The small aromatic compound SynuClean-D inhibits the aggregation and seeded polymerization of multiple α-synuclein strains

Parkinson’s disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, as well as the accumulation of intraneuronal proteinaceous inclusions known as Lewy bodies and Lewy neurites. The major protein component of Lewy inclusions is the intrinsically disordered protein α-synuclein (α-Syn), which can adopt diverse amyloid structures. Different conformational strains of α-Syn have been proposed to be related to the onset of distinct synucleinopathies; however, how specific amyloid fibrils cause distinctive pathological traits is not clear. Here, we generated three different α-Syn amyloid conformations at different pH and salt concentrations and analyzed the activity of SynuClean-D (SC-D), a small aromatic molecule, on these strains. We show that incubation of α-Syn with SC-D reduced the formation of aggregates and the seeded polymerization of α-Syn in all cases. Moreover, we found that SC-D exhibited a general fibril disaggregation activity. Finally, we demonstrate that treatment with SC-D also reduced strain-specific intracellular accumulation of phosphorylated α-Syn inclusions. Taken together, we conclude that SC-D may be a promising hit compound to inhibit polymorphic α-Syn aggregation.     

p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions

α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson’s disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated in vitro. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.     

SMG1 Identified as a Regulator of Parkinson’s Disease-Associated alpha-Synuclein through siRNA Screening

Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson’s disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.     

alpha-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells

α-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimer’s disease patients. Moreover, a proteolytic fragment of α-synuclein, the so-called non-amyloid component of Alzheimer’s disease amyloid, was found to be an integral part of Alzheimer’s dementia related plaques. However, the role of α-synuclein in pathomechanism of Alzheimer’s disease remains elusive. In particular, the relationship between α-synuclein and amyloid beta is unknown. In the present study we showed the involvement of α-synuclein in amyloid beta secretion and in the mechanism of amyloid beta evoked mitochondria dysfunction and cell death. Rat pheochromocytoma PC12 cells transfected with amyloid beta precursor protein bearing Swedish double mutation (APPsw) and control PC12 cells transfected with empty vector were used in this study. α-Synuclein (10 μM) was found to increase by twofold amyloid beta secretion from control and APPsw PC12 cells. Moreover, α-synuclein decreased the viability of PC12 cells by about 50% and potentiated amyloid beta toxicity leading to mitochondrial dysfunction and caspase-dependent programmed cell death. Inhibitor of caspase-3 (Z-DEVD-FMK, 100 μM), and a mitochondrial permeability transition pore blocker, cyclosporine A (2 μM) protected PC12 cells against α-synuclein or amyloid beta evoked cell death. In contrast Z-DEVD-FMK and cyclosporine A were ineffective in APPsw cells containing elevated amount of amyloid beta treated with α-synuclein. It was found that the inhibition of neuronal and inducible nitric oxide synthase reversed the toxic effect of α-synuclein in control but not in APPsw cells. Our results indicate that α-synuclein enhances the release and toxicity of amyloid beta leading to nitric oxide mediated irreversible mitochondria dysfunction and caspase-dependent programmed cell death.     

Interaction between coxsackievirus B3 infection and α-synuclein in models of Parkinson’s disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. This mechanism has been investigated to reveal factors that initiate Lewy pathology with the aim of preventing further progression of PD. Here, we demonstrate that coxsackievirus B3 (CVB3) infection can induce α-syn-associated inclusion body formation in neurons which might act as a trigger for PD. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In CVB3-infected mice brains, α-syn aggregates were observed in the cell body of midbrain neurons. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and exhibited neuronal cell death, including that of dopaminergic neurons in the substantia nigra. These results may be attributed to distinct autophagy-related pathways engaged by CVB3 and α-syn. This study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.     

α-synuclein impairs autophagosome maturation through abnormal actin stabilization

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson’s disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson’s disease and related α-synucleinopathies.     

p21-activated kinase 4 controls the aggregation of α-synuclein by reducing the monomeric and aggregated forms of α-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1

Aggregation of misfolded alpha-synuclein (α-synuclein) is a central player in the pathogenesis of neurodegenerative diseases. Therefore, the regulatory mechanism underlying α-synuclein aggregation has been intensively studied in Parkinson’s disease (PD) but remains poorly understood. Here, we report p21-activated kinase 4 (PAK4) as a key regulator of α-synuclein aggregation. Immunohistochemical analysis of human PD brain tissues revealed an inverse correlation between PAK4 activity and α-synuclein aggregation. To investigate their causal relationship, we performed loss-of-function and gain-of-function studies using conditional PAK4 depletion in nigral dopaminergic neurons and the introduction of lentivirus expressing a constitutively active form of PAK4 (caPAK4; PAK4^S445N/S474E), respectively. For therapeutic relevance in the latter setup, we injected lentivirus into the striatum following the development of motor impairment and analyzed the effects 6 weeks later. In the loss-of-function study, Cre-driven PAK4 depletion in dopaminergic neurons enhanced α-synuclein aggregation, intracytoplasmic Lewy body-like inclusions and Lewy-like neurites, and reduced dopamine levels in PAK4^DAT-CreER mice compared to controls. Conversely, caPAK4 reduced α-synuclein aggregation, as assessed by a marked decrease in both proteinase K-resistant and Triton X100-insoluble forms of α-synuclein in the AAV-α-synuclein-induced PD model. Mechanistically, PAK4 specifically interacted with the NEDD4-1 E3 ligase, whose pharmacological inhibition and knockdown suppressed the PAK4-mediated downregulation of α-synuclein. Collectively, these results provide new insights into the pathogenesis of PD and suggest PAK4-based gene therapy as a potential disease-modifying therapy in PD.Subject terms: Parkinson''s disease, Cell death in the nervous system     

Neurodegenerative Disorders: The Role of Genetic Factors in Their Origin and the Efficiency of Treatment

The review considers the main molecular physiological causes of neurodegenerative disorders. The genetic factors involved in Parkinson’s and Alzheimer’s diseases are conventionally divided into pharmacodynamic and pharmacokinetic. The former are analyzed at the levels of dopamine (DA) neurons and polymorphism of the D₁, D₂, and D₃ DA receptors. The role of polymorphisms of some proteins such as parkin (PARK1-PARK10) and α-synuclein in generation of Lewy bodies is described. The pharmacokinetic factors play a role in Parkinson’s disease (PD) at the level of metabolism of DA, dioxyphenylalanine, and tyrosine and include polymorphisms of enzymes and proteins involved in the relevant metabolic reactions. The profile of DA metabolites may contribute to neurotoxicity and the development of PD. Prospects of drug therapy of PD and the risk of adverse drug effects such as mental disorders and dyskinesia are considered in terms of polymorphisms of enzymes and transport proteins.__________Translated from Fiziologiya Cheloveka, Vol. 31, No. 4, 2005, pp. 119–130.Original Russian Text Copyright © 2005 by Sukhanov, Ionov, Piruzyan.     

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson’s disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson’s disease pathology.     

Temperature-Dependent Structural Changes of Parkinson's Alpha-Synuclein Reveal the Role of Pre-Existing Oligomers in Alpha-Synuclein Fibrillization

Amyloid fibrils of α-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson''s disease brains. α-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study α-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, >80°C, α-synuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that α-synuclein was fully reversible and the NMR studies showed no observable structural changes of α-synuclein after 95°C treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing α-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of α-synuclein also occurred. The reduction of pre-existing oligomers of α-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to α-synuclein fibrillization. Our results facilitate the understanding of α-synuclein fibrillization.     

Investigation of Intramolecular Dynamics and Conformations of α-, β- and γ-Synuclein

The synucleins are a family of natively unstructured proteins consisting of α-, β-, and γ-synuclein which are primarily expressed in neurons. They have been linked to a wide variety of pathologies, including neurological disorders, such as Parkinson’s disease (α-synuclein) and dementia with Lewy bodies (α- and β-synuclein), as well as various types of cancers (γ-synuclein). Self-association is a key pathological feature of many of these disorders, with α-synuclein having the highest propensity to form aggregates, while β-synuclein is the least prone. Here, we used a combination of fluorescence correlation spectroscopy and single molecule Förster resonance energy transfer to compare the intrinsic dynamics of different regions of all three synuclein proteins to investigate any correlation with putative functional or dysfunctional interactions. Despite a relatively high degree of sequence homology, we find that individual regions sample a broad range of diffusion coefficients, differing by almost a factor of four. At low pH, a condition that accelerates aggregation of α-synuclein, on average smaller diffusion coefficients are measured, supporting a hypothesis that slower intrachain dynamics may be correlated with self-association. Moreover, there is a surprising inverse correlation between dynamics and bulkiness of the segments. Aside from this observation, we could not discern any clear relationship between the physico-chemical properties of the constructs and their intrinsic dynamics. This work suggests that while protein dynamics may play a role in modulating self-association or interactions with other binding partners, other factors, particularly the local cellular environment, may be more important.     

Pale Body-Like Inclusion Formation and Neurodegeneration following Depletion of 26S Proteasomes in Mouse Brain Neurones are Independent of α-Synuclein

Parkinson’s disease (PD) is characterized by the progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurones and the formation of Lewy bodies (LB) in a proportion of the remaining neurones. α-synuclein is the main component of LB, but the pathological mechanisms that lead to neurodegeneration associated with LB formation remain unclear. Three pivotal elements have emerged in the development of PD: α-synuclein, mitochondria and protein degradation systems. We previously reported a unique model, created by conditional genetic depletion of 26S proteasomes in the SNpc of mice, which mechanistically links these three elements with the neuropathology of PD: progressive neurodegeneration and intraneuronal inclusion formation. Using this model, we tested the hypothesis that α-synuclein was essential for the formation of inclusions and neurodegeneration caused by 26S proteasomal depletion. We found that both of these processes were independent of α-synuclein. This provides an important insight into the relationship between the proteasome, α-synuclein, inclusion formation and neurodegeneration. We also show that the autophagy-lysosomal pathway is not activated in 26S proteasome-depleted neurones. This leads us to suggest that the paranuclear accumulation of mitochondria in inclusions in our model may reflect a role for the ubiquitin proteasome system in mitochondrial homeostasis and that neurodegeneration may be mediated through mitochondrial factors linked to inclusion biogenesis.     

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

Background

The pathology of Parkinson''s disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated α-synuclein are demonstrated in the signature inclusions of Parkinson''s disease. However, whether the nitration of α-synuclein is relevant to the pathogenesis of PD is unknown.

Methodology/Principal Findings

In this study, effect of nitrated α-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-α-synuclein intracellular. We provide evidence to show that the nitrated α-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated α-synuclein. Moreover, we show that administration of nitrated α-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D₂R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated α-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified α-synuclein had significantly less severe behavioral effects.

Conclusions/Significance

Our results provide evidence that α-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD.     

Review: Formation and Properties of Amyloid-like Fibrils Derived from α-Synuclein and Related Proteins

Synucleinsare small proteins that are highly expressed in brain tissue and are localised at presynaptic terminals in neurons. α-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms of α-synuclein have been associated with autosomal-dominant Parkinson's Disease. A fragment of α-synuclein has also been identified as the non-Aβ component of Alzheimer's Disease amyloid. In this review we describe some structural properties of α-synuclein and the two mutant forms, as well as α-synuclein fragments, with particular emphasis on their ability to form β-sheet on ageing and aggregate to form amyloid-like fibrils. Differences in the rates of aggregation and morphologies of the fibrils formed by α-synuclein and the two mutant proteins are highlighted. Interactions between α-synuclein and other proteins, especially those that are components of amyloid or Lewy bodies, are considered. The toxicity of α-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases.     

α-Synuclein and Anti-α-Synuclein Antibodies in Parkinson’s Disease,Atypical Parkinson Syndromes,REM Sleep Behavior Disorder,and Healthy Controls

α-synuclein is thought to play a key role in Parkinson’s disease (PD) because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (n = 14), atypical Parkinson syndromes (n = 11), idiopathic rapid eye movement sleep behavior disorder (n = 10), and healthy controls (n = 9), to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power) of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rho = 0.16). In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.     

α-Synuclein Levels in Blood Plasma from LRRK2 Mutation Carriers

The diagnosis of Parkinson’s disease (PD) remains primarily a clinical issue, based mainly on phenotypic patterns. The identification of biomarkers capable of permitting the preclinical detection of PD is critically needed. α-Synuclein is a key protein in PD, with missense and multiplication mutations in the gene encoding α-synuclein (SNCA) having been reported in familial cases of PD, and accumulation of the protein identified in Lewy bodies (LBs) and Lewy neurites (LNs) in affected brain regions. With the objective of validating the use of α-synuclein as a clinical or progressive biomarker in an accessible tissue, we used an enzyme-linked immunosorbent assay (ELISA) to measure α-synuclein levels in the peripheral blood plasma of idiopathic PD and LRRK2 mutation carrier patients and compared our findings with healthy control subjects. Compared to healthy controls, we found a significant decrease in plasma total α-synuclein levels in idiopathic PD (iPD) patients (n = 134, p = 0.010). However, the reduction was less significant in patients who were LRRK2 mutation carriers (n = 32, p = 0.133). This lack of significance could be due to the small number of individuals employed in this group. No predictive value of total α-synuclein in the diagnosis of PD was found in a receiver operating characteristic (ROC) curve analysis. Although this is a pilot study requiring corroboration on a larger cohort of patients, our results highlight the possible use of plasma α-synuclein as a biomarker for PD.     

Methamphetamine binds to α-synuclein and causes a conformational change which can be detected by nanopore analysis

α-Synuclein is an intrinsically disordered protein of 140 amino acids which is abundant in dopaminergic neurons. Misfolding and aggregation of α-synuclein leads to the formation of Lewy bodies inside the neurons which is the hallmark of Parkinson’s disease and related dementias. Here we show by nanopore analysis that the recreational drug, methamphetamine, binds to the N-terminus of α-synuclein and causes a conformational change which cannot be detected by circular dichroism spectroscopy. The results suggest a mechanism for the psychoactivity of methamphetamine as well as an increased incidence of Parkinson’s disease amongst users of the drug.     

A small molecule toll-like receptor antagonist rescues α-synuclein fibril pathology

The propagation and accumulation of pathological α-synuclein protein is thought to underlie the clinical symptoms of the neurodegenerative movement disorder Parkinson’s disease (PD). Consequently, there is significant interest in identifying the mechanisms that contribute to α-synuclein pathology, as these may inform therapeutic targets for the treatment of PD. One protein that appears to contribute to α-synuclein pathology is the innate immune pathogen recognition receptor, toll-like receptor 2 (TLR2). TLR2 is expressed on neurons, and its activation results in the accumulation of α-synuclein protein; however, the precise mechanism by which TLR2 contributes to α-synuclein pathology is unclear. Herein we demonstrate using human cell models that neuronal TLR2 activation acutely impairs the autophagy lysosomal pathway and markedly potentiates α-synuclein pathology seeded with α-synuclein preformed fibrils. Moreover, α-synuclein pathology could be ameliorated with a novel small molecule TLR2 inhibitor, including in induced pluripotent stem cell-derived neurons from a patient with PD. These results provide further insight into how TLR2 activation may promote α-synuclein pathology in PD and support that TLR2 may be a potential therapeutic target for the treatment of PD.     

Structural and Dynamical Insights into the Membrane-Bound α-Synuclein

Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson’s disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES), which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å) layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could potentially be related to its physiological functions as well as to the onset of Parkinson’s diseases.