Interaction Of Α-syn Research Articles

Alpha-Synuclein Binding and Translocation by Alpha-Hemolysin Channel

Gauging the pathways of a natively unfolded Parkinson disease-related protein, α-synuclein (α-syn), between and within cells, is important for understanding pathogenesis. Here, we use a robust β-barrel ion channel, α-hemolysin (α-HL), as a model system to investigate the possibility of channel-assisted membrane transport of α-syn. The interaction of α-syn with the channel was observed as a transient, ∼95 % block of the channel current when (i) α-syn was applied from the membrane side where the shorter (stem) part of the α-HL heptamer is exposed and (ii) the applied potential is lower on the side of α-syn application. While the on-rate of α-syn binding to the channel exponentially increases with the applied field, the off-rate displays a crossover behavior. Statistical analysis of the blockage events indicates that at voltages >80 mV a significant portion of α-HL-bound α-syn undergoes subsequent translocation. The observed on-rate varies by more than 100 times, depending on bilayer lipid composition. To delineate the role of specific domains, N-terminal membrane-interacting and acidic C-terminal tail, we produced and examined 1) a C-terminal truncation variant lacking the last 25 amino acids (α-syn115) and 2) a synthetic peptide composed of the last 23 residues (C23). In the case of α-syn115, the absence of the highly negatively charged C-terminal tail leads to a significant decrease in the rates of both binding and translocation events. For C23, no time-resolvable blockades of the channel current could be detected, suggesting that membrane binding of α-syn plays an important role in the translocation process. Surprisingly, we found that α-syn can also effectively block mitochondrial channel VDAC, an endogenous β-barrel protein, suggesting that “large” membrane pores may serve as gateways for α-synuclein transport in vivo.

The gamma chain subunit of Fc receptors is required for alpha-synuclein-induced pro-inflammatory signaling in microglia

BackgroundThe protein alpha-synuclein (α-SYN), which is found in the Lewy bodies of dopamine-producing (DA) neurons in the substantia nigra (SN), has an important role in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that neuroinflammation plays a key role in PD pathogenesis. In an AAV-synuclein mouse model of PD, we have found that over-abundance of α-SYN triggers the expression of NF-κB p65, and leads to microglial activation and DA neurodegeneration. We also have observed that Fcγ receptors (FcγR), proteins present on the surface of microglia that bind immunoglobulin G (IgG) and other ligands, are key modulators of α-SYN-induced neurodegeneration.MethodsIn order to study the role of FcγRs in the interactions of α-SYN and microglia, we treated the primary microglial cultures from wild-type (WT) and FcγR−/− mice with aggregated human α-SYN in vitro.ResultsUsing immunocytochemistry, we found that α-SYN was taken up by both WT and FcγR−/− microglia, however, their patterns of internalization were different, with aggregation in autophagosomes in WT cells and more diffuse localization in FcγR−/− microglia. In WT microglia, α-SYN induced the nuclear accumulation of NF-κB p65 protein and downstream chemokine expression while in FcγR−/− mouse microglia, α-SYN failed to trigger the enhancement of nuclear NF-κB p65, and the pro-inflammatory signaling was reduced.ConclusionsOur results suggest that α-SYN can interact directly with microglia and can be internalized and trafficked to autophagosomes. FcγRs mediate this interaction, and in the absence of the gamma chain, there is altered intracellular trafficking and attenuation of pro-inflammatory NF-κB signaling. Therefore, blocking either FcγR signaling or downstream NF-κB activation may be viable therapeutic strategies in PD.

Characterization of a disordered protein during micellation: interactions of α-synuclein with sodium dodecyl sulfate.

To better understand the interaction of α-synuclein (αSyn) with lipid membranes, we carried out self-assembly molecular dynamics simulations of αSyn with monomeric and micellar sodium dodecyl sulfate (SDS), a widely used membrane mimic. We find that both electrostatic and hydrophobic forces contribute to the interactions of αSyn with SDS. In the presence of αSyn, our simulations suggest that SDS aggregates along the protein chain and forms small-size micelles at very early times. Aggregation is followed by formation of a collapsed protein-SDS micelle complex, which is consistent with experimental results. Finally, interaction of αSyn with preformed micelles induces alterations in the shape of the micelle, and the N-terminal helix (residues 3 through 37) tends to associate with micelles. Overall, our simulations provide an atomistic description of the early time scale αSyn-SDS interaction during the self-assembly of SDS into micelles.

Comparison of α-Synuclein and Amyloid Beta Membrane Interactions

α-synuclein (αSyn) and amyloid beta (1-42) peptide (Aβ) are the main constituents of pathological deposits in the midbrain of individuals affected by Parkinson’s disease and Alzheimer’s disease respectively. Defining the interactions of αSyn and Aβ with membranes is essential for understanding the neurotoxicity caused by mutations and/or overexpression.View Large Image | View Hi-Res Image | Download PowerPoint SlideWe performed a systematic in vitro study of the effects of membrane charge, phase, curvature, defects and lipid unsaturation on αSyn and Aβ binding using model vesicles and proteins labeled with a new solvatochromic fluorescent probe. The probe’s emission spectrum strongly depends on the membrane properties, allowing clear discrimination of the protein bound to vesicles of different composition that enables measurements of the kinetics of αSyn migration between membranes of different compositions [1]. The interaction of αSyn with vesicular membranes is fast and reversible while the membrane binding of Aβ is mainly kinetically controlled and competes with aggregation. By introducing the probe at different positions of αSyn and Aβ we were able to estimate the relative immersion of different protein domains into membrane, and its changes depending on membrane composition and lipid-to-protein ratio.[1] Shvadchak, et al., J. Biol. Chem., (2011).

Interference of alpha-synuclein with cAMP/PKA-dependent CREB signaling for tyrosine hydroxylase gene expression in SK-N-BE(2)C cells

Alpha-synuclein (α-SYN) is a small and highly conserved presynaptic protein that is implicated in both normal brain function and the pathogenesis of neurodegenerative diseases. Although the physiological function of α-SYN has not been fully evaluated, several lines of evidence suggest that it plays an important role in the regulation of dopamine homeostasis in the brain. Early studies have demonstrated that interaction of α-SYN and tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine synthesis, is functionally significant for dopaminergic neurotransmission and the pathophysiology of Parkinson's disease. In the present study, we would like to evaluate whether overexpression of wild-type or mutant α-SYN might affect cAMP/PKA-dependent TH activation in DA-producing SK-N-BE(2)C cells. Here we show that wild-type and mutant A30P and A53T α-SYN attenuate forskolin-induced TH up-regulation, but do not suppress TH basal expression in SK-N-BE(2)C cells. Forskolin-induced increase in TH promoter activity and CRE-dependent transcription are significantly suppressed in α-SYN-overexpressing cells. Alpha-SYN enters the nucleus, but does not bind to CREB or interfere with forskolin-induced CREB phosphorylation. These data indicate that elevated levels of α-SYN due to a specific disease or the normal aging process could be associated with dopaminergic neuronal dysfunction through interference with TH regulation.

Coordination Features and Affinity of the Cu2+ Site in the α-Synuclein Protein of Parkinson’s Disease

Parkinson's disease (PD) is the second most prevalent age-related, neurodegenerative disorder, affecting >1% of the population over the age of 60. PD pathology is marked by intracellular inclusions composed primarily of the protein α-synuclein (α-syn). These inclusions also contain copper, and the interaction of Cu(2+) with α-syn may play an important role in PD fibrillogenesis. Here we report the stoichiometry, affinity, and coordination structure of the Cu(2+)-α-syn complex. Electron paramagnetic resonance (EPR) titrations show that monomeric α-syn binds 1.0 equiv of Cu(2+) at the protein N-terminus. Next, an EPR competition technique demonstrates that α-syn binds Cu(2+) with a K(d) of ≈0.10 nM. Finally, EPR and electron spin echo modulation (ESEEM) applied to a suite of mutant and truncated α-syn constructs reveal a coordination sphere arising from the N-terminal amine, the Asp2 amide backbone and side chain carboxyl group, and the His50 imidazole. The high binding affinity identified here, in accord with previous measurements, suggests that copper uptake and sequestration may be a part of α-syn's natural function, perhaps modulating copper's redox properties. The findings further suggest that the long-range interaction between the N-terminus and His50 may have a weakening effect on the interaction of α-syn with lipid membranes, thereby mobilizing monomeric α-syn and hastening fibrillogenesis.

Voltage-Dependent Membrane Insertion and Translocation of α-Synuclein: Liposome Studies

Alpha-synuclein (αSyn), a 140 amino acid protein enriched in neuronal tissue, is implicated in the pathogenesis of Parkinson's Disease (PD), one of the most common neurodegenerative disorders. The membrane appears to be a potential target of αSyn. Studies with oligomeric or aggregated “protofibrillar” or amyloid forms of αSyn, in which β-structure is dominant, have been shown to permeabilize membranes in cell-free systems.1,2 A monomeric αSyn preparation, which could allow determination of a defined structure in membrane provided an alternative perspective.3 αSyn, disordered in solution, assumes a predominately α-helical conformation when bound to liposomes with an anionic surface and high degree of curvature.3,4 Using cysteine scanning mutagenesis to probe the membrane interaction of different regions of αSyn, the interaction of monomeric αSyn with membranes has been studied, thus far with mutants that span the polypeptide, from Val3 to Ala124. Two membrane systems, planar bilayer membranes and liposomes, have utilized an array of tagged cysteines to determine voltage-dependent topology of αSyn in membranes. Voltage-dependent membrane insertion into, and translocation across, liposome anionic phospholipid membranes, was studied through the response of the fluorescence probe 7-nitro-2,1-benzoxadiazole (NBD), covalently attached to individual Cys residues, to a potassium diffusion potential generated by addition of valinomycin to K+-loaded liposomes. Translocation was assessed through quenching of NBD fluorescence by entrapped bromide. A map of the voltage-dependent membrane topology of αSyn was obtained, in response to a potential of −118 mV, in which a segment of αSyn containing Val48Cys is translocated. In liposomes and planar bilayer membranes, translocation with the PD familial mutant E46K was greater than that of wild type. (Support: Michael J. Fox Foundation for Parkinson's Research).

Role of TPPP/p25 on α-synuclein-mediated oligodendroglial degeneration and the protective effect of SIRT2 inhibition in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder presenting variable combinations of parkinsonism, cerebellar ataxia, corticospinal and autonomic dysfunction. Alpha-synuclein (α-SYN)-immunopositive glial cytoplasmic inclusions (GCIs) represent the neuropathological hallmark of MSA, and tubulin polymerization promoting protein (TPPP)/p25 in oligodendroglia has been known as a potent stimulator of α-SYN aggregation. To gain insight into the molecular pathomechanisms of GCI formation and subsequent oligodendroglial degeneration, we ectopically expressed α-SYN and TPPP in HEK293T and oligodendroglial KG1C cell lines. Here we showed that TPPP specifically accelerated α-SYN oligomer formation and co-immunoprecipitation analysis revealed the specific interaction of TPPP and α-SYN. Moreover, phosphorylation of α-SYN at Ser-129 facilitated the TPPP-mediated α-SYN oligomerization. TPPP facilitated α-SYN-positive cytoplasmic perinuclear inclusions mimicking GCI in both cell lines; however, apoptotic cell death was only observed in KG1C cells. This apoptotic cell death was partly rescued by sirtuin 2 (SIRT2) inhibition. Together, our results provide further insight into the molecular pathogenesis of MSA and potential therapeutic approaches.

RETRACTED ARTICLE: Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors

BackgroundAlpha-Synuclein (α-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the α-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of α-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.ResultsIn the present study, we analysed the ability of cytosolic factors to regulate α-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant α-syn. To characterize cytosolic factor(s) that modulate α-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate α-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T α-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance α-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P α-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.ConclusionThese findings suggest that endogenous brain cytosolic factors regulate Wt and mutant α-syn membrane binding, and could represent potential targets to influence α-syn solubility in brain.

CHIP Targets Toxic α-Synuclein Oligomers for Degradation

alpha-Synuclein (alphaSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric alphaSyn intermediates may represent the most toxic alphaSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on alphaSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced alphaSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic alphaSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of alphaSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric alphaSyn may contribute to the successful development of drug therapies that target oligomeric alphaSyn by mimicking or enhancing the powerful effects of CHIP.

Formation of a High Affinity Lipid-Binding Intermediate during the Early Aggregation Phase of α-Synuclein

The alpha-synuclein (alpha-syn) protein is clearly implicated in Parkinson's disease (PD). Mutations or triplication of the alpha-syn gene leads to early onset PD, possibly by accelerating alpha-syn oligomerization. alpha-syn interacts with lipids, and this membrane binding activity may relate to its toxic activity. To understand how the alpha-syn aggregation state affects its lipid binding activity we used surface plasmon resonance to study the interaction of wild-type and mutant alpha-syn with a charged phospholipid membrane, as a function of its aggregation state. Apparent dissociation constants for alpha-syn indicated that an intermediate species, present during the lag phase of amyloid formation, binds with an increased affinity to the membrane surface. Formation of this species was dependent upon the rate of fibril formation. Fluorescence anisotropy studies indicate that only upon the formation of amyloid material can alpha-syn perturb the acyl-chain region of the lipid bilayer. Circular dichroism spectroscopy showed that upon aging, both wild-type and mutant alpha-syn lose their ability to form lipid-bound alpha-helical species once they become fibrillar. These results indicate that alpha-syn forms a high affinity lipid binding intermediate species during fibril formation. Oligomeric alpha-syn is known to be toxic, and it is feasible that the high affinity binding species described here may correspond to a toxic species involved in PD.

A Novel Mechanism of Interaction between α-Synuclein and Biological Membranes

Conformational abnormalities and aggregation of α-synuclein (α-syn) have been linked to the pathogenesis of Parkinson’s (PD) and related diseases. It has been shown that α-syn can stably bind artificial phospholipid vesicles through α-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of α-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within α-syn, we demonstrated that the membrane interaction of α-syn in cells is also mediated by α-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of α-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of α-syn similar to the interaction with biological membranes. These results suggest that in cells, α-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of α-syn itself and by the cytoplasmic context.

Defective membrane interactions of familial Parkinson’s disease mutant A30P α-synuclein

α-Synuclein (α-Syn) is an abundant presynaptic protein of unknown function, which has been implicated in the pathogenesis of Parkinson’s disease. α-Syn has been suggested to play a role in lipid transport and synaptogenesis, and growing evidence suggests that α-Syn interactions with cellular membranes are physiologically important. In the current study, we demonstrate that the familial Parkinson’s disease-linked A30P mutant α-Syn is defective in binding to phospholipid vesicles in vitro as determined by vesicle ultracentrifugation, circular dichroism spectroscopy, and low-angle X-ray diffraction. Interestingly, our data also suggest that α-Syn may bind to the lipid vesicles as a dimer, which suggest that this species could be a physiologically relevant and functional entity. In contrast, the naturally occurring murine A53T substitution, which is also linked to Parkinson’s disease, displayed a normal membrane-binding activity that was comparable to wild-type α-Syn. A double mutant A53T/A30P α-Syn showed defective membrane binding similar to the A30P protein, indicating that the proline mutation is dominant in terms of impairing the membrane-binding activity. With these observations, we suggest that the A53T and A30P mutants may have different physiological consequences in vivo and could possibly contribute to early onset Parkinson’s disease via unique mechanisms.