Aggregation and beyond: alpha-synuclein-based biomarkers in synucleinopathies.

Parkinson's disease is clinically known for the loss of dopaminergic neurons in the substantia nigra pars compacta and accumulation of intraneuronal cytoplasmic inclusions rich in alpha-synuclein called 'Lewy bodies' and 'Lewy neurites'. Together with dementia with Lewy bodies and multiple system atrophy, Parkinson's disease is part of a group of disorders called synucleinopathies. Currently, diagnosis of synucleinopathies is based on the clinical assessment which often takes place in advanced disease stages. While the causal role of alpha-synuclein aggregates in these disorders is still debatable, measuring the levels, types or seeding properties of different alpha-synuclein species hold great promise as biomarkers. Recent studies indicate significant differences in peptide, protein and RNA levels in blood samples from patients with Parkinson's disease. Seed amplification assays using CSF, blood, skin biopsy, olfactory swab samples show great promise for detecting synucleinopathies and even for discriminating between different synucleinopathies. Interestingly, small extracellular vesicles, such as exosomes, display differences in their cargoes in Parkinson's disease patients versus controls. In this update, we focus on alpha-synuclein aggregation and possible sources of disease-related species released in extracellular vesicles, which promise to revolutionize the diagnosis and the monitoring of disease progression.

The lysosomal ß-glucocerebrosidase strikes mitochondria: implications for Parkinson's therapeutics.

Parkinson's disease is a neurodegenerative disorder primarily known for typical motor features that arise due to the loss of dopaminergic neurons in the substantia nigra. However, the precise molecular aetiology of the disease is still unclear. Several cellular pathways have been linked to Parkinson's disease, including the autophagy-lysosome pathway, α-synuclein aggregation and mitochondrial function. Interestingly, the mechanistic link between GBA1, the gene that encodes for lysosomal ß-glucocerebrosidase (GCase), and Parkinson's disease lies in the interplay between GCase functions in the lysosome and mitochondria. GCase mutations alter mitochondria-lysosome contact sites. In the lysosome, reduced GCase activity leads to glycosphingolipid build-up, disrupting lysosomal function and autophagy, thereby triggering α-synuclein accumulation. Additionally, α-synuclein aggregates reduce GCase activity, creating a self-perpetuating cycle of lysosomal dysfunction and α-synuclein accumulation. GCase can also be imported into the mitochondria, where it promotes the integrity and function of mitochondrial complex I. Thus, GCase mutations that impair its normal function increase oxidative stress in mitochondria, the compartment where dopamine is oxidized. In turn, the accumulation of oxidized dopamine adducts further impairs GCase activity, creating a second cycle of GCase dysfunction. The oxidative state triggered by GCase dysfunction can also induce mitochondrial DNA damage which, in turn, can cause dopaminergic cell death. In this review, we highlight the pivotal role of GCase in Parkinson's disease pathogenesis and discuss promising examples of GCase-based therapeutics, such as gene and enzyme replacement therapies, small molecule chaperones and substrate reduction therapies, among others, as potential therapeutic interventions.

In silico analysis of alpha-synuclein protein variants and posttranslational modifications related to Parkinson's disease.

Parkinson's disease (PD) is among the most prevalent neurodegenerative disorders, affecting over 10 million people worldwide. The protein encoded by the SNCA gene, alpha-synuclein (ASYN), is the major component of Lewy body (LB) aggregates, a histopathological hallmark of PD. Mutations and posttranslational modifications (PTMs) in ASYN are known to influence protein aggregation and LB formation, possibly playing a crucial role in PD pathogenesis. In this work, we applied computational methods to characterize the effects of missense mutations and PTMs on the structure and function of ASYN. Missense mutations in ASYN were compiled from the literature/databases and underwent a comprehensive predictive analysis. Phosphorylation and SUMOylation sites of ASYN were retrieved from databases and predicted by algorithms. ConSurf was used to estimate the evolutionary conservation of ASYN amino acids. Molecular dynamics (MD) simulations of ASYN wild-type and variants A30G, A30P, A53T, and G51D were performed using the GROMACS package. Seventy-seven missense mutations in ASYN were compiled. Although most mutations were not predicted to affect ASYN stability, aggregation propensity, amyloid formation, and chaperone binding, the analyzed mutations received relatively high rates of deleterious predictions and predominantly occurred at evolutionarily conserved sites within the protein. Moreover, our predictive analyses suggested that the following mutations may be possibly harmful to ASYN and, consequently, potential targets for future investigation: K6N, T22I, K34E, G36R, G36S, V37F, L38P, G41D, and K102E. The MD analyses pointed to remarkable flexibility and essential dynamics alterations at nearly all domains of the studied variants, which could lead to impaired contact between NAC and the C-terminal domain triggering protein aggregation. These alterations may have functional implications for ASYN and provide important insight into the molecular mechanism of PD, supporting the design of future biomedical research and improvements in existing therapies for the disease.

Aging and neurodegeneration: From molecular mechanisms to therapeutic interventions.

Protein aggregation is a common age-associated process and can be a pathological hallmark of various neurodegenerative conditions, possibly because of an age-associated decline in the activity of components of the proteostasis network. The specific molecular drivers of protein aggregation in certain cell types are not well understood, posing tremendous challenges to current research aimed at devising strategies to treat neurodegenerative diseases. This preface introduces the special issue "Aging and Neurodegeneration: from molecular mechanisms to therapeutic interventions," featuring articles that assess the drivers of pathology in the aging cell, including oxidative stress, protein glycation/aggregation, and mitochondrial impairment.

Altered hippocampal doublecortin expression in Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by motor and non-motor symptoms. Motor symptoms include bradykinesia, resting tremors, muscular rigidity, and postural instability, while non-motor symptoms include cognitive impairments, mood disturbances, sleep disturbances, autonomic dysfunction, and sensory abnormalities. Some of these symptoms may be influenced by the proper hippocampus functioning, including adult neurogenesis. Doublecortin (DCX) is a microtubule-associated protein that plays a pivotal role in the development and differentiation of migrating neurons. This study utilized postmortem human brain tissue of PD and age-matched control individuals to investigate DCX expression in the context of adult hippocampal neurogenesis. Our findings demonstrate a significant reduction in the number of DCX-expressing cells within the subgranular zone (SGZ), as well as a decrease in the nuclear area of these DCX-positive cells in postmortem brain tissue obtained from PD cases, suggesting an impairment in the adult hippocampal neurogenesis. Additionally, we found that the nuclear area of DCX-positive cells correlates with pH levels. In summary, we provide evidence supporting that the process of hippocampal adult neurogenesis is likely to be compromised in PD patients before cognitive dysfunction, shedding light on potential mechanisms contributing to the neuropsychiatric symptoms observed in affected individuals. Understanding these mechanisms may offer novel insights into the pathophysiology of PD and possible therapeutic avenues.

Synthesis and Semi-Synthesis of Alpha-Synuclein: Insight into the Chemical Complexity of Synucleinopathies.

The chemical rules governing protein folding have intrigued generations of researchers for decades. With the advent of artificial intelligence (AI), prediction of protein structure has improved tremendously. However, there is still a level of analysis that is only possible through wet laboratory experiments, especially in respect to the investigation of the pathological effect of mutations and posttranslational modifications (PTMs) on proteins of interest. This requires the availability of pure peptides and proteins in sufficient quantities for biophysical, biochemical, and functional studies. In this context, chemical protein synthesis and semi-synthesis are powerful tools in protein research, which help to enlighten the role of protein modification in the physiology and pathology of proteins. A protein of high interest in the field of biomedicine is alpha-synuclein (aSyn), a protein deeply associated with several devastating neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple systems atrophy (MSA). Here, we describe several methods and pathways to synthesize native or modified aSyn, and discuss how these approaches enable us to address pathological mechanisms that may open novel perspectives for therapeutic intervention.

Chemical synthesis of site-selective advanced glycation end products in α-synuclein and its fragments.

Advanced glycation end products (AGEs) arise from the Maillard reaction between dicarbonyls and proteins, nucleic acids, or specific lipids. Notably, AGEs are linked to aging and implicated in various disorders, spanning from cancer to neurodegenerative diseases. While dicarbonyls like methylglyoxal preferentially target arginine residues, lysine-derived AGEs, such as N(6)-(1-carboxymethyl)lysine (CML) and N(6)-(1-carboxyethyl)lysine (CEL), are also abundant. Predicting protein glycation in vivo proves challenging due to the intricate nature of glycation reactions. In vitro, glycation is difficult to control, especially in proteins that harbor multiple glycation-prone amino acids. α-Synuclein (aSyn), pivotal in Parkinson's disease and synucleinopathies, has 15 lysine residues and is known to become glycated at multiple lysine sites. To understand the influence of glycation in specific regions of aSyn on its behavior, a strategy for site-specific glycated protein production is imperative. To fulfill this demand, we devised a synthetic route integrating solid-phase peptide synthesis, orthogonal protection of amino acid side-chain functionalities, and reductive amination strategies. This methodology yielded two disease-related N-terminal peptide fragments, each featuring five and six CML and CEL modifications, alongside a full-length aSyn protein containing a site-selective E46CEL modification. Our synthetic approach facilitates the broad introduction of glycation motifs at specific sites, providing a foundation for generating glycated forms of synucleinopathy-related and other disease-relevant proteins.

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition.

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson's disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.

PrP meets alpha-synuclein: Molecular mechanisms and implications for disease.

The discovery of prions has challenged dogmas and has revolutionized our understanding of protein-misfolding diseases. The concept of self-propagation via protein conformational changes, originally discovered for the prion protein (PrP), also applies to other proteins that exhibit similar behavior, such as alpha-synuclein (aSyn), a central player in Parkinson's disease and in other synucleinopathies. aSyn pathology appears to spread from one cell to another during disease progression, and involves the misfolding and aggregation of aSyn. How the transfer of aSyn between cells occurs is still being studied, but one important hypothesis involves receptor-mediated transport. Interestingly, recent studies indicate that the cellular prion protein (PrPC ) may play a crucial role in this process. PrPC has been shown to act as a receptor/sensor for protein aggregates in different neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis. Here, we provide a comprehensive overview of the current state of knowledge regarding the interaction between aSyn and PrPC and discuss its role in synucleinopathies. We examine the properties of PrP and aSyn, including their structure, function, and aggregation. Additionally, we discuss the current understanding of PrPC 's role as a receptor/sensor for aSyn aggregates and identify remaining unanswered questions in this area of research. Ultimately, we posit that exploring the interaction between aSyn and PrPC may offer potential treatment options for synucleinopathies.

Is it all the RAGE? Defining the role of the receptor for advanced glycation end products in Parkinson's disease.

The receptor for advanced glycation end products (RAGE) is a transmembrane receptor that belongs to the immunoglobulin superfamily and is extensively associated with chronic inflammation in non-transmissible diseases. As chronic inflammation is consistently present in neurodegenerative diseases, it was largely assumed that RAGE could act as a critical modulator of neuroinflammation in Parkinson's disease (PD), similar to what was reported for Alzheimer's disease (AD), where RAGE is postulated to mediate pro-inflammatory signaling in microglia by binding to amyloid-ß peptide. However, accumulating evidence from studies of RAGE in PD models suggests a less obvious scenario. Here, we review physiological aspects of RAGE and address the current questions about the potential involvement of this receptor in the cellular events that may be critical for the development and progression of PD, exploring possible mechanisms beyond the classical view of the microglial activation/neuroinflammation/neurodegeneration axis that is widely assumed to be the general mechanism of RAGE action in the adult brain.

Increased alpha-synuclein and neuroinflammation in the substantia nigra triggered by systemic inflammation are reversed by targeted inhibition of the receptor for advanced glycation end products (RAGE).

The receptor for advanced glycation end products (RAGE) is a protein of the immunoglobulin superfamily capable of regulating inflammation. Considering the role of this receptor in the initiation and establishment of neuroinflammation, and the limited understanding of the function of RAGE in the maintenance of this condition, this study describes the effects of RAGE inhibition in the brain, through an intranasal treatment with the antagonist FPS-ZM1, in an animal model of chronic neuroinflammation induced by acute intraperitoneal injection of lipopolysaccharide (LPS). Seventy days after LPS administration (2 mg/kg, i.p.), Wistar rats received, intranasally, 1.2 mg of FPS-ZM1 over 14 days. On days 88 and 89, the animals were submitted to the open-field test and were killed on day 90 after the intraperitoneal injection of LPS. Our results indicate that blockade of encephalic RAGE attenuates LPS-induced chronic neuroinflammation in different brain regions. Furthermore, we found that intranasal FPS-ZM1 administration reduced levels of gliosis markers, RAGE ligands, and α-synuclein in the substantia nigra pars compacta. Additionally, the treatment also reversed the increase in S100 calcium-binding protein B (RAGE ligand) in the cerebrospinal fluid and the cognitive-behavioral deficits promoted by LPS-less time spent in the central zone of the open-field arena (more time in the lateral zones), decreased total distance traveled, and increased number of freezing episodes. In summary, our study demonstrates the prominent role of RAGE in the maintenance of a chronic neuroinflammatory state triggered by a single episode of systemic inflammation and also points to possible future RAGE-based therapeutic approaches to treat conditions in which chronic neuroinflammation and increased α-synuclein levels could play a relevant role, such as in Parkinson's disease.

The alpha-synuclein oligomers activate nuclear factor of activated T-cell (NFAT) modulating synaptic homeostasis and apoptosis.

BACKGROUND: Soluble oligomeric forms of alpha-synuclein (aSyn-O) are believed to be one of the main toxic species in Parkinson's disease (PD) leading to degeneration. aSyn-O can induce Ca2+ influx, over activating downstream pathways leading to PD phenotype. Calcineurin (CN), a phosphatase regulated by Ca2+ levels, activates NFAT transcription factors that are involved in the regulation of neuronal plasticity, growth, and survival. METHODS: Here, using a combination of cell toxicity and gene regulation assays performed in the presence of classical inhibitors of the NFAT/CN pathway, we investigate NFAT's role in neuronal degeneration induced by aSyn-O. RESULTS: aSyn-O are toxic to neurons leading to cell death, loss of neuron ramification and reduction of synaptic proteins which are reversed by CN inhibition with ciclosporin-A or VIVIT, a NFAT specific inhibitor. aSyn-O induce NFAT nuclear translocation and transactivation. We found that aSyn-O modulates the gene involved in the maintenance of synapses, synapsin 1 (Syn 1). Syn1 mRNA and protein and synaptic puncta are drastically reduced in cells treated with aSyn-O which are reversed by NFAT inhibition. CONCLUSIONS: For the first time a direct role of NFAT in aSyn-O-induced toxicity and Syn1 gene regulation was demonstrated, enlarging our understanding of the pathways underpinnings synucleinopathies.

More than meets the eye in Parkinson's disease and other synucleinopathies: from proteinopathy to lipidopathy.

The accumulation of proteinaceous inclusions in the brain is a common feature among neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease (PD), and dementia with Lewy bodies (DLB). The main neuropathological hallmark of PD and DLB are inclusions, known as Lewy bodies (LBs), enriched not only in α-synuclein (aSyn), but also in lipid species, organelles, membranes, and even nucleic acids. Furthermore, several genetic risk factors for PD are mutations in genes involved in lipid metabolism, such as GBA1, VSP35, or PINK1. Thus, it is not surprising that mechanisms that have been implicated in PD, such as inflammation, altered intracellular and vesicular trafficking, mitochondrial dysfunction, and alterations in the protein degradation systems, may be also directly or indirectly connected through lipid homeostasis. In this review, we highlight and discuss the recent evidence that suggests lipid biology as important drivers of PD, and which require renovated attention by neuropathologists. Particularly, we address the implication of lipids in aSyn accumulation and in the spreading of aSyn pathology, in mitochondrial dysfunction, and in ER stress. Together, this suggests we should broaden the view of PD not only as a proteinopathy but also as a lipidopathy.

Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation.

Alpha-Synuclein (α-Synuclein) is a 140 amino acid protein implicated in neurodegenerative disorders known as synucleinopathies, where it accumulates in proteinaceous inclusions in the brain. The normal physiological function of α-Synuclein remains obscure, as it exists in several non-neuronal cells in which its function has not been studied. Given the tremendous interest in studying α-Synuclein, and the existing limitations in the production of modified forms of the protein, we developed a method for the chemical synthesis of α-Synuclein by combining peptide fragment synthesis via automated microwave-assisted solid-phase peptide synthesis and ligation strategies. Our synthetic pathway enables the synthesis of protein variants of interest, carrying either mutations or posttranslational modifications, for further investigations of the effects on the structure and aggregation behavior of the protein. Ultimately, our study forms the foundation for future syntheses and studies of other custom-made α-Synuclein variants with a single or several modifications, as necessary.

Type 2 Diabetes and Parkinson's Disease: A Focused Review of Current Concepts.

Highly reproducible epidemiological evidence shows that type 2 diabetes (T2D) increases the risk and rate of progression of Parkinson's disease (PD), and crucially, the repurposing of certain antidiabetic medications for the treatment of PD has shown early promise in clinical trials, suggesting that the effects of T2D on PD pathogenesis may be modifiable. The high prevalence of T2D means that a significant proportion of patients with PD may benefit from personalized antidiabetic treatment approaches that also confer neuroprotective benefits. Therefore, there is an immediate need to better understand the mechanistic relation between these conditions and the specific molecular pathways affected by T2D in the brain. Although there is considerable evidence that processes such as insulin signaling, mitochondrial function, autophagy, and inflammation are involved in the pathogenesis of both PD and T2D, the primary aim of this review is to highlight the evidence showing that T2D-associated dysregulation of these pathways occurs not only in the periphery but also in the brain and how this may facilitate neurodegeneration in PD. We also discuss the challenges involved in disentangling the complex relationship between T2D, insulin resistance, and PD, as well as important questions for further research. © 2022 International Parkinson and Movement Disorder Society.

Changes in α-Synuclein Posttranslational Modifications in an AAV-Based Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) pathology is characterized by the loss of dopaminergic neurons of the nigrostriatal system and accumulation of Lewy bodies (LB) and Lewy neurites (LN), inclusions mainly composed of alpha-synuclein (α-Syn) fibrils. Studies linking the occurrence of mutations and multiplications of the α-Syn gene (SNCA) to the onset of PD support that α-Syn deposition may play a causal role in the disease, in line with the hypothesis that disease progression may correlate with the spreading of LB pathology in the brain. Interestingly, LB accumulate posttranslationally modified forms of α-Syn, suggesting that α-Syn posttranslational modifications impinge on α-Syn aggregation and/or toxicity. Here, we aimed at investigating changes in α-Syn phosphorylation, nitration and acetylation in mice subjected to nigral stereotaxic injections of adeno-associated viral vectors inducing overexpression of human α-Syn (AAV-hα-Syn), that model genetic PD with SNCA multiplications. We detected a mild increase of serine (Ser) 129 phosphorylated α-Syn in the substantia nigra (SN) of AAV-hα-Syn-injected mice in spite of the previously described marked accumulation of this PTM in the striatum. Following AAV-hα-Syn injection, tyrosine (Tyr) 125/136 nitrated α-Syn accumulation in the absence of general 3-nitrotirosine (3NT) or nitrated-Tyr39 α-Syn changes and augmented protein acetylation abundantly overlapping with α-Syn immunopositivity were also detected.

Beyond Motor Deficits: Environmental Enrichment Mitigates Huntington's Disease Effects in YAC128 Mice.

Huntington's disease (HD) is a neurodegenerative genetic disorder characterized by motor, psychiatric, cognitive, and peripheral symptoms without effective therapy. Evidence suggests that lifestyle factors can modulate disease onset and progression, and environmental enrichment (EE) has emerged as a potential approach to mitigate the progression and severity of neurodegenerative processes. Wild-type (WT) and yeast artificial chromosome (YAC) 128 mice were exposed to different EE conditions. Animals from cohort 1 were exposed to EE between postnatal days 21 and 60, and animals from cohort 2 were exposed to EE between postnatal days 60 and 120. Motor and non-motor behavioral tests were employed to evaluate the effects of EE on HD progression. Monoamine levels, hippocampal cell proliferation, neuronal differentiation, and dendritic arborization were also assessed. Here we show that EE had an antidepressant-like effect and slowed the progression of motor deficits in HD mice. It also reduced monoamine levels, which correlated with better motor performance, particularly in the striatum. EE also modulated neuronal differentiation in the YAC128 hippocampus. These results confirm that EE can impact behavior, hippocampal neuroplasticity, and monoamine levels in YAC128 mice, suggesting this could be a therapeutic strategy to modulate neuroplasticity deficits in HD. However, further research is needed to fully understand EE's mechanisms and long-term effects as an adjuvant therapy for this debilitating condition.

The courage to change science.

Bureaucracy, performance assessment and other pressures increasingly encroach on scientists' ability to do science. The research community as a whole needs to address these perils.

Characterization of the activity, aggregation, and toxicity of heterodimers of WT and ALS-associated mutant Sod1.

Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.

Doxycycline inhibits α-synuclein-associated pathologies in vitro and in vivo.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn). Doxycycline, a tetracyclic antibiotic shows neuroprotective effects, initially proposed to be due to its anti-inflammatory properties. More recently, an additional mechanism by which doxycycline may exert its neuroprotective effects has been proposed as it has been shown that it inhibits amyloid aggregation. Here, we studied the effects of doxycycline on aSyn aggregation in vivo, in vitro and in a cell free system using real-time quaking induced conversion (RT-QuiC). Using H4, SH-SY5Y and HEK293 cells, we found that doxycycline decreases the number and size of aSyn aggregates in cells. In addition, doxycycline inhibits the aggregation and seeding of recombinant aSyn, and attenuates the production of mitochondrial-derived reactive oxygen species. Finally, we found that doxycycline induces a cellular redistribution of aggregates in a C.elegans animal model of PD, an effect that is associated with a recovery of dopaminergic function. In summary, we provide strong evidence that doxycycline treatment may be an effective strategy against synucleinopathies.