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The Journal of Biological Chemistry Dec 2020Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the...
Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.
Topics: Animals; Cells, Cultured; Dopaminergic Neurons; Endosomes; Ferroptosis; Humans; Lysosomes; Mice, Inbred C57BL; Mice, Knockout; Parkinson Disease; alpha-Synuclein
PubMed: 33453994
DOI: 10.1074/jbc.RA120.013428 -
Movement Disorders : Official Journal... Oct 2022The α-Synuclein (α-Syn) V15A variant has been found in two Caucasian families with Parkinson's disease (PD). However, the significance of this missense variant...
BACKGROUND
The α-Synuclein (α-Syn) V15A variant has been found in two Caucasian families with Parkinson's disease (PD). However, the significance of this missense variant remained unclear.
OBJECTIVE
We sought to elucidate whether V15A could increase aggregation or change phospholipid affinity.
METHODS
A sequencing analysis for the SNCA encoding α-Syn from 875 patients with PD and 324 control subjects was performed. Comparing with known pathogenic missense variants of α-Syn, A30P, and A53T, we analyzed the effects of V15A on binding to phospholipid membrane, self-aggregation, and seed-dependent aggregation in cultured cells.
RESULTS
Genetic screening identified SNCA c.44 T>C (p.V15A) from two Japanese PD families. The missense variant V15A was extremely rare in several public databases and predicted as pathogenic using in silico tools. The amplification activity of α-Syn V15A fibrils was stronger than that of wild-type α-Syn fibrils.
CONCLUSIONS
The discovery of the V15A variant from Japanese families reinforces the possibility that the V15A variant may be a causative variant for developing PD. V15A had a reduced affinity for phospholipids and increased propagation activity compared with wild-type. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Topics: Humans; alpha-Synuclein; Cell Line; Mutation, Missense; Parkinson Disease; Phospholipids
PubMed: 35894540
DOI: 10.1002/mds.29162 -
Biochimica Et Biophysica Acta. Proteins... 2019Aggregation of the neuronal protein α-synuclein into amyloid fibrils is a hallmark of Parkinson's disease. The propensity of α-synuclein to aggregate increases with...
BACKGROUND
Aggregation of the neuronal protein α-synuclein into amyloid fibrils is a hallmark of Parkinson's disease. The propensity of α-synuclein to aggregate increases with the protein concentration. For the development of efficient inhibitors of α-synuclein aggregation, it is important to know the critical concentration of aggregation (the concentration of monomeric protein, below which the protein does not aggregate).
METHODS
We performed in vitro aggregation studies of α-synuclein at low concentrations (0.11-20 μM). Aggregation kinetics was measured by ThT fluorescence. Obtained aggregates were characterized using CD-spectroscopy, fluorescent spectroscopy, dynamic light scattering and AFM imaging.
RESULTS
Monomeric α-synuclein at concentrations 0.45 μM and above was able to bind to fibril ends resulting in fibril growth. At the protein concentrations below 0.4 μM, monomers did not fibrillize, and fibrils disaggregated. In the absence of seeds, fibrils were formed only at monomer concentrations higher than 10 μM. At low micromolar concentrations, we observed formation of prefibrillar amyloid aggregates, which are able to induce fibril formation in α-synuclein solutions of high concentrations.
CONCLUSIONS
The critical concentration of α-synuclein fibril growth is ~0.4 μM. Prefibrillar amyloid aggregates appear at concentrations between 0.45 and 3 μM and are an intermediate state between monomers and fibrils. Although morphologically different from fibrils, prefibrillar aggregates have similar properties to those of fibrils.
GENERAL SIGNIFICANCE
We determined the critical concentration of α-synuclein fibril growth. We showed that fibrils can grow at much lower monomer concentrations than that required for de novo fibril formation. We characterized a prefibrillar intermediate species formed upon aggregation of α-synuclein at low micromolar concentration.
Topics: Amyloid; Circular Dichroism; Humans; Protein Aggregates; Spectrometry, Fluorescence; alpha-Synuclein
PubMed: 31096048
DOI: 10.1016/j.bbapap.2019.05.003 -
Molecules (Basel, Switzerland) Jan 2020The object of our analysis is the structure of alpha-synuclein (ASyn), which, under in vivo conditions, associates with presynaptic vesicles. Misfolding of ASyn is known...
The object of our analysis is the structure of alpha-synuclein (ASyn), which, under in vivo conditions, associates with presynaptic vesicles. Misfolding of ASyn is known to be implicated in Parkinson's disease. The availability of structural information for both the micelle-bound and amyloid form of ASyn enables us to speculate on the specific mechanism of amyloid transformation. This analysis is all the more interesting given the fact that-Unlike in Aβ(1-42) amyloids-only the central fragment (30-100) of ASyn has a fibrillar structure, whereas, its N- and C-terminal fragments (1-30 and 100-140, respectively) are described as random coils. Our work addresses the following question: Can the ASyn chain-as well as the aforementioned individual fragments-adopt globular conformations? In order to provide an answer, we subjected the corresponding sequences to simulations carried out using Robetta and I-Tasser, both of which are regarded as accurate protein structure predictors. In addition, we also applied the fuzzy oil drop (FOD) model, which, in addition to optimizing the protein's internal free energy, acknowledges the presence of an external force field contributed by the aqueous solvent. This field directs hydrophobic residues to congregate near the center of the protein body while exposing hydrophilic residues on its surface. Comparative analysis of the obtained models suggests that fragments which do not participate in forming the amyloid fibril (i.e., 1-30 and 100-140) can indeed attain globular conformations. We also explain the influence of mutations observed in vivo upon the susceptibility of ASyn to undergo amyloid transformation. In particular, the 30-100 fragment (which adopts a fibrillar structure in PDB) is not predicted to produce a centralized hydrophobic core by any of the applied toolkits (Robetta, I-Tasser, and FOD). This means that in order to minimize the entropically disadvantageous contact between hydrophobic residues and the polar solvent, ASyn adopts the form of a ribbonlike micelle (rather than a spherical one). In other words, the ribbonlike micelle represents a synergy between the conformational preferences of the protein chain and the influence of its environment.
Topics: Amyloid; Humans; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Mutation; Peptide Fragments; Protein Conformation; alpha-Synuclein
PubMed: 32019169
DOI: 10.3390/molecules25030600 -
Trends in Neurosciences Jun 2022A recent study by Tripathi et al. used a protein engineering approach to demonstrate that cellular stress caused by familial α-synuclein mutations can be alleviated by...
A recent study by Tripathi et al. used a protein engineering approach to demonstrate that cellular stress caused by familial α-synuclein mutations can be alleviated by altering the monounsaturated fatty acid equilibrium in neuronal cells. This work supports the notion that metabolic perturbation of lipids may be involved in the pathogenesis of Parkinson's disease.
Topics: Fatty Acids; Humans; Mutation; Parkinson Disease; alpha-Synuclein
PubMed: 35379479
DOI: 10.1016/j.tins.2022.03.006 -
Journal of Medicinal Chemistry Nov 2019Aggregation of the neuronal protein α-synuclein into amyloid fibrils plays a central role in the development of Parkinson's disease. Growth of fibrils can be suppressed...
Aggregation of the neuronal protein α-synuclein into amyloid fibrils plays a central role in the development of Parkinson's disease. Growth of fibrils can be suppressed by blocking fibril ends from their interaction with monomeric proteins. In this work, we constructed inhibitors that bind to the ends of α-synuclein amyloid fibrils with very high affinity. They are based on synthetic α-synuclein dimers and interact with fibrils via two monomeric subunits adopting conformation that efficiently blocks fibril elongation. By tuning the charge of dimers, we further enhanced the binding affinity and prepared a construct that inhibits fibril elongation at nanomolar concentration (IC ≈ 20 nM). To the best of our knowledge, it is the most efficient inhibitor of α-synuclein fibrillization.
Topics: Amyloid; Central Nervous System Agents; Circular Dichroism; Disulfides; Humans; Protein Multimerization; Structure-Activity Relationship; alpha-Synuclein
PubMed: 31625739
DOI: 10.1021/acs.jmedchem.9b01400 -
Chemphyschem : a European Journal of... Mar 2021Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However,...
Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However, α-synuclein is prone to form aggregates, and these inclusions, known as Lewy bodies, are the hallmark of Parkinson's disease. α-synuclein can alter its conformation and acquire aggregating capacity, forming aggregates containing β-sheets. This protein's pathogenic importance is based on its ability to form oligomers that impair synaptic transmission and neuronal function by increasing membrane permeability and altering homeostasis, generating a deleterious effect over cells. First, we establish that oligomers interfere with the mechanical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane, as demonstrated by nanoindentation curves. In contrast, nanoindentation revealed that the α-synuclein monomer's presence leads to a much more resistant lipid bilayer. Moreover, the oligomers' interaction with cell membranes can promote lactate dehydrogenase (LDH) release, suggesting the activation of cytotoxic events.
Topics: Animals; Cell Line, Tumor; Cell Membrane; Hydrophobic and Hydrophilic Interactions; L-Lactate Dehydrogenase; Lipid Bilayers; Mice; Phosphatidylcholines; Protein Aggregates; Protein Multimerization; alpha-Synuclein
PubMed: 33482036
DOI: 10.1002/cphc.202000831 -
Journal of Thrombosis and Thrombolysis May 2022Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and...
Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets.
Topics: Antibodies, Monoclonal; Blood Platelets; Flow Cytometry; Humans; alpha-Synuclein
PubMed: 34797472
DOI: 10.1007/s11239-021-02607-z -
Current Alzheimer Research Sep 2007Alpha-synuclein is the main constituent of intra-neuronal Lewy bodies, which are characteristic of Parkinson's disease, but aggregates are also found as axonal... (Review)
Review
Alpha-synuclein is the main constituent of intra-neuronal Lewy bodies, which are characteristic of Parkinson's disease, but aggregates are also found as axonal inclusions. Alpha-synuclein pathology is found together with beta-amyloid plaques and neurofibrillary tangles in Alzheimer's disease and other neurodegenerative disorders. In spite of the fact that the biological function of this synaptic protein is not known so far, there is an increasing body of evidence indicating an interaction with amyloid peptides, but also with tau-hyperphosphorylation. A high proportion of alpha-synuclein purified from Lewy bodies is phosphorylated on Ser129. There are still different opinions about the toxicity of the alpha-synuclein aggregates. Alpha-synuclein seems to influence different intracellular signaling pathways which are in direct relation to defense mechanisms against reactive oxygen species or apoptosis. It is obvious that overproduction of alpha-synuclein, but also different mutations, are inducing the formation of aggregates. Because of the possible link to neurodegeneration, different attempts have been made to counteract alpha-synuclein aggregation. An interesting approach is utilizing beta-synuclein, a biological factor, with an aminoacid sequence closely resembling that of alpha-synuclein. Proof of concept studies indicated that overexpression of beta-synuclein is able to counteract alpha-synuclein aggregation in a transgenic animal model, while also ameliorating functional deficits. As an alternative approach, the use of low molecular beta-synuclein N-terminal peptide derivatives has been considered. Several of these structures displayed clear neuroprotective activities in tissue culture models of neurodegeneration, including beta-amyloid toxicity. Therefore it has been speculated that these compounds might have a broad therapeutic efficacy in different neurodegenerative disorders. A proof of concept study in hAPP-transgenic animals resulted in a highly significant decrease in beta-amyloid plaque load, an increase in soluble beta-amyloid peptides and a decrease in insoluble forms. There was also significant improvement of cognitive deficits in this APP transgenic mouse model following intranasal but also peripheral treatment with three of these compounds. From this study it is concluded that the observed effects of the peptides derived from beta-synuclein N-terminus are depending on both, a direct interaction with aggregation of proteins, but also with stimulation of anti-apoptotic and anti-oxidative intracellular signaling pathways.
Topics: Animals; Enzyme Inhibitors; Humans; Neurodegenerative Diseases; Serine; alpha-Synuclein
PubMed: 17908049
DOI: 10.2174/156720507781788783 -
Biochemical Society Transactions Nov 2005There is strong evidence for the involvement of alpha-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease).... (Review)
Review
There is strong evidence for the involvement of alpha-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which alpha-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of alpha-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce alpha-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-synuclein (A53T), a familial PD-associated mutation.
Topics: Amino Acid Sequence; Humans; Molecular Sequence Data; Neurodegenerative Diseases; alpha-Synuclein
PubMed: 16246056
DOI: 10.1042/BST20051106