Structure-Toxicity Relationship in Intermediate Fibrils from α-Synuclein Condensates.

The aberrant aggregation of α-synuclein (αS) into amyloid fibrils is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson's disease. Although the structural properties of mature amyloids of αS are currently understood, the nature of transient protofilaments and fibrils that appear during αS aggregation remains elusive. Using solid-state nuclear magnetic resonance (ssNMR), cryogenic electron microscopy (cryo-EM), and biophysical methods, we here characterized intermediate amyloid fibrils of αS forming during the aggregation from liquid-like spherical condensates to mature amyloids adopting the structure of pathologically observed aggregates. These transient amyloid intermediates, which induce significant levels of cytotoxicity when incubated with neuronal cells, were found to be stabilized by a small core in an antiparallel ß-sheet conformation, with a disordered N-terminal region of the protein remaining available to mediate membrane binding. In contrast, mature amyloids that subsequently appear during the aggregation showed different structural and biological properties, including low levels of cytotoxicity, a rearranged structured core embedding also the N-terminal region, and a reduced propensity to interact with the membrane. The characterization of these two fibrillar forms of αS, and the use of antibodies and designed mutants, enabled us to clarify the role of critical structural elements endowing intermediate amyloid species with the ability to interact with membranes and induce cytotoxicity.

Region-specific changes in gene expression are associated with cognitive deficits in the alpha-synuclein-induced model of Parkinson's disease: A transcriptomic profiling study.

Mild cognitive impairment (MCI) is a common trait of Parkinson's disease (PD), often associated with early motor deficits, eventually evolving to PD with dementia in later disease stages. The neuropathological substrate of MCI is poorly understood, which weakens the development and administration of proper therapies. In an α-synuclein (αSyn)-based model of PD featuring early motor and cognitive impairments, we investigated the transcriptome profile of brain regions involved in PD with cognitive deficits, via a transcriptomic analysis based on RNA sequencing (RNA-seq) technology. Rats infused in the substantia nigra with human α-synuclein oligomers (H-SynOs) developed mild cognitive deficits after three months, as measured by the two-trial recognition test in a Y-maze and the novel object recognition test. RNA-seq analysis showed that 17,436 genes were expressed in the anterior cingulate cortex (ACC) and 17,216 genes in the hippocampus (HC). In the ACC, 51 genes were differentially expressed between vehicle and H-αSynOs treated samples, which showed N= 21 upregulated and N = 30 downregulated genes. In the HC, 104 genes were differentially expressed, the majority of them not overlapping with DEGs in the ACC, with N = 41 upregulated and N = 63 downregulated in H-αSynOs-treated samples. The Gene Ontology (GO) and the Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis, followed by the protein-protein interaction (PPI) network inspection of DEGs, revealed that in the ACC most enriched terms were related with immune functions, specifically with antigen processing/presentation via the major histocompatibility complex (MHC) class II and phagocytosis via CD68, supporting a role for dysregulated immune responses in early PD cognitive dysfunction. Immunofluorescence analysis confirmed the decreased expression of CD68 within microglial cells. In contrast, the most significantly enriched terms in the HC were mainly involved in mitochondrial homeostasis, potassium voltage-gated channel, cytoskeleton and fiber organisation, suggesting that the gene expression in the neuronal population was mostly affected in this region in early disease stages. Altogether results show that H-αSynOs trigger a region-specific dysregulation of gene expression in ACC and HC, providing a pathological substrate for MCI associated with early PD.

MetalHawk: Enhanced Classification of Metal Coordination Geometries by Artificial Neural Networks.

The chemical properties of metal complexes are strongly dependent on the number and geometrical arrangement of ligands coordinated to the metal center. Existing methods for determining either coordination number or geometry rely on a trade-off between accuracy and computational costs, which hinders their application to the study of large structure data sets. Here, we propose MetalHawk (https://github.com/vrettasm/MetalHawk), a machine learning-based approach to perform simultaneous classification of metal site coordination number and geometry through artificial neural networks (ANNs), which were trained using the Cambridge Structural Database (CSD) and Metal Protein Data Bank (MetalPDB). We demonstrate that the CSD-trained model can be used to classify sites belonging to the most common coordination numbers and geometry classes with balanced accuracy equal to 96.51% for CSD-deposited metal sites. The CSD-trained model was also found to be capable of classifying bioinorganic metal sites from the MetalPDB database, with balanced accuracy equal to 84.29% on the whole PDB data set and to 91.66% on manually reviewed sites in the PDB validation set. Moreover, we report evidence that the output vectors of the CSD-trained model can be considered as a proxy indicator of metal-site distortions, showing that these can be interpreted as a low-dimensional representation of subtle geometrical features present in metal site structures.

α-Synuclein and biological membranes: the danger of loving too much.

The aberrant aggregation of α-Synuclein (αS), a disordered protein primarily localised at the neuronal synapses, is associated with a number of neurodegenerative disorders including Parkinson's disease (PD). The biological properties of αS are strictly connected with its ability to bind synaptic membranes under both physiological and pathological conditions. Here we overview the recent studies on the structural and biological properties of the membrane interaction by αS. The characterisation of this state is particularly challenging as the membrane binding of αS is weak, transient and features a considerable degree of conformational disorder. Advancements in this area have been achieved through combinations of nuclear magnetic resonance (NMR), super-resolution microscopy, cryo-EM and cellular biophysics. Current data clarified the central role of the equilibrium between ordered and disordered states of αS at the membrane surface, which regulates the membrane affinity, the aggregation into amyloid fibrils and the promotion of vesicle clustering. Recent results on toxic oligomeric species of αS also revealed common features in the membrane interaction of functional and aberrant forms of this protein. These findings therefore evidence the challenging nature of identifying suitable therapeutics to target the aberrant aggregation of αS in PD while leaving its normal physiological form unperturbed.

Metal interactions of α-synuclein probed by NMR amide-proton exchange.

The aberrant aggregation of α-synuclein (αS), a disordered protein primarily expressed in neuronal cells, is strongly associated with the underlying mechanisms of Parkinson's disease. It is now established that αS has a weak affinity for metal ions and that these interactions alter its conformational properties by generally promoting self-assembly into amyloids. Here, we characterised the nature of the conformational changes associated with metal binding by αS using nuclear magnetic resonance (NMR) to measure the exchange of the backbone amide protons at a residue specific resolution. We complemented these experiments with 15N relaxation and chemical shift perturbations to obtain a comprehensive map of the interaction between αS and divalent (Ca2+, Cu2+, Mn2+, and Zn2+) and monovalent (Cu+) metal ions. The data identified specific effects that the individual cations exert on the conformational properties of αS. In particular, binding to calcium and zinc generated a reduction of the protection factors in the C-terminal region of the protein, whereas both Cu(II) and Cu(I) did not alter the amide proton exchange along the αS sequence. Changes in the R2/R1 ratios from 15N relaxation experiments were, however, detected as a result of the interaction between αS and Cu+ or Zn2+, indicating that binding to these metals induces conformational perturbations in distinctive regions of the protein. Collectively our data suggest that multiple mechanisms of enhanced αS aggregation are associated with the binding of the analysed metals.

Thermal tuning of protein hydration in a hyperthermophilic enzyme.

Water at the protein surface is an active biological molecule that plays a critical role in many functional processes. Using NMR-restrained MD simulations, we here addressed how protein hydration is tuned at high biological temperatures by analysing homologous acylphosphatase enzymes (AcP) possessing similar structure and dynamics under very different thermal conditions. We found that the hyperthermophilic Sso AcP at 80°C interacts with a lower number of structured waters in the first hydration shell than its human homologous mt AcP at 37°C. Overall, the structural and dynamical properties of waters at the surface of the two enzymes resulted similar in the first hydration shell, including solvent molecules residing in the active site. By contrast the dynamical content of water molecules in the second hydration shell was found to diverge, with higher mobility observed in Sso AcP at 80°C. Taken together the results delineate the subtle differences in the hydration properties of mt AcP and Sso AcP, and indicate that the concept of corresponding states with equivalent dynamics in homologous mesophilic and hyperthermophylic proteins should be extended to the first hydration shell.

Enhancing Biomolecular Simulations with Hybrid Potentials Incorporating NMR Data.

Some recent advances in biomolecular simulation and global optimization have used hybrid restraint potentials, where harmonic restraints that penalize conformations inconsistent with experimental data are combined with molecular mechanics force fields. These hybrid potentials can be used to improve the performance of molecular dynamics, structure prediction, energy landscape sampling, and other computational methods that rely on the accuracy of the underlying force field. Here, we develop a hybrid restraint potential based on NapShift, an artificial neural network trained to predict protein nuclear magnetic resonance (NMR) chemical shifts from sequence and structure. In addition to providing accurate predictions of experimental chemical shifts, NapShift is fully differentiable with respect to atomic coordinates, which allows us to use it for structural refinement. By employing NapShift to predict chemical shifts from the protein conformation at each simulation step, we can compute an energy penalty and the corresponding hybrid restraint forces based on the difference between the predicted values and the experimental chemical shifts. The performance of the hybrid restraint potential was benchmarked using both basin-hopping global optimization and molecular dynamics simulations. In each case, the NapShift hybrid potential improved the accuracy, leading to better structure prediction via basin-hopping and increased local stability in molecular dynamics simulations. Our results suggest that neural network hybrid potentials based on NMR observables can enhance a broad range of molecular simulation methods, and the prediction accuracy will improve as more experimental training data become available.

The role of structural dynamics in the thermal adaptation of hyperthermophilic enzymes.

Proteins from hyperthermophilic organisms are evolutionary optimised to adopt functional structures and dynamics under conditions in which their mesophilic homologues are generally inactive or unfolded. Understanding the nature of such adaptation is of crucial interest to clarify the underlying mechanisms of biological activity in proteins. Here we measured NMR residual dipolar couplings of a hyperthermophilic acylphosphatase enzyme at 80°C and used these data to generate an accurate structural ensemble representative of its native state. The resulting energy landscape was compared to that obtained for a human homologue at 37°C, and additional NMR experiments were carried out to probe fast (15N relaxation) and slow (H/D exchange) backbone dynamics, collectively sampling fluctuations of the two proteins ranging from the nanosecond to the millisecond timescale. The results identified key differences in the strategies for protein-protein and protein-ligand interactions of the two enzymes at the respective physiological temperatures. These include the dynamical behaviour of a ß-strand involved in the protection against aberrant protein aggregation and concerted motions of loops involved in substrate binding and catalysis. Taken together these results elucidate the structure-dynamics-function relationship associated with the strategies of thermal adaptation of protein molecules.

The Intranigral Infusion of Human-Alpha Synuclein Oligomers Induces a Cognitive Impairment in Rats Associated with Changes in Neuronal Firing and Neuroinflammation in the Anterior Cingulate Cortex.

Parkinson's disease (PD) is a complex pathology causing a plethora of non-motor symptoms besides classical motor impairments, including cognitive disturbances. Recent studies in the PD human brain have reported microgliosis in limbic and neocortical structures, suggesting a role for neuroinflammation in the development of cognitive decline. Yet, the mechanism underlying the cognitive pathology is under investigated, mainly for the lack of a valid preclinical neuropathological model reproducing the disease's motor and non-motor aspects. Here, we show that the bilateral intracerebral infusion of pre-formed human alpha synuclein oligomers (H-αSynOs) within the substantia nigra pars compacta (SNpc) offers a valid model for studying the cognitive symptoms of PD, which adds to the classical motor aspects previously described in the same model. Indeed, H-αSynOs-infused rats displayed memory deficits in the two-trial recognition task in a Y maze and the novel object recognition (NOR) test performed three months after the oligomer infusion. In the anterior cingulate cortex (ACC) of H-αSynOs-infused rats the in vivo electrophysiological activity was altered and the expression of the neuron-specific immediate early gene (IEG) Npas4 (Neuronal PAS domain protein 4) and the AMPA receptor subunit GluR1 were decreased. The histological analysis of the brain of cognitively impaired rats showed a neuroinflammatory response in cognition-related regions such as the ACC and discrete subareas of the hippocampus, in the absence of any evident neuronal loss, supporting a role of neuroinflammation in cognitive decline. We found an increased GFAP reactivity and the acquisition of a proinflammatory phenotype by microglia, as indicated by the increased levels of microglial Tumor Necrosis Factor alpha (TNF-α) as compared to vehicle-infused rats. Moreover, diffused deposits of phospho-alpha synuclein (p-αSyn) and Lewy neurite-like aggregates were found in the SNpc and striatum, suggesting the spreading of toxic protein within anatomically interconnected areas. Altogether, we present a neuropathological rat model of PD that is relevant for the study of cognitive dysfunction featuring the disease. The intranigral infusion of toxic oligomeric species of alpha-synuclein (α-Syn) induced spreading and neuroinflammation in distant cognition-relevant regions, which may drive the altered neuronal activity underlying cognitive deficits.

Local Backbone Geometry Plays a Critical Role in Determining Conformational Preferences of Amino Acid Residues in Proteins.

The definition of the structural basis of the conformational preferences of the genetically encoded amino acid residues is an important yet unresolved issue of structural biology. In order to gain insights into this intricate topic, we here determined and compared the amino acid propensity scales for different (φ, ψ) regions of the Ramachandran plot and for different secondary structure elements. These propensities were calculated using the Chou-Fasman approach on a database of non-redundant protein chains retrieved from the Protein Data Bank. Similarities between propensity scales were evaluated by linear regression analyses. One of the most striking and unexpected findings is that distant regions of the Ramachandran plot may exhibit significantly similar propensity scales. On the other hand, contiguous regions of the Ramachandran plot may present anticorrelated propensities. In order to provide an interpretative background to these results, we evaluated the role that the local variability of protein backbone geometry plays in this context. Our analysis indicates that (dis)similarities of propensity scales between different regions of the Ramachandran plot are coupled with (dis)similarities in the local geometry. The concept that similarities of the propensity scales are dictated by the similarity of the NCαC angle and not necessarily by the similarity of the (φ, ψ) conformation may have far-reaching implications in the field.

Cryptic binding properties of a transient folding intermediate in a PDZ tandem repeat.

PDZ domains are the most diffused protein-protein interaction modules of the human proteome and are often present in tandem repeats. An example is PDZD2, a protein characterized by the presence of six PDZ domains that undergoes a proteolytic cleavage producing sPDZD2, comprising a tandem of two PDZ domains, namely PDZ5 and PDZ6. Albeit the physiopathological importance of sPDZD2 is well-established, the interaction with endogenous ligands has been poorly characterized. To understand the determinants of the stability and function of sPDZD2, we investigated its folding pathway. Our data highlights the presence of a complex scenario involving a transiently populated folding intermediate that may be accumulated from the concurrent denaturation of both PDZ5 and PDZ6 domains. Importantly, double jump kinetic experiments allowed us to pinpoint the ability of this transient intermediate to bind the physiological ligand of sPDZD2 with increased affinity compared to the native state. In summary, our results provide an interesting example of a functionally competent misfolded intermediate, which may exert a cryptic function that is not captured from the analysis of the native state only.

Plasticity of Membrane Binding by the Central Region of α-Synuclein.

Membrane binding by α-synuclein (αS), an intrinsically disordered protein whose aggregation is associated with Parkinson's disease, is a key step in determining its biological properties under both physiological and pathological conditions. Upon membrane interaction, αS retains a partial level of structural disorder despite acquiring α-helical content. In the membrane-bound state, the equilibrium between the helical-bound and disordered-detached states of the central region of αS (residues 65-97) has been involved in a double-anchor mechanism that promotes the clustering of synaptic vesicles. Herein, we investigated the underlying molecular bases of this equilibrium using enhanced coarse-grained molecular dynamics simulations. The results enabled clarifying the conformational dependencies of the membrane affinity by this protein region that, in addition to playing a role in physiological membrane binding, has key relevance for the aggregation of αS and the mechanisms of the toxicity of the resulting assemblies.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

Repurposing Pomalidomide as a Neuroprotective Drug: Efficacy in an Alpha-Synuclein-Based Model of Parkinson's Disease.

Marketed drugs for Parkinson's disease (PD) treat disease motor symptoms but are ineffective in stopping or slowing disease progression. In the quest of novel pharmacological approaches that may target disease progression, drug-repurposing provides a strategy to accelerate the preclinical and clinical testing of drugs already approved for other medical indications. Here, we targeted the inflammatory component of PD pathology, by testing for the first time the disease-modifying properties of the immunomodulatory imide drug (IMiD) pomalidomide in a translational rat model of PD neuropathology based on the intranigral bilateral infusion of toxic preformed oligomers of human α-synuclein (H-αSynOs). The neuroprotective effect of pomalidomide (20 mg/kg; i.p. three times/week 48 h apart) was tested in the first stage of disease progression by means of a chronic two-month administration, starting 1 month after H-αSynOs infusion, when an already ongoing neuroinflammation is observed. The intracerebral infusion of H-αSynOs induced an impairment in motor and coordination performance that was fully rescued by pomalidomide, as assessed via a battery of motor tests three months after infusion. Moreover, H-αSynOs-infused rats displayed a 40-45% cell loss within the bilateral substantia nigra, as measured by stereological counting of TH + and Nissl-stained neurons, that was largely abolished by pomalidomide. The inflammatory response to H-αSynOs infusion and the pomalidomide treatment was evaluated both in CNS affected areas and peripherally in the serum. A reactive microgliosis, measured as the volume occupied by the microglial marker Iba-1, was present in the substantia nigra three months after H-αSynOs infusion as well as after H-αSynOs plus pomalidomide treatment. However, microglia differed for their phenotype among experimental groups. After H-αSynOs infusion, microglia displayed a proinflammatory profile, producing a large amount of the proinflammatory cytokine TNF-α. In contrast, pomalidomide inhibited the TNF-α overproduction and elevated the anti-inflammatory cytokine IL-10. Moreover, the H-αSynOs infusion induced a systemic inflammation with overproduction of serum proinflammatory cytokines and chemokines, that was largely mitigated by pomalidomide. Results provide evidence of the disease modifying potential of pomalidomide in a neuropathological rodent model of PD and support the repurposing of this drug for clinical testing in PD patients.

A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca2+-ATPase.

SERCA is a P-type ATPase embedded in the sarcoplasmic reticulum and plays a central role in muscle relaxation. SERCA's function is regulated by single-pass membrane proteins called regulins. Unlike other regulins, dwarf open reading frame (DWORF) expressed in cardiac muscle has a unique activating effect. Here, we determine the structure and topology of DWORF in lipid bilayers using a combination of oriented sample solid-state NMR spectroscopy and replica-averaged orientationally restrained molecular dynamics. We found that DWORF's structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64°, and a transmembrane C-terminal helix with an angle of 32°. A kink induced by Pro15, unique to DWORF, separates the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORF's activating effect on SERCA. Overall, our findings directly link DWORF's structural topology to its activating effect on SERCA.

Protein kinase R dependent phosphorylation of α-synuclein regulates its membrane binding and aggregation.

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases.

Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation.

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to ß-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN's cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme's active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins.

Membrane Interactions and Toxicity by Misfolded Protein Oligomers.

The conversion of otherwise soluble proteins into insoluble amyloid aggregates is associated with a range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, as well as non-neuropathic conditions such as type II diabetes and systemic amyloidoses. It is increasingly evident that the most pernicious species among those forming during protein aggregation are small prefibrillar oligomers. In this review, we describe the recent progress in the characterization of the cellular and molecular interactions by toxic misfolded protein oligomers. A fundamental interaction by these aggregates involves biological membranes, resulting in two major model mechanisms at the onset of the cellular toxicity. These include the membrane disruption model, resulting in calcium imbalance, mitochondrial dysfunction and intracellular reactive oxygen species, and the direct interaction with membrane proteins, leading to the alteration of their native function. A key challenge remains in the characterization of transient interactions involving heterogeneous protein aggregates. Solving this task is crucial in the quest of identifying suitable therapeutic approaches to suppress the cellular toxicity in protein misfolding diseases.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.