (SV) markers from the NMJ. Notably, the level of Rab3, which is important for the allocation of AZ proteins to individual release sites, was severely reduced at unc-104(bris) mutant NMJs. Overexpression of Rab3 partially ameliorates synaptic phenotypes of unc-104(bris) larvae, suggesting that lack of presynaptic Rab3 contributes to defects in synapse maturation.

The Yin and Yang of protein folding.

The study of protein aggregation saw a renaissance in the last decade, when it was discovered that aggregation is the cause of several human diseases, making this field of research one of the most exciting frontiers in science today. Building on knowledge about protein folding energy landscapes, determined using an array of biophysical methods, theory and simulation, new light is now being shed on some of the key questions in protein-misfolding diseases. This review will focus on the mechanisms of protein folding and amyloid fibril formation, concentrating on the role of partially folded states in these processes, the complexity of the free energy landscape, and the potentials for the development of future therapeutic strategies based on a full biophysical description of the combined folding and aggregation free-energy surface.