Cytosolic sequestration of spatacsin by Protein Kinase A and 14-3-3 proteins.

Mutations in SPG11, encoding spatacsin, constitute the major cause of autosomal recessive Hereditary Spastic Paraplegia (HSP) with thinning of the corpus callosum. Previous studies showed that spatacsin orchestrates cellular traffic events through the formation of a coat-like complex and its loss of function results in lysosomal and axonal transport impairments. However, the upstream mechanisms that regulate spatacsin trafficking are unknown. Here, using proteomics and CRISPR/Cas9-mediated tagging of endogenous spatacsin, we identified a subset of 14-3-3 proteins as physiological interactors of spatacsin. The interaction is modulated by Protein Kinase A (PKA)-dependent phosphorylation of spatacsin at Ser1955, which initiates spatacsin trafficking from the plasma membrane to the intracellular space. Our study provides novel insight in understanding spatacsin physio-pathological roles with mechanistic dissection of its associated pathways.

Extracellular clusterin limits the uptake of α-synuclein fibrils by murine and human astrocytes.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α-synuclein. Clearance of extracellular α-synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α-synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α-synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α-synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human-induced pluripotent stem cell (iPSC)-derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α-synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α-synuclein preformed fibrils (pffs) through dynamin-dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α-synuclein pffs exposure. Specifically, we found that clusterin interacts with α-synuclein pffs in the extracellular compartment and the clusterin/α-synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock-out primary astrocytes and clusterin knock-down hiPSC-derived astrocytes we observed that clusterin limits the uptake of α-synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno-associated virus- and the α-synuclein pffs- injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α-synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology.

Transcriptome analysis of LRRK2 knock-out microglia cells reveals alterations of inflammatory- and oxidative stress-related pathways upon treatment with α-synuclein fibrils.

Several previous studies have linked the Parkinson's disease (PD) gene LRRK2 to the biology of microglia cells. However, the precise ways in which LRRK2 affects microglial function have not been fully resolved. Here, we used the RNA-Sequencing to obtain transcriptomic profiles of LRRK2 wild-type (WT) and knock-out (KO) microglia cells treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS) as a general inflammatory insult. We observed that, although α-synuclein PFFs and LPS mediate overlapping gene expression profiles in microglia, there are also distinct responses to each stimulus. α-Synuclein PFFs trigger alterations of oxidative stress-related pathways with the mitochondrial dismutase Sod2 as a strongly differentially regulated gene. We validated SOD2 at mRNA and protein levels. Furthermore, we found that LRRK2 KO microglia cells reported attenuated induction of mitochondrial SOD2 in response to α-synuclein PFFs, indicating a potential contribution of LRRK2 to oxidative stress-related pathways. We validate several genes in vivo using single-cell RNA-Seq from acutely isolated microglia after striatal injection of LPS into the mouse brain. Overall, these results suggest that microglial LRRK2 may contribute to the pathogenesis of PD via altered oxidative stress signaling.

Fibrils of α-Synuclein Abolish the Affinity of Cu2+-Binding Site to His50 and Induce Hopping of Cu2+ Ions in the Termini.

The effect of Cu2+ on α-synuclein (AS) aggregation is important because clinical studies of patients with Parkinson's disease have shown elevated levels of Cu2+ in the cerebrospinal fluid. So far, the molecular architectures of Cu2+-AS fibril complexes at atomic resolution are unknown. The current work identifies for the first time that His50 cannot bind Cu2+ ions in mature fibrils. Moreover, it shows hopping of Cu2+ ions between residues in AS fibrils and changes in the Cu2+ coordination mode in Cu2+ ions that bind in the termini of AS. The current study combines extensive experimental techniques, density functional theory calculations, and computational modeling tools to provide a complete description of the Cu2+ binding site in AS fibrils. Our findings illustrate for the first time the specific interactions between Cu2+ ions and AS fibrils, suggesting a new mechanistic perspective on the effect of Cu2+ ions on AS aggregation.

High-Pressure-Driven Reversible Dissociation of α-Synuclein Fibrils Reveals Structural Hierarchy.

The analysis of the α-synuclein (aS) aggregation process, which is involved in Parkinson's disease etiopathogenesis, and of the structural feature of the resulting amyloid fibrils may shed light on the relationship between the structure of aS aggregates and their toxicity. This may be considered a paradigm of the ground work needed to tackle the molecular basis of all the protein-aggregation-related diseases. With this aim, we used chemical and physical dissociation methods to explore the structural organization of wild-type aS fibrils. High pressure (in the kbar range) and alkaline pH were used to disassemble fibrils to collect information on the hierarchic pathway by which distinct ß-sheets sequentially unfold using the unique possibility offered by high-pressure Fourier transform infrared spectroscopy. The results point toward the formation of kinetic traps in the energy landscape of aS fibril disassembly and the presence of transient partially folded species during the process. Since we found that the dissociation of wild-type aS fibrils by high pressure is reversible upon pressure release, the disassembled molecules likely retain structural information that favors fibril reformation. To deconstruct the role of the different regions of aS sequence in this process, we measured the high-pressure dissociation of amyloids formed by covalent chimeric dimers of aS (syn-syn) and by the aS deletion mutant that lacks the C-terminus, i.e., aS (1-99). The results allowed us to single out the role of dimerization and that of the C-terminus in the complete maturation of fibrillar aS.

Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature.

α-synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α-synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α-synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α-synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α-synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs.

The chaperone-like protein 14-3-3η interacts with human α-synuclein aggregation intermediates rerouting the amyloidogenic pathway and reducing α-synuclein cellular toxicity.

Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to ß-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.

DJ-1 is a copper chaperone acting on SOD1 activation.

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis.

Dopamine-derived quinones affect the structure of the redox sensor DJ-1 through modifications at Cys-106 and Cys-53.

The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.

Alpha-synuclein pore forming activity upon membrane association.

Alpha-synuclein is a natively unfolded protein widely expressed in neurons at the presynaptic level. It is linked to Parkinson's disease by two lines of evidence: amyloid fibrils of the protein accumulate in patients' brains and three genetic mutants cause autosomal dominant forms of the disease. The biological role of the protein and the mechanisms involved in the etiopathogenesis of Parkinson's disease are still unknown. Membrane binding causes the formation of an amphipathic alpha-helix, which lies on the surface without crossing the bilayer. Recent observations however reported that the application of a voltage induces a pore-like activity of alpha-synuclein. This study aims to characterize the pore forming activity of the protein starting from its monomeric form. In particular, experiments with planar lipid membranes allowed recording of conductance activity bursts with a defined and reproducible fingerprint. Additional experiments with deletion mutants and covalently bound alpha-synuclein dimers were performed to understand both pore assembly and stoichiometry. The information acquired allowed formulation of a model for pore formation at different conductance levels.

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties.

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4-6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H2O2, MPP+ and MnCl2 were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.

Copper(I)-α-synuclein interaction: structural description of two independent and competing metal binding sites.

The aggregation of α-synuclein (αS) is a critical step in the etiology of Parkinson's disease. Metal ions such as copper and iron have been shown to bind αS, enhancing its fibrillation rate in vitro. αS is also susceptible to copper-catalyzed oxidation that involves the reduction of Cu(II) to Cu(I) and the conversion of O(2) into reactive oxygen species. The mechanism of the reaction is highly selective and site-specific and involves interactions of the protein with both oxidation states of the copper ion. The reaction can induce oxidative modification of the protein, which generally leads to extensive protein oligomerization and precipitation. Cu(II) binding to αS has been extensively characterized, indicating the N terminus and His-50 as binding donor residues. In this study, we have investigated αS-Cu(I) interaction by means of NMR and circular dichroism analysis on the full-length protein (αS(1-140)) and on two, designed ad hoc, model peptides: αS(1-15) and αS(113-130). In order to identify and characterize the metal binding environment in full-length αS, in addition to Cu(I), we have also used Ag(I) as a probe for Cu(I) binding. Two distinct Cu(I)/Ag(I) binding domains with comparable affinities have been identified. The structural rearrangements induced by the metal ions and the metal coordination spheres of both sites have been extensively characterized.

Copper Binding and Redox Activity of α-Synuclein in Membrane-Like Environment.

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson's disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations. For these reasons, the ternary interaction between αSyn, copper, and membranes needs to be elucidated in detail. Here, we investigated the structural properties of copper-αSyn binding through NMR, EPR, and XAS analyses, with particular emphasis on copper(I) coordination since the reduced state is particularly relevant for oxygen activation chemistry. The analysis was performed in different membrane model systems, such as micellar sodium dodecyl sulfate (SDS) and unilamellar vesicles, comparing the binding of full-length αSyn and N-terminal peptide fragments. The presence of membrane-like environments induced the formation of a copper:αSyn = 1:2 complex where Cu+ was bound to the Met1 and Met5 residues of two helical peptide chains. In this coordination, Cu+ is stabilized and is unreactive in the presence of O2 in catechol substrate oxidation.

Worm-like Ising model for protein mechanical unfolding under the effect of osmolytes.

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.

The Roc domain of LRRK2 as a hub for protein-protein interactions: a focus on PAK6 and its impact on RAB phosphorylation.

Leucine-rich repeat kinase 2 (LRRK2) has taken center stage in Parkinson's disease (PD) research as mutations cause familial PD and more common variants increase lifetime risk for disease. One unique feature in LRRK2 is the coexistence of GTPase/Roc (Ras of complex) and kinase catalytic functions, bridged by a COR (C-terminal Of Roc) platform for dimerization. Multiple PD mutations are located within the Roc/GTPase domain and concomitantly lead to defective GTPase activity and augmented kinase activity in cells, supporting a crosstalk between GTPase and kinase domains. In addition, biochemical and structural data highlight the importance of Roc as a molecular switch modulating LRRK2 monomer-to-dimer equilibrium and building the interface for interaction with binding partners. Here we review the effects of PD Roc mutations on LRRK2 function and discuss the importance of Roc as a hub for multiple molecular interactions relevant for the regulation of cytoskeletal dynamics and intracellular trafficking pathways. Among the well-characterized Roc interactors, we focused on the cytoskeletal-related kinase p21-activated kinase 6 (PAK6). We report the affinity between LRRK2-Roc and PAK6 measured by microscale thermophoresis (MST). We further show that PAK6 can modulate LRRK2-mediated phosphorylation of RAB substrates in the presence of LRRK2 wild-type (WT) or the PD G2019S kinase mutant but not when the PD Roc mutation R1441G is expressed. These findings support a mechanism whereby mutations in Roc might affect LRRK2 activity through impaired protein-protein interaction in the cell.

Observing the osmophobic effect in action at the single molecule level.

Protecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single-molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising-like protein model with support for group-transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single-molecule experiments.

Conformational equilibria in monomeric alpha-synuclein at the single-molecule level.

Human alpha-Synuclein (alphaSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of alphaSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alphaSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy-based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant alphaSyn. The conformational heterogeneity of monomeric alphaSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and "beta-like" structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the "beta-like" structures significantly increased in different conditions promoting the aggregation of alphaSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric alphaSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric alphaSyn.

Parkinson's Disease-Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance.

Parkinson's disease (PD) is a neurodegenerative, progressive disease without a cure. To prevent PD onset or at least limit neurodegeneration, a better understanding of the underlying cellular and molecular disease mechanisms is crucial. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent one of the most common causes of familial PD. In addition, LRRK2 variants are risk factors for sporadic PD, making LRRK2 an attractive therapeutic target. Mutations in LRRK2 have been linked to impaired alpha-synuclein (α-syn) degradation in neurons. However, in which way pathogenic LRRK2 affects α-syn clearance by astrocytes, the major glial cell type of the brain, remains unclear. The impact of astrocytes on PD progression has received more attention and recent data indicate that astrocytes play a key role in α-syn-mediated pathology. In the present study, we aimed to compare the capacity of wild-type astrocytes and astrocytes carrying the PD-linked G2019S mutation in Lrrk2 to ingest and degrade fibrillary α-syn. For this purpose, we used two different astrocyte culture systems that were exposed to sonicated α-syn for 24 h and analyzed directly after the α-syn pulse or 6 days later. To elucidate the impact of LRRK2 on α-syn clearance, we performed various analyses, including complementary imaging, transmission electron microscopy, and proteomic approaches. Our results show that astrocytes carrying the G2019S mutation in Lrrk2 exhibit a decreased capacity to internalize and degrade fibrillar α-syn via the endo-lysosomal pathway. In addition, we demonstrate that the reduction of α-syn internalization in the Lrrk2 G2019S astrocytes is linked to annexin A2 (AnxA2) loss of function. Together, our findings reveal that astrocytic LRRK2 contributes to the clearance of extracellular α-syn aggregates through an AnxA2-dependent mechanism.

Dopamine quinones interact with alpha-synuclein to form unstructured adducts.

alpha-Synuclein (alphasyn) 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 beta-sheet-rich species, called protofibrils, rather than fibrils themselves, may be the neurotoxic species. The oxidation products of dopamine (DAQ) can inhibit alphasyn fibril formation supporting the idea that DAQ might stabilize alphasyn protofibrils. In the present work, through different biochemical and biophysical techniques, we isolated and structurally characterized alphasyn/DAQ adducts. Contrary to protofibrils, we demonstrated that alphasyn/DAQ adducts retain an unfolded conformation. We then investigated the nature of the modifications induced on alphasyn by DAQ. Our results indicate that only a small fraction of alphasyn interacts with DAQ in a covalent way, so that non-covalent interaction appears to be the major modification induced by DAQ on alphasyn.

Pathogenic mutations shift the equilibria of alpha-synuclein single molecules towards structured conformers.

Alpha-synuclein (alpha-Syn) is an abundant brain protein whose mutations have been linked to early-onset Parkinson's disease (PD). We recently demonstrated, by means of a single-molecule force spectroscopy (SMFS) methodology, that the conformational equilibrium of monomeric wild-type (WT) alpha-Syn shifts toward beta-containing structures in several unrelated conditions linked to PD pathogenicity. Herein, we follow the same methodology previously employed for WT alpha-Syn to characterize the conformational heterogeneity of pathological alpha-Syn mutants A30P, A53T, and E46K. Contrary to the bulk ensemble-averaged spectroscopies so far employed to this end by different authors, our single-molecule methodology monitored marked differences in the conformational behaviors of the mutants with respect to the WT sequence. We found that all the mutants have a much higher propensity than the WT to adopt a monomeric compact conformation that is compatible with the acquiring of beta structure. Mutants A30P and A53T show a similar conformational equilibrium that is significantly different from that of E46K. Another class of conformations, stabilized by mechanically weak interactions (MWI), shows a higher variety in the mutants than in the WT protein. In the A30P mutant these interactions are relatively stronger, and therefore the corresponding conformations are possibly more structured. The more structured and globular conformations of the mutants can explain their higher propensity to aggregate with respect to the WT.