Pharmacological Modulators of Tau Aggregation and Spreading

Tauopathies are neurodegenerative disorders characterized by the deposition of aggregates composed of abnormal tau protein in the brain. Additionally, misfolded forms of tau can propagate from cell to cell and throughout the brain. This process is thought to lead to the templated misfolding of the native forms of tau, and thereby, to the formation of newer toxic aggregates, thereby propagating the disease. Therefore, modulation of the processes that lead to tau aggregation and spreading is of utmost importance in the fight against tauopathies. In recent years, several molecules have been developed for the modulation of tau aggregation and spreading. In this review, we discuss the processes of tau aggregation and spreading and highlight selected chemicals developed for the modulation of these processes, their usefulness, and putative mechanisms of action. Ultimately, a stronger understanding of the molecular mechanisms involved, and the properties of the substances developed to modulate them, will lead to the development of safer and better strategies for the treatment of tauopathiesA.D.-M. is supported by a postdoctoral fellowship from the Galician Government (Programa de axuda á etapa posdoutoral, XUGA, GAIN, ED481B 2017/053). T.F.O. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC 2067/1-390729940S

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

Altres ajuts: ICREA (ICREA-Academia 2015 and 2020); Fundació la Marató de TV3 (20144330)Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, as well as the accumulation of intraneuronal proteinaceous inclusions known as Lewy bodies and Lewy neurites. The major protein component of Lewy inclusions is the intrinsically disordered protein α-synuclein (α-Syn), which can adopt diverse amyloid structures. Different conformational strains of α-Syn have been proposed to be related to the onset of distinct synucleinopathies; however, how specific amyloid fibrils cause distinctive pathological traits is not clear. Here, we generated three different α-Syn amyloid conformations at different pH and salt concentrations and analyzed the activity of SynuClean-D (SC-D), a small aromatic molecule, on these strains. We show that incubation of α-Syn with SC-D reduced the formation of aggregates and the seeded polymerization of α-Syn in all cases. Moreover, we found that SC-D exhibited a general fibril disaggregation activity. Finally, we demonstrate that treatment with SC-D also reduced strain-specific intracellular accumulation of phosphorylated α-Syn inclusions. Taken together, we conclude that SC-D may be a promising hit compound to inhibit polymorphic α-Syn aggregation

Spreading of α-Synuclein and Tau: A Systematic Comparison of the Mechanisms Involved

Alzheimer's disease (AD) and Parkinson's disease (PD) are age-associated neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn) and tau, respectively. The coexistence of aSyn and tau aggregates suggests a strong overlap between tauopathies and synucleinopathies. Interestingly, misfolded forms of aSyn and tau can propagate from cell to cell, and throughout the brain, thereby templating the misfolding of native forms of the proteins. The exact mechanisms involved in the propagation of the two proteins show similarities, and are reminiscent of the spreading characteristic of prion diseases. Recently, several models were developed to study the spreading of aSyn and tau. Here, we discuss the mechanisms involved, the similarities and differences between the spreading of the two proteins and that of the prion protein, and the different cell and animal models used for studying these processes. Ultimately, a deeper understanding of the molecular mechanisms involved may lead to the identification of novel targets for therapeutic intervention in a variety of devastating neurodegenerative diseases

Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid

α-, β-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson’s disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member

Investigation of the interplay between mitochondria and α-synuclein for the development of neuropathological events related to Parkinson’s disease: a new role for the mitochondrial protease LonP1

Parkinson’s disease (PD) is a progressive, neurodegenerative movement disorder that affects approximately 1% of adults aged 60 and over. It is characterized by the selective degeneration of dopaminergic neurons in the substantia nigra, accumulation of α-synuclein fibrils, and impaired mitochondrial function. Mitochondrial dysfunction induces α-syn aggregation, which constitutes the main component of Lewy bodies. Moreover, α-syn can lead to a dose-dependent loss of mitochondrial transmembrane potential (Δψm). The mechanisms underlying the interplay between α-syn and mitochondrial dysfunction, as well as their potential involvement in neurodegeneration are not yet clear. The aim of this thesis was the generation of relevant cellular models for investigating molecular interactions and signaling pathways that are involved in the progressive appearance of PD-related cell phenotypes, focusing on the influence of α-synuclein in mitochondrial function. As such models, we considered the exposure of neuronal cells in conditions that lead to mitochondrial impairment, namely the presence of neurotoxins that increase the load of free radicals, and the generation cytoplasmic hybrid cells (cybrids) by replacing the mitochondria of healthy neuronal cells with the mitochondria from patients with inherited forms of the disease. We developed and characterized both models. In order to develop the chronic model, the SHSY5Y cells were cultured under conditions of mild but continuous (chronic) oxidative stress challenge with the neurotoxin 6-OHDA. By using a series of biochemical and cell biology approaches we demonstrated that this system can faithfully recapitulate many of the key PD features that have been reported in previous in vitro and in vivo studies, in a chronological order. In this model we have identified a new role of the mitochondrial protease LonP1 in the nucleus and specifically in the chromatin, probably important for the progression of Parkinson's disease. Focusing on the mitochondrial function, we developed cybrid cell lines containing mitochondria from patients with the inherited form of PD as well as from healthy relatives. We characterized basic cellular properties such as mitochondria function, proliferation and differentiation potential, and examined possible nucleotide changes using next generation sequencing approaches (deep sequencing). Although no differences were detected between «PD» and «healthy» cybrids, these cell lines constitute a valuable experimental model, which in conjuction with the chronic stress model could be explored in future studies to contribute to the understanding of the role of mitochondria dysfunction in Parkinson's disease.Η νόσος του Parkinson (PD) είναι μια προοδευτική κινητική νευροεκφυλιστική διαταραχή η οποία προσβάλλει περίπου το 1% των ενηλίκων ηλικίας άνω των 60 ετών. Οφείλεται στην εκλεκτική εκφύλιση των ντοπαμινεργικών νευρώνων της μέλαινας ουσίας και χαρακτηρίζεται από συσσώρευση συσσωματωμάτων ινιδιακής μορφής της πρωτεΐνης α-synuclein και εξασθένηση της μιτοχονδριακής λειτουργίας. Η μιτοχονδριακή δυσλειτουργία επάγει τη συσσωμάτωση της α-synuclein, η οποία αποτελεί το κύριο συστατικό των σωματίων του Lewy. Επιπλέον η α-synuclein μπορεί να οδηγήσει σε δοσοεξαρτώμενη απώλεια του δυναμικού της μιτοχονδριακής μεμβράνης Δψm. Οι μηχανισμοί που διέπουν την αλληλεπίδραση μεταξύ της α-synuclein και τη μιτοχονδριακή δυσλειτουργία καθώς και η πιθανή συμμετοχή τους στη νευροεκφύλιση δεν είναι ακόμη σαφείς. Στόχος της διατριβής ήταν η δημιουργία κατάλληλων κυτταρικών μοντέλων για τη διερεύνηση των μοριακών αλληλεπιδράσεων και των σηματοδοτικών μονοπατιών που εμπλέκονται στην προοδευτική εκδήλωση της νόσου του Parkinson, με επίκεντρο την επίδραση της α-synuclein στη μιτοχονδριακή λειτουργία. Ως τέτοια μοντέλα επιλέξαμε την ανάπτυξη ενός in vitro συστήματος για τη μελέτη του προοδευτικού χαρακτήρα της νόσου του Parkinson κάτω από συνθήκες οξειδωτικού stress, καθώς και τη δημιουργία μιας ιδιαίτερης κατηγορίας υβριδικών κυττάρων (cybrids) αντικαθιστώντας τα μιτοχόνδρια υγιών νευρικών κυττάρων με τα μιτοχόνδρια ασθενών με κληρονομικές μορφές της νόσου. Αναπτύξαμε και χαρακτηρίσαμε και τα δύο μοντέλα. Για την ανάπτυξη του χρόνιου μοντέλου, καλλιεργήθηκαν τα κύτταρα SHSY5Y, υπό συνθήκες ήπιας συνεχούς (χρόνιας) πρόκλησης οξειδωτικού stress με τη νευροτοξίνη 6-OHDA. Με μια σειρά βιοχημικών και κυτταροβιολογικών μεθόδων αποδείξαμε ότι στο σύστημα αυτό αναπαράγονται πολλά από τα παρκινσονιακά χαρακτηριστικά που έχουν κατά καιρούς αναφερθεί σε in vitro και in vivo μελέτες, και μάλιστα με μια συγκεκριμένη χρονική ακολουθία. Τα πειράματά μας αποκάλυψαν για πρώτη φορά, μια πιθανή σύνδεση της πρωτεΐνης LonP1 με την εμφάνιση παθολογικών χαρακτηριστικών της νόσου στα νευρικά κύτταρα, όπως η συσσωμάτωση της α-synuclein και η μιτοχονδριακή δυσλειτουργία. Σε αυτό ταυτοποιήσαμε έναν νέο ρόλο για την μιτοχονδριακή πρωτεάση LonP1 στον πυρήνα και συγκεκριμένα στη χρωματίνη, πιθανά σημαντικό για την πορεία της νόσου Parkinson. Εστιάζοντας στη μιτοχονδριακή λειτουργία, εγκαθιδρύσαμε και αναπτύξαμε υβριδικές κυτταρικές σειρές, οι οποίες περιέχουν μιτοχόνδρια ασθενών με την κληρονομική μορφή της νόσου του Parkinson, καθώς και από υγιή συγγενικά τους άτομα. Ακολούθησε πλήρης χαρακτηρισμός αυτών των σειρών, όσον αφορά βασικές κυτταρικές ιδιότητες όπως είναι η λειτουργικότητα των μιτοχονδρίων, ο πολλαπλασιασμός και η ικανότητα διαφοροποίησής τους, και έγινε έλεγχος του βαθμού των νουκλεοτιδικών αλλαγών που έχουν συσσωρευτεί στη μιτοχονδριακή αλληλουχία, με μεθοδολογία αλληλούχισης νέας γενιάς (deep sequencing). Παρότι δεν ανιχνεύθηκαν διαφορές μεταξύ «PD» και «υγιών» cybrids, τα κύτταρα αυτά παραμένουν στη διάθεσή μας ως ένα πολύτιμο πειραματικό μοντέλο το οποίο σε συνδυασμό με το μοντέλο του χρόνιου stress θα μπορούσε μελλοντικά να συμβάλλει στην κατανόηση του ρόλου της μιτοχονδριακής δυσλειτουργίας στην νόσο του Parkinson

Endogenous levels of alpha-synuclein modulate seeding and aggregation in cultured cells

International audienceParkinson's disease is a progressive neurodegenerative disorder characterized by the accumulation of misfolded alphasynuclein in intraneuronal inclusions known as Lewy bodies and Lewy neurites. Multiple studies strongly implicate the levels of alpha-synuclein as a major risk factor for the onset and progression of Parkinson's disease. Alpha-synuclein pathology spreads progressively throughout interconnected brain regions but the precise molecular mechanisms underlying the seeding of alpha-synuclein aggregation are still unclear. Here, using stable cell lines expressing alpha-synuclein, we examined the correlation between endogenous alpha-synuclein levels and the seeding propensity by exogenous alpha-synuclein preformed fibrils. We applied biochemical approaches and imaging methods in stable cell lines expressing alpha-synuclein and in primary neurons to determine the impact of alpha-synuclein levels on seeding and aggregation. Our results indicate that the levels of alpha-synuclein define the pattern and severity of aggregation and the extent of p-alpha-synuclein deposition, likely explaining the selective vulnerability of different cell types in synucleinopathies. The elucidation of the cellular processes involved in the pathological aggregation of alpha-synuclein will enable the identification of novel targets and the development of therapeutic strategies for Parkinson's disease and other synucleinopathies

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

International audienceParkinson'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

α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials