Synapsin III is a key component of α‐synuclein fibrils in Lewy bodies of PD brains

Longhena, Francesca; Faustini, Gaia; Varanita, Tatiana; Zaltieri, Michela; Porrini, Vanessa; Tessari, Isabella; Poliani, Pietro Luigi; Missale, Cristina; Borroni, Barbara; Padovani, Alessandro; Bubacco, Luigi; Pizzi, Marina; Spano, PierFranco; Bellucci, Arianna

doi:10.1111/bpa.12587

Cited by 38 publications

(36 citation statements)

References 67 publications

(95 reference statements)

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“…[3][4][5] We recently described that the synaptic phosphoprotein synapsin III (Syn III) interacts and cooperates with aSyn in the control of DA release from mesencephalic neurons. [6] Moreover, we showed that Syn III is another key component of aSyn insoluble fibrils extracted from the post-mortem brains of sporadic PD patients [7] and that Syn III is a crucial mediator of aSyn aggregation and toxicity. [8] Strikingly, our most recent findings suggest that threo-methylphenidate (MPH), a monoamine re-uptake inhibitor clinically approved for the treatment of attention deficits and hyperactivity disorder (ADHD) [9] and for ameliorating gait freezing in advanced PD, [10][11][12][13][14] is able to stimulate an aSyn/Syn III-mediated locomotor response, specifically in aSyn transgenic mice at a pathological stage that exhibit severe dopaminergic functional deficits.…”

Section: Introductionmentioning

confidence: 93%

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Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET‐Based Assay of Synapsin III Binding

et al. 2020

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We previously described synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with α‐synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxicity. Our recent observations point to threo‐methylphenidate (MPH), a monoamine re‐uptake inhibitor that efficiently counteracts the freezing‐gait characteristic of advanced PD, as a ligand for Syn III. We have designed and synthesised two different fluorescently labelled MPH derivatives, one with Rhodamine Red (RHOD) and one with 5‐carboxytetramethylrhodamine (TAMRA), to be used for assessing MPH binding to Syn III by FRET. TAMRA‐MPH exhibited the ideal characteristics to be used as a FRET acceptor, as it was able to enter into the SK‐N‐SH cells and could interact specifically with human green fluorescent protein (GFP)‐tagged Syn III but not with GFP alone. Moreover, the uptake of TAMRA‐MPH and co‐localization with Syn III was also observed in primary mesencephalic neurons. These findings support that MPH is a Syn III ligand and that TAMRA‐conjugated drug molecules might be valuable tools to study drug‐ligand interactions by FRET or to detect Syn III in cytological and histological samples.

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Section: Introductionmentioning

confidence: 93%

“…For the synthesis of 2, the PEG linker is introduced using Mitsunobu reaction conditions with 4 to give N-Boc-amino-PEG4-alcohol (7). Both the amines of 7 are then deprotected under acidic conditions, to give 8, which is then reacted with commercial 6-COOH-TAMRA, using a TSTU-based amide coupling protocol.…”

Section: Chemistrymentioning

confidence: 99%

Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET‐Based Assay of Synapsin III Binding

et al. 2020

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“…In this context, our results suggest that droplet formation by a-synuclein may be involved in a physiological mechanism to cluster synaptic vesicles, possibly in conjunction with other proteins such as synapsins, which have also been shown to phase-separate into liquid-like assemblies [55]. Indeed, synapsins are implicated in synaptic function in conjunction with synuclein [57,58] and are components of Lewy bodies [59].…”

Section: Discussionmentioning

confidence: 67%

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Hardenberg

Sinnige

Casford

et al. 2020

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Misfolded a-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson's disease. A large body of evidence shows that a-synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinson's disease, that a-synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of a-synuclein condensates in the presence of lipids and other cellular components.Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over 2% of the world's population over 65 years of age [1,2]. The molecular origins of this disease are not fully understood, although they have been closely associated with dysregulation of the behaviour of a-synuclein [1, 3, 4], a disordered protein whose function appears to involve the regulation of synaptic vesicle trafficking [5][6][7][8]. This gap in our knowledge of the disease aetiology is no doubt partly responsible for the lack of disease modifying therapies for PD [9][10][11][12].The pathological hallmark of PD is the presence of Lewy bodies within dopaminergic neurons in the brains of affected patients [13]. Although misfolded a-synuclein is a major constituent of Lewy bodies [14,15], ultrastructural and proteome studies have indicated that these aberrant deposits contain a plethora of cellular components, including lipid membranes and organelle fragments [16][17][18][19][20]. These findings have led to the suggestion that the processes associated with the formation of Lewy bodies could be major drivers of neurotoxicity in PD [16,18,21].Since it has also been shown that a-synuclein can aggregate into ordered amyloid fibrils in vitro [22][23][24][25][26] and in vivo [27][28][29][30][31][32], we asked how such a-synuclein aggregation can be reconciled with the formation of highly complex and heterogeneous assemblies such as Lewy bodies. We hypothesised that a-synuclein may be capable of irreversibly capturing cellular components through liquid-liquid phase separation, as this mechanism has been shown to drive the self-assembly of various disease-associated proteins on-pathway to the formation of solid aggregates [33][34][35][36]. Under healthy conditions, the condensation of proteins into a dense liquid droplet state through liquid-liquid phase separation is normally reversible, and exploited in a variety of ways to carry out cellular functions, including RNA metabolism, ribosome biogenesis, DNA damage response and signal transduction [33,34,37]. Upon dysregulation, however, liquid droplets can mature into gel-like deposits, which irreversibly sequester important cellular components and lead to pathological processes [38...

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“…Previous studies have demonstrated the use of PLA to study αSyn interacting with other proteins, such as the dopamine transporter and αSyn complexes in a tg mouse overexpressing αSyn 1–120 ( 20 ). Interactions with TOM20 ( 21 ) and the synaptic protein synapsin III ( 22 , 23 ) have also been observed with PLA. In addition, PLA has been used to specifically detect oligomeric αSyn in brain tissue from PD patients ( 24 ).…”

Section: Discussionmentioning

confidence: 91%

In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons

et al. 2018

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The aggregation of alpha-synuclein (αSyn) is the pathological hallmark of Parkinson’s disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of αSyn is to promote the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between αSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human αSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human αSyn, a PLA signal indicating close proximity between αSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human αSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of αSyn and the SNARE proteins in primary neuronal cultures.

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Synapsin III is a key component of α‐synuclein fibrils in Lewy bodies of PD brains

Cited by 38 publications

References 67 publications

Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET‐Based Assay of Synapsin III Binding

Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET‐Based Assay of Synapsin III Binding

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons

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