Α-syn Fibril Formation Research Articles

Inhibition of α‐synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.

Comparative Analysis of Different Peptidyl-Prolyl Isomerases Reveals FK506-binding Protein 12 as the Most Potent Enhancer of α-Synuclein Aggregation

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.

Conserved C-Terminal Charge Exerts a Profound Influence on the Aggregation Rate of α-Synuclein

α-Synuclein (α-syn) is the major component of filamentous Lewy bodies found in the brains of patients diagnosed with Parkinson's disease (PD). Recent studies demonstrate that, in addition to the wild-type sequence, α-syn is found in several modified forms, including truncated and phosphorylated species. Although the mechanism by which the neuronal loss in PD occurs is unknown, aggregation and fibril formation of α-syn are considered to be key pathological features. In this study, we analyze the rates of fibril formation and the monomer–fibril equilibrium for eight disease-associated truncated and phosphorylated α-syn variants. Comparison of the relative rates of aggregation reveals a strong monotonic relationship between the C-terminal charge of α-syn and the lag time prior to the observation of fibril formation, with truncated species exhibiting the fastest aggregation rates. Moreover, we find that a decrease in C-terminal charge shifts the equilibrium to favor the fibrillar species. An analysis of these findings in the context of linear growth theories suggests that the loss of the charge-mediated stabilization of the soluble state is responsible for the enhanced aggregation rate and increased extent of fibril fraction. Therefore, C-terminal charge is kinetically and thermodynamically protective against α-syn polymerization and may provide a target for the treatment of PD.

Baicalein Inhibits Formation of α‐Synuclein Oligomers within Living Cells and Prevents Aβ Peptide Fibrillation and Oligomerisation

Abnormal protein aggregation in the brain is linked to the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Recent studies revealed that the oligomeric form of aggregates is most likely the toxic species, and thus could be a good therapeutic target. To screen for potent inhibitors that can inhibit both oligomerisation and fibrillation of α-synuclein (α-syn), we systematically compared the antioligomeric and antifibrillar activities of eight compounds that were extracted from Chinese herbal medicines through three platforms that can monitor the formation of α-syn fibrils and oligomers in cell-free or cellular systems. Our results revealed that baicalein, a flavonoid extracted from the Chinese herbal medicine Scutellaria baicalensis Georgi ("huang qin" in Chinese), is a potent inhibitor of α-syn oligomerisation both in cell-free and cellular systems, and is also an effective inhibitor of α-syn fibrillation in cell-free systems. We further tested the protective effect of baicalein against α-syn-oligomer-induced toxicity in neuronal cells. Our data showed that baicalein inhibited the formation of α-syn oligomers in SH-SY5Y and Hela cells, and protected SH-SY5Y cells from α-syn-oligomer-induced toxicity. We also explored the effect of baicalein on amyloid-β peptide (Aβ) aggregation and toxicity. We found that baicalein can also inhibit Aβ fibrillation and oligomerisation, disaggregate pre-formed Aβ amyloid fibrils and prevent Aβ fibril-induced toxicity in PC12 cells. Our study indicates that baicalein is a good inhibitor of amyloid protein aggregation and toxicity. Given the role of these processes in neurodegenerative diseases such as AD and PD, our results suggest that baicalein has potential as a therapeutic agent for the treatment of these devastating disorders.

Dopamine quinones interact with α-synuclein to form unstructured adducts

α-Synuclein (αsyn) fibril formation is considered a central event in the pathogenesis of Parkinson’s disease (PD). In recent years, it has been proposed that prefibrillar annular oligomeric β-sheet-rich species, called protofibrils, rather than fibrils themselves, may be the neurotoxic species. The oxidation products of dopamine (DAQ) can inhibit αsyn fibril formation supporting the idea that DAQ might stabilize αsyn protofibrils. In the present work, through different biochemical and biophysical techniques, we isolated and structurally characterized αsyn/DAQ adducts. Contrary to protofibrils, we demonstrated that αsyn/DAQ adducts retain an unfolded conformation. We then investigated the nature of the modifications induced on αsyn by DAQ. Our results indicate that only a small fraction of αsyn interacts with DAQ in a covalent way, so that non-covalent interaction appears to be the major modification induced by DAQ on αsyn.

Methionine oxidation stabilizes non-toxic oligomers of α-synuclein through strengthening the auto-inhibitory intra-molecular long-range interactions

Oxidative stress and aggregation of the presynaptic protein α-synuclein (α-Syn) are implied in the pathogenesis of Parkinson's disease and several other neurodegenerative diseases. Various posttranslational modifications, such as oxidation, nitration and truncation, have significant effects on the kinetics of α-Syn fibrillation in vitro. α-Syn is a typical natively unfolded protein, which possesses some residual structure. The existence of long-range intra-molecular interactions between the C-terminal tail (residues 120–140) and the central part of α-Syn (residues 30–100) was recently established (Bertoncini et al. (2005) Proc Natl Acad Sci U S A 102, 1430–1435). Since α-Syn has four methionines, two of which (Met 1 and 5) are at the N-terminus and the other two (Met 116 and 127) are in the hydrophobic cluster at the C-terminus of protein, the perturbation of these residues via their oxidation represents a good model for studying the effect of long-range interaction on α-Syn fibril formation. In this paper we show that Met 1, 116, and 127 are more protected from the oxidation than Met 5 likely due to the residual structure in the natively unfolded α-Syn. In addition to the hydrophobic interactions between the C-terminal hydrophobic cluster and hydrophobic central region of α-Syn, there are some long-range electrostatic interactions in this protein. Both of these interactions likely serve as auto-inhibitors of α-Syn fibrillation. Methionine oxidation affects both electrostatic and hydrophobic long-range interactions in α-Syn. Finally, oxidation of methionines by H 2O 2 greatly inhibited α-Syn fibrillation in vitro, leading to the formation of relatively stable oligomers, which are not toxic to dopaminergic and GABAergic neurons.

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

Cytosolic Catechols Inhibit α-Synuclein Aggregation and Facilitate the Formation of Intracellular Soluble Oligomeric Intermediates

Aberrant aggregation of alpha-synuclein (alpha-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of alpha-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on alpha-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form alpha-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of alpha-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant alpha-syn revealed the presence of oligomeric alpha-syn in nondegenerating dopaminergic neurons that do contain insoluble alpha-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of alpha-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.

Fibrillization of α-synuclein and tau in familial Parkinson's disease caused by the A53T α-synuclein mutation

Mutations in the α-synuclein ( α-syn) gene are responsible for a rare familial parkinsonism syndrome, a finding that has led to extensive characterization of altered α-syn structure in sporadic Parkinson's disease (PD) and other neurodegenerative disorders. We report here the immunohistochemical, biochemical and ultrastructural characterization of α-syn neuropathology in a case of familial PD with the A53T α-syn gene mutation. Insoluble filamentous α-syn lesions were detected in almost all brain regions examined and as in sporadic PD, we observed the accumulation of insoluble nitrated α-syn in this familial disorder. Significant accumulations of filamentous insoluble tau protein also were detected in some brain regions of this patient, suggesting a role for A53T mutant α-syn in tau fibrillization. Indeed, in vitro studies of tau and α-syn fibrillization showed that the A53T mutation accelerated α-syn fibril formation, initiated tau assembly into filaments and synergistically enhanced fibrillization of both tau and α-syn. Our data implicate fibrillization of α-syn and tau in the pathogenesis of PD, and suggest that distinct amyloidogenic proteins may cross-seed each other in neurodegenerative diseases.