Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein

Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.     

Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson''s disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.     

The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation

Human α-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of α-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of α-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in α-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form α-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.     

E46K Parkinson’s-Linked Mutation Enhances C-Terminal-to-N-Terminal Contacts in α-Synuclein

Parkinson's disease (PD) is associated with the deposition of fibrillar aggregates of the protein α-synuclein (αS) in neurons. Intramolecular contacts between the acidic C-terminal tail of αS and its N-terminal region have been proposed to regulate αS aggregation, and two originally described PD mutations, A30P and A53T, reportedly reduce such contacts. We find that the most recently discovered PD-linked αS mutation E46K, which also accelerates the aggregation of the protein, does not interfere with C-terminal-to-N-terminal contacts and instead enhances such contacts. Furthermore, we do not observe a substantial reduction in such contacts in the two previously characterized mutants. Our results suggest that C-terminal-to-N-terminal contacts in αS are not strongly protective against aggregation, and that the dominant mechanism by which PD-linked mutations facilitate αS aggregation may be altering the physicochemical properties of the protein such as net charge (E46K) and secondary structure propensity (A30P and A53T).     

Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils: IMPLICATIONS FOR CANCER

Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.     

Cataract-linked mutation R188H promotes βB2-crystallin aggregation and fibrillization during acid denaturation

Cataract is characterized by the formation of light-scattering protein aggregates in the lens. β/γ-Crystallins are the predominant structural proteins in the cytosol of lens fiber cells, and more than fifty β/γ-crystallin mutations have been linked to autosomal dominant congenital cataract. However, the structural role of these mutations in the formation of the core structures of amorphous aggregates or amyloid-like fibrils has not been elucidated yet. In this research, we studied the effects of the V187M and R188H mutations on the aggregation and fibrillization of βB2-crystallin during acid denaturation. The behavior of V187M was the same as the WT protein, suggesting that the residue at position 187 contributed little to the aggregation/fibrillization process. R188H promoted the formation of amorphous aggregates at pH above 3 and accelerated fibrillization at pH 3. The distinct behaviors of the mutants suggested that the residue at position 188 might play a regulatory role in βB2-crystallin aggregation/fibrillization but not reside in the core of the aggregates/fibrils.     

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.     

The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein alpha-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K alpha-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K alpha-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K alpha-synuclein fibrils possess a typical amyloid ultrastructure. E46K alpha-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of alpha-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type alpha-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of alpha-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT alpha-synuclein that are observed in vitro.     

Defects in very long chain fatty acid synthesis enhance alpha-synuclein toxicity in a yeast model of Parkinson's disease

We identified three S. cerevisiae lipid elongase null mutants (elo1Δ, elo2Δ, and elo3Δ) that enhance the toxicity of alpha-synuclein (α-syn). These elongases function in the endoplasmic reticulum (ER) to catalyze the elongation of medium chain fatty acids to very long chain fatty acids, which is a component of sphingolipids. Without α-syn expression, the various elo mutants showed no growth defects, no reactive oxygen species (ROS) accumulation, and a modest decrease in survival of aged cells compared to wild-type cells. With (WT, A53T or E46K) α-syn expression, the various elo mutants exhibited severe growth defects (although A30P had a negligible effect on growth), ROS accumulation, aberrant protein trafficking, and a dramatic decrease in survival of aged cells compared to wild-type cells. Inhibitors of ceramide synthesis, myriocin and FB1, were extremely toxic to wild-type yeast cells expressing (WT, A53T, or E46K) α-syn but much less toxic to cells expressing A30P. The elongase mutants and ceramide synthesis inhibitors enhance the toxicity of WT α-syn, A53T and E46K, which transit through the ER, but have a negligible effect on A30P, which does not transit through the ER. Disruption of ceramide-sphingolipid homeostasis in the ER dramatically enhances the toxicity of α-syn (WT, A53T, and E46K).     

Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants

High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.     

Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines,in Yeast,and in Vivo

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.     

Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson’s disease

Understanding α-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson’s disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in α-synuclein acquire β-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of α-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, ⁴⁰Val and ⁷⁴Val, which play key roles in β-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D_V74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.     

The aggregation of mutant p53 produces prion-like properties in cancer

The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.     

Structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites

α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD—A30P, E46K and A53T—are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.     

Residual structure and dynamics in Parkinson's disease-associated mutants of alpha-synuclein

alpha-Synuclein (alpha S) is a pre-synaptic protein that has been implicated as a possible causative agent in the pathogenesis of Parkinson's disease (PD). Two autosomal dominant missense mutations in the alpha S gene are associated with early onset PD. Because alpha S is found in an aggregated fibrillar form in the Lewy body deposits characteristic of Parkinson's patients, aggregation of the protein is believed to be related to its involvement in the disease process. The wild type (WT) and early onset mutants A30P and A53T display diverse in vitro aggregation kinetics even though the gross physicochemical and morphological properties of the mutants are highly similar. We used high resolution solution NMR spectroscopy to compare the structural and dynamic properties of the A53T and A30P mutants with those of WT alpha S in the free state. We found that the A30P mutation disrupts a region of residual helical structure that exists in the WT protein, whereas the A53T mutation results in a slight enhancement of a small region around the site of mutation with a preference for extended conformations. Based on these results and on the anticipated effects of these mutations on elements of secondary structure, we proposed a model of how these two PD-linked mutations influence alpha S fibril formation that is consistent with the documented differences in the fibrillization kinetics of the two mutants.     

E46K human alpha-synuclein transgenic mice develop Lewy-like and tau pathology associated with age-dependent, detrimental motor impairment

Synucleinopathies are a group of neurodegenerative disorders associated with the formation of aberrant amyloid inclusions composed of the normally soluble presynaptic protein α-synuclein (α-syn). Parkinson disease is the most well known of these disorders because it bears α-syn pathological inclusions known as Lewy bodies (LBs). Mutations in the gene for α-syn, including the E46K missense mutation, are sufficient to cause Parkinson disease as well as other synucleinopathies like dementia with LBs. Herein, we describe transgenic mice expressing E46K human α-syn in CNS neurons that develop detrimental age-dependent motor impairments. These animals accumulate age-dependent intracytoplasmic neuronal α-syn inclusions that parallel disease and recapitulate the biochemical, histological, and morphological properties of LBs. Surprisingly, the morphology of α-syn inclusions in E46K human α-syn transgenic mice more closely resemble LBs than the previously described transgenic mice (line M83) that express neuronal A53T human α-syn. E46K human α-syn mice also develop abundant neuronal tau inclusions that resemble neurofibrillary tangles. Subsequent studies on the ability of E46K α-syn to induce tau inclusions in cellular models suggest that both direct and indirect mechanisms of protein aggregation are probably involved in the formation of the tau inclusions observed here in vivo. Re-evaluation of presymptomatic transgenic mice expressing A53T human α-syn reveals that the formation of α-syn inclusions in mice must be synchronized; however, inclusion formation is diffuse within affected areas of the neuroaxis such that there was no clustering of inclusions. Collectively, these findings provide insights in the mechanisms of formation of these aberrant proteinaceous inclusions and support the notion that α-syn aggregates are involved in the pathogenesis of human diseases.     

Rapid self-assembly of alpha-synuclein observed by in situ atomic force microscopy

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.     

The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson's disease

α-Synuclein (α-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson's disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How α-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of α-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic α-syn oligomers. In this study, we have generated α-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of α-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of α-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to α-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like α-syn aggregates in the substantia nigra. These results provide new insights into how α-syn-induced toxicity is related to its aggregation.     

Differences in the pathways of proteins unfolding induced by urea and guanidine hydrochloride: molten globule state and aggregates

It was shown that at low concentrations guanidine hydrochloride (GdnHCl) can cause aggregation of proteins in partially folded state and that fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates rather than with hydrophobic clusters on the surface of protein in molten globule state. That is why the increase in ANS fluorescence intensity is often recorded in the pathway of protein denaturation by GdnHCl, but not by urea. So what was previously believed to be the molten globule state in the pathway of protein denaturation by GdnHCl, in reality, for some proteins represents the aggregates of partially folded molecules.