Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential

AbstractPathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a “tropism” for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

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Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

10.1101/2020.03.12.989103 ◽

2020 ◽

Author(s):

Jace Jones-Tabah ◽

Hanan Mohammad ◽

Shadi Hadj-Youssef ◽

Lucy Kim ◽

Ryan D. Martin ◽

...

Keyword(s):

Resonance Energy Transfer ◽

Resonance Energy ◽

Dopamine D1 Receptor ◽

D1 Receptor ◽

Striatal Neurons ◽

Dynamic Cell ◽

Cell Type Specific ◽

Fret Biosensors ◽

Optically Detected

AbstractLike many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell-type specific, and can change in response to disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson’s disease (PD), alterations in this pathway lead to activation of extracellular regulated kinases (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

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The Molecular Chaperone DNAJB6, but Not DNAJB1, Suppresses the Seeded Aggregation of Alpha-Synuclein in Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20184495 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4495 ◽

Cited By ~ 2

Author(s):

Natasja Deshayes ◽

Sertan Arkan ◽

Christian Hansen

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Lewy Bodies ◽

Resonance Energy ◽

Alpha Synuclein ◽

Hek293 Cells ◽

Induced Aggregation ◽

In Cells

Alpha-synuclein (α-Syn) can misfold and aggregate, causing the degeneration of dopaminergic neurons, as seen in Parkinson’s disease (PD). We recently demonstrated that DNAJB6, a co-chaperone found in Lewy bodies (LB), suppresses the aggregation of α-Syn in cells and in vitro. In this study, we compared the capacities of DNAJB1 and DNAJB6 to suppress the seeded α-Syn aggregation in HEK293 cells expressing α-Syn tagged with cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). The aggregation of α-Syn was seeded by the transfection of the cells with recombinant α-Syn pre-formed fibrils (PFFs), following the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated knockout (KO) of these two genes, respectively. We quantified the α-Syn aggregation by fluorescence microscopy and fluorescence resonance energy transfer (FRET) analysis. We detected significantly more aggregates in the DNAJB6 KO cells compared with the parental cells, whereas the DNAJB1 KO had no effect on the α-Syn aggregation. This is the first evidence that DNAJB6 can suppress α-Syn aggregation, induced by exogenous α-Syn seeds, in cells. Next, we explored whether this mechanism could be dependent on protein degradation pathways. We observed that the increase in the α-Syn PFF-induced aggregation in the DNAJB6 KO cells compared with the parental cells was strongly diminished upon the incubation of the cells with the proteasomal inhibitor MG132. These results consolidate that DNAJB6 is a suppressor of α-Syn aggregation, and suggest that DNAJB6 may target misfolded and/or aggregated α-Syn for proteasomal degradation.

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Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum

Life ◽

10.3390/life10080147 ◽

2020 ◽

Vol 10 (8) ◽

pp. 147 ◽

Cited By ~ 2

Author(s):

Fabiana Miraglia ◽

Verdiana Valvano ◽

Lucia Rota ◽

Cristina Di Primio ◽

Valentina Quercioli ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Alpha Synuclein ◽

Stable Cell Line ◽

Fret Biosensors ◽

Alpha Synuclein Aggregation ◽

Small Subpopulation

Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (αS) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of αS resulted in the formation of compact αS-positive structures in a small subpopulation of cells, resembling αS inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of αS FRET biosensors (AFBs) able to track αS conformation in cells. In native conditions, expression in i36, a stable cell line for ER αS, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of αS (αS-CFP/αS-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with αS N- or C- termini (YFP-αS-CFP), showed a positive FRET signal. These data confirmed that αS has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of αS oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in αS conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track αS conformational changes in vivo.

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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Biological nanoscale fluorescent probes: From structure and performance to bioimaging

Reviews in Analytical Chemistry ◽

10.1515/revac-2020-0119 ◽

2020 ◽

Vol 39 (1) ◽

pp. 209-221

Author(s):

Jiafeng Wan ◽

Xiaoyuan Zhang ◽

Kai Zhang ◽

Zhiqiang Su

Keyword(s):

Fluorescent Probes ◽

Organic Dyes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

High Sensitivity ◽

Biological Imaging ◽

Fluorescent Materials ◽

And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

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Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

Nuclear Receptor Signaling ◽

10.1621/nrs.04021 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04021 ◽

Cited By ~ 14

Author(s):

Kristen L. Koterba ◽

Brian G. Rowan

Keyword(s):

Energy Transfer ◽

Protein Interactions ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Renilla Luciferase ◽

Receptor Protein ◽

Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507121112 ◽

2015 ◽

Vol 112 (21) ◽

pp. 6718-6723 ◽

Cited By ~ 53

Author(s):

Akihiro Goto ◽

Ichiro Nakahara ◽

Takashi Yamaguchi ◽

Yuji Kamioka ◽

Kenta Sumiyama ◽

...

Keyword(s):

D2 Receptor ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Signaling Cascades ◽

Male Mice ◽

Indirect Pathway ◽

Dynamic Activity ◽

Reward Seeking ◽

Electric Shocks

The selection of reward-seeking and aversive behaviors is controlled by two distinct D1 and D2 receptor-expressing striatal medium spiny neurons, namely the direct pathway MSNs (dMSNs) and the indirect pathway MSNs (iMSNs), but the dynamic modulation of signaling cascades of dMSNs and iMSNs in behaving animals remains largely elusive. We developed an in vivo methodology to monitor Förster resonance energy transfer (FRET) of the activities of PKA and ERK in either dMSNs or iMSNs by microendoscopy in freely moving mice. PKA and ERK were coordinately but oppositely regulated between dMSNs and iMSNs by rewarding cocaine administration and aversive electric shocks. Notably, the activities of PKA and ERK rapidly shifted when male mice became active or indifferent toward female mice during mating behavior. Importantly, manipulation of PKA cascades by the Designer Receptor recapitulated active and indifferent mating behaviors, indicating a causal linkage of a dynamic activity shift of PKA and ERK between dMSNs and iMSNs in action selection.

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Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713959115 ◽

2018 ◽

Vol 115 (46) ◽

pp. E10859-E10868 ◽

Cited By ~ 5

Author(s):

Yuwei Li ◽

Jason A. Junge ◽

Cosimo Arnesano ◽

Garrett G. Gross ◽

Jeffrey H. Miner ◽

...

Keyword(s):

Cell Polarity ◽

Protein Interactions ◽

Protein Function ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Explant Culture ◽

Scaffolding Protein ◽

Fluorescence Lifetime Imaging ◽

Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

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Multiple system atrophy: genetic risks and alpha-synuclein mutations

F1000Research ◽

10.12688/f1000research.12193.1 ◽

2017 ◽

Vol 6 ◽

pp. 2072 ◽

Cited By ~ 7

Author(s):

Heather T Whittaker ◽

Yichen Qui ◽

Conceição Bettencourt ◽

Henry Houlden

Keyword(s):

Multiple System Atrophy ◽

Protein Misfolding ◽

Research Collaboration ◽

Late Onset ◽

Alpha Synuclein ◽

Common Mechanism ◽

International Research Collaboration ◽

Cytoplasmic Inclusions ◽

Genetic Risks

Multiple system atrophy (MSA) is one of the few neurodegenerative disorders where we have a significant understanding of the clinical and pathological manifestations but where the aetiology remains almost completely unknown. Research to overcome this hurdle is gaining momentum through international research collaboration and a series of genetic and molecular discoveries in the last few years, which have advanced our knowledge of this rare synucleinopathy. In MSA, the discovery of α-synuclein pathology and glial cytoplasmic inclusions remain the most significant findings. Families with certain types of α-synuclein mutations develop diseases that mimic MSA, and the spectrum of clinical and pathological features in these families suggests a spectrum of severity, from late-onset Parkinson’s disease to MSA. Nonetheless, controversies persist, such as the role of common α-synuclein variants in MSA and whether this disorder shares a common mechanism of spreading pathology with other protein misfolding neurodegenerative diseases. Here, we review these issues, specifically focusing on α-synuclein mutations.

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