Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation
2018; Springer Nature; Volume: 19; Issue: 5 Linguagem: Inglês
10.15252/embr.201744617
ISSN1469-3178
AutoresCristine Betzer, Louise Berkhoudt Lassen, Anders Olsen, Rikke Hahn Kofoed, Lasse Reimer, Emil Gregersen, Jin Zheng, Tito Calì, Wei‐Ping Gai, Tong Chen, Arne Moeller, Marisa Brini, YuHong Fu, Glenda M. Halliday, Tomasz Brudek, Susana Aznar, Bente Pakkenberg, Jens Peter Andersen, Poul Henning Jensen,
Tópico(s)Alzheimer's disease research and treatments
ResumoArticle29 March 2018Open Access Transparent process Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation Cristine Betzer Cristine Betzer orcid.org/0000-0001-5429-3548 Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Louise Berkhoudt Lassen Louise Berkhoudt Lassen Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Anders Olsen Anders Olsen Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark Search for more papers by this author Rikke Hahn Kofoed Rikke Hahn Kofoed Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Lasse Reimer Lasse Reimer Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Emil Gregersen Emil Gregersen Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Jin Zheng Jin Zheng Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Tito Calì Tito Calì Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Wei-Ping Gai Wei-Ping Gai Neuropathological Laboratory, Department of Medicine, Center for Neurological Diseases, University of Adelaide, Adelaide, SA, Australia Search for more papers by this author Tong Chen Tong Chen Department of Medical Biochemistry, School of Medicine, Flinders University, Bedford Park, SA, Australia Search for more papers by this author Arne Moeller Arne Moeller Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Structural Biology, Max Plank Institute of Biophysics, Frankfurt, Germany Search for more papers by this author Marisa Brini Marisa Brini Department of Biology, University of Padova, Padova, Italy Search for more papers by this author Yuhong Fu Yuhong Fu Brain & Mind Centre, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Glenda Halliday Glenda Halliday Brain & Mind Centre, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Tomasz Brudek Tomasz Brudek Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark Search for more papers by this author Susana Aznar Susana Aznar Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark Search for more papers by this author Bente Pakkenberg Bente Pakkenberg Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark Search for more papers by this author Jens Peter Andersen Jens Peter Andersen Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Poul Henning Jensen Corresponding Author Poul Henning Jensen [email protected] orcid.org/0000-0002-4439-9020 Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Cristine Betzer Cristine Betzer orcid.org/0000-0001-5429-3548 Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Louise Berkhoudt Lassen Louise Berkhoudt Lassen Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Anders Olsen Anders Olsen Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark Search for more papers by this author Rikke Hahn Kofoed Rikke Hahn Kofoed Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Lasse Reimer Lasse Reimer Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Emil Gregersen Emil Gregersen Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Jin Zheng Jin Zheng Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Tito Calì Tito Calì Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Wei-Ping Gai Wei-Ping Gai Neuropathological Laboratory, Department of Medicine, Center for Neurological Diseases, University of Adelaide, Adelaide, SA, Australia Search for more papers by this author Tong Chen Tong Chen Department of Medical Biochemistry, School of Medicine, Flinders University, Bedford Park, SA, Australia Search for more papers by this author Arne Moeller Arne Moeller Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Structural Biology, Max Plank Institute of Biophysics, Frankfurt, Germany Search for more papers by this author Marisa Brini Marisa Brini Department of Biology, University of Padova, Padova, Italy Search for more papers by this author Yuhong Fu Yuhong Fu Brain & Mind Centre, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Glenda Halliday Glenda Halliday Brain & Mind Centre, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia Search for more papers by this author Tomasz Brudek Tomasz Brudek Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark Search for more papers by this author Susana Aznar Susana Aznar Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark Search for more papers by this author Bente Pakkenberg Bente Pakkenberg Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark Search for more papers by this author Jens Peter Andersen Jens Peter Andersen Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Poul Henning Jensen Corresponding Author Poul Henning Jensen [email protected] orcid.org/0000-0002-4439-9020 Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Author Information Cristine Betzer1,2, Louise Berkhoudt Lassen1,2, Anders Olsen3, Rikke Hahn Kofoed1,2, Lasse Reimer1,2, Emil Gregersen1,2, Jin Zheng1,2, Tito Calì4, Wei-Ping Gai5, Tong Chen6, Arne Moeller1,7, Marisa Brini8, Yuhong Fu9, Glenda Halliday9, Tomasz Brudek10, Susana Aznar10, Bente Pakkenberg10, Jens Peter Andersen2 and Poul Henning Jensen *,1,2 1Danish Research Institute of Translational Neuroscience – DANDRITE, Aarhus University, Aarhus, Denmark 2Department of Biomedicine, Aarhus University, Aarhus, Denmark 3Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark 4Department of Biomedical Sciences, University of Padova, Padova, Italy 5Neuropathological Laboratory, Department of Medicine, Center for Neurological Diseases, University of Adelaide, Adelaide, SA, Australia 6Department of Medical Biochemistry, School of Medicine, Flinders University, Bedford Park, SA, Australia 7Department of Structural Biology, Max Plank Institute of Biophysics, Frankfurt, Germany 8Department of Biology, University of Padova, Padova, Italy 9Brain & Mind Centre, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia 10Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, Copenhagen, Denmark *Corresponding author. Tel: +45 28992056; E-mail: [email protected] EMBO Reports (2018)19:e44617https://doi.org/10.15252/embr.201744617 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+. CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo. Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies. Synopsis Aggregated α-synuclein stimulates the calcium pump SERCA thereby reducing cytosolic Ca2+ and contributing to neurodegeneration. Pharmacological SERCA inhibition protects against α-synuclein aggregate stress with potential therapeutic implications. α-synuclein aggregates bind the calcium pump SERCA in cells and in brain tissue affected by dementia with Lewy bodies. α-synuclein aggregates reduce cytosolic Ca2+ by activating SERCA. SERCA inhibition counteracts calcium efflux and protects cells from α-synuclein aggregate stress. Introduction The small unfolded neuronal protein α-synuclein (AS) is closely linked to Parkinson's disease (PD). This is evidenced by the findings that autosomal-dominant familial PD can be caused by duplications and triplications of the normal SNCA gene encoding AS, by missense mutations causing exchange of single-amino acid residues (A30P, E46K, H50Q, G51D and A53T, A53E), and by variations in the SNCA gene, which represents the greatest genetic risk factor for sporadic PD. Aggregated amyloid-type fibrillar forms of AS accumulate in intra-neuronal Lewy body inclusions, which are the pathological hallmark of PD. Similar AS-containing intracellular inclusions also exist in other neurodegenerative diseases, so-called α-synucleinopathies, that besides PD are dominated by dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). On the pathway from native protein to aggregated amyloid species, soluble oligomeric species are hypothesised to represent cytotoxic forms. The molecular mechanisms whereby AS aggregates contribute to the degeneration of neuronal populations are still unclear, but it has been proposed that they may be linked to perturbations of homeostatic mechanisms, for example proteostasis, mitochondrial functions, and direct toxic actions on membranes (reviewed in 1). Disturbances in brain Ca2+ regulation have recently been linked to PD because treatment of hypertension with antagonists of L-type Ca2+ CaV1 channels reduces the risk of PD 2-4 and the expression of CaV channels in the brain is changed in PD 4, 5. Average cytosolic Ca2+ concentrations are kept in the nM range, contrasting with the mM concentrations present outside cells and in the endoplasmic reticulum. The steep gradient makes Ca2+ an ideal signalling molecule because its cytosolic concentrations can be regulated precisely with spatio-temporal precision by opening of Ca2+ channels. Once in the cytosol, Ca2+ has to be removed by active transporting pumps in ER, Golgi and the plasma membrane, by Na+/Ca2+ exchanger in the plasma membrane, and by mitochondrial buffering. The mechanism whereby Ca2+ channel antagonists modulate the disease course of PD is still unknown, but it is hypothesised that the mechanism reduces the oxidant stress of dopaminergic neurons of substantia nigra that display an energy-consuming pacemaking firing pattern driven by Ca2+ influx via CaV1 channels 6. How this localised effect in dopaminergic neurons is related to the progressive nature of PD remains unclear, but recent data indicate a complex interplay between AS, cytosolic Ca2+ and CaV1 channels in dopaminergic substantia nigra neurons 7. Braak hypothesised that PD arises in the deep brainstem nuclei and spreads via the midbrain to neocortical regions 8. This spreading pattern can be clinically observed in some patients, where REM sleep behaviour disorder (RBD) represents a prodromal phase of PD, and most PD patients develop cognitive impairment and dementia in their later phases due to involvement of neocortex. Ca2+ deregulation has also been hypothesised as playing a general role in several neurodegenerative diseases like Alzheimer's disease and Huntington's disease. In these diseases, increased cytosolic Ca2+ represents a common theme which suggests the presence of mechanisms that lead to the influx of Ca2+ from extracellular space, endoplasmic reticulum and mitochondria as targets for therapeutic intervention 9. We have previously demonstrated that AS aggregate-dependent degenerative processes are initiated at very early time points in cell models when no overt phenotypes are present 10. In the present study, we investigated the temporal development of changes in cytosolic Ca2+ in cellular and neuronal models of AS aggregate-dependent degeneration. Surprisingly, we observed early reduction in cytosolic Ca2+ across models. This reduction was later followed by increased cytosolic Ca2+ when degeneration became apparent. Inhibitors of AS aggregation blocked both the early and late Ca2+ changes. This suggests activation of mechanisms whereby cytosolic Ca2+ is removed against large gradients. Co-immunoprecipitation experiments demonstrated that soluble and insoluble AS aggregates bind to the Ca2+ pump SERCA in contrast to monomers. SERCA is located in the endoplasmic reticulum. Their interaction was validated in cells using proximity ligation assays (PLA) where the antibody pair against SERCA and AS only produced a signal when the aggregation was not prevented. In vitro biochemical experiments demonstrate that AS aggregates activate the transmembrane Ca2+ pumping and ATP hydrolysis by SERCA. Counteracting the activation of SERCA in cells with the SERCA inhibitor cyclopiazonic acid (CPA) abrogated both the early reduction and later increase in cytosolic Ca2+ and protected cells and neurons against AS aggregate-dependent cell death. Treatment of AS-transgenic Caenorhabditis elegans with CPA protected the dopaminergic neurons against AS-dependent degeneration. Analyses of human brains by proximity ligation assay demonstrated the interaction between SERCA and aggregated AS in patients with DLB but not in controls. Moreover, SERCA was present in purified Lewy bodies from DLB patients and in glial cytoplasmic inclusions from MSA patients and SERCA copurified with insoluble AS in the detergent-insoluble fraction of MSA brain. We hypothesise that a slow build-up of AS aggregates occurs within individual neurons during the process of disease spreading throughout the nervous system. We posit that the accumulating aggregates in the early stages activate SERCA and cause a reduction in cytosolic Ca2+, triggering decisive pathophysiological changes and leading to ensuing cell death (Fig 8). Counteracting this early phase or its down-stream processes holds promise for increasing the vitality of the affected cells, thereby slowing and modifying the disease course. Results Neuronal Ca2+ homeostasis has been linked to PD pathogenesis by virtue of a reduced prevalence of sporadic PD among patients treated for hypertension with Ca2+-antagonists 2, 3 and genomewide association studies linking the protective effect of caffeine against PD to the GRIN2A gene, which encodes a subunit of the N-methyl-d-aspartate receptor (NMDA)-activated Ca2+ channel 11, 12. Using the Ca2+ indicator Fura-2, we investigated the temporal changes in cytosolic Ca2+ concentration in three cell models of AS aggregation-dependent cytotoxicity, the mitotic oligodendrocytic cell model OLN-t40-AS, the non-mitotic retinoic acid-differentiated neuroblastoma SH-SY5Y model with inducible AS expression and primary hippocampal neuron cultures from mThy1-αSyn ("line 61") mice (ASO) overexpressing human AS (Fig 1) 13-15. The OLN-t40-AS model revealed a surprising reduction in cytosolic Ca2+ from approximately 195 to 180 nM when measured 8 h after inducing AS aggregation by co-expression of the aggregation-inducing protein p25α (Fig 1A). At this early time point, no overt morphological changes are observed, but early AS aggregation-dependent gene expression responses have been detected like increases in the NF-κB inhibitor, IκBiα 10. Not surprisingly, an increased cytosolic Ca2+ level was measured 24 h after induction of AS aggregation, where cells show visible degenerative changes such as perinuclear microtubule retraction and nuclear NF-κB translocation (Fig EV4B) 10, 15. Both the early decrease and later increase in basal intracellular Ca2+ concentration were due to AS aggregation as they could be inhibited by the AS aggregation inhibitor ASI-1D (Fig 1A) 16. Differentiated non-mitotic SH-SY5Y cells with inducible AS expression displayed a reduction of cytosolic Ca2+ from 170 to 150 nM after 5 days of AS expression and an increase after 10 days when compared to b-gal-expressing control cells. Aggregate inhibitor ASI-1D rescued both the initial reduction and late increase (Fig 1B). The biphasic AS aggregation-dependent cytosolic Ca2+ change also occurs in primary cultures of mouse hippocampal neurons expressing human AS that display a reduction in cytosolic Ca2+ from 150 to 125 nM after 5 days of culture compared to non-transgenic controls and an increase from 150 to 175 nM after 14 days with both phases blocked by ASI-1D (Fig 1C). Hence, using different cell models, we demonstrate a surprising link between early stages of intracellular AS aggregation and cytosolic Ca2+ reduction. In order to gain more insight to Ca2+ dynamics upon accumulation of AS, we measured Ca2+ over time in both the SH-SY5Y model (Fig 1D) and primary hippocampal neurons (Fig 1E). We found that the early phase of decreased cytosolic Ca2+ in SH-SY5Y cells is significant from day 4 until day 8, whereas the later Ca2+ increase is pronounced after day 10. This closely resembles what we see in primary hippocampal neurons where the early phase of reduced Ca2+ is significant between day 5 and 8 and the later phase, with increased Ca2+ occurring after day 12 and being significant at day 15 (Fig 1C and E). The OLN-T40-AS cells, the SH-SY5Y AS cells and primary neurons isolated from AS-transgenic mice are based on over-expression of AS but their protein levels of AS are not many fold higher than in total brain lysate from C57BL/6J wt mice (Fig EV1). In the OLN model, the AS level is double as high as in brain homogenate and in the SH-SY5Y model levels after 5 and 10 days of AS expression are approx. 75 and 150% of total brain homogenate, respectively. Primary neurons from wt mice have approx. 50% of the AS in adult mice brain whereas primary neurons from AS-transgenic mice has approx. 80 and 95% at DIV 5 and DIV 14, respectively. Figure 1. Cellular stress from AS aggregates causes early reduction in cytosolic calcium followed by later increaseCytosolic Ca2+ levels were quantified by the Ca2+ sensor Fura-2 and converted to absolute concentrations using the Fura-2 Calcium Imaging Calibration Kit. The AS aggregation inhibitor ASI-1D (20 μM) was used to validate that phenotypes were aggregate-dependent. Statistical analyses were performed using one-way ANOVA multiple comparisons with Sidak post hoc test. Mitotic OLN-t40-AS cells were transfected with p25α and the fluorescent transfection marker tdTomato. OLN-t40-AS cells transfected with tdTomato and empty expression vectors served as negative controls. Bars display Ca2+ concentrations as mean ± SD, N = 3 (*P = 0.0001, **P = 0.0002, #P = 0.0011, ##P = 0.0033). The average Ca2+ level of individual experiments was calculated by measuring > 50 or more tdTomato expressing cells. Non-mitotic SH-SY5Y cells were generated by treatment with retinoic acid (RA; 10 μM) for 2 days, after which AS expression was induced by removal of doxycycline (dox) and cytosolic Ca2+ measured after 5 days and 10 days of AS expression. See timeline under bars. Cells induced to express β-galactosidase (b-gal) upon dox removal were used as negative controls. Bars display Ca2+ concentrations as mean ± SD, N = 4 (*P = 0.0005, **P = 0.0005, #P = 0.0062, ##P = 0.0055). The average Ca2+ level was calculated by measuring > 200 randomly selected cells in each experiment. Primary hippocampal neurons were isolated from new-born (P0) mice expressing human AS under the mThy1 promoter and wild-type (wt) littermates. Cytosolic Ca2+ was measured after 5 days in vitro (5 DIV) and 14 days in vitro culture (14 DIV). See timeline under graphs. Bars display Ca2+ concentrations as mean ± SD, N = 3 (*P = 0.002, **P = 0.0007, #P = 0.0071, ##P = 0.0455). The average cellular Ca2+ is based on > 500 neurons per experiment. Cytosolic Ca2+ in SHSY5Y cells as in (B) measured every second day. Points represent Ca2+ concentrations as mean ± SD, N = 3 (*P = 0.0377, **P = 0.0057, ***P = 0.03, #P = 0.045, ##P = 0.0229). Cytosolic Ca2+ in primary hippocampal neurons as in (C) measured every third day. Points represent Ca2+ concentrations as mean ± SD, N = 3 (*P = 0.01, **P = 0.0073, #P = 0.0058). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Evaluation of the AS level in cell models used for study About 10 μg total lysate from the different cell models, primary mouse hippocampal neurons from wild-type and AS-transgenic mice (ASO), and total brain homogenate from a wild-type C57BL/6J mouse were resolved by 10–16% SDS–PAGE probed for α-syn (ASY-1), and actin. The level of AS was quantified and normalised to the actin level. The data are presented with the level in total brain homogenate from 4-month-old C57BL/6J mouse arbitrarily numbered as 100. Download figure Download PowerPoint Toxic AS oligomers associate with ER of presymptomatic AS-transgenic mice and in brain fractions from PD brain tissue enriched in ER 17. The free Ca2+ concentration in the ER is approximately 0.5 mM, compared to a concentration of 100 nM in cytosol 18. This steep gradient is maintained by the high-capacity transmembrane P-type Ca2+ ATPase SERCA and is critical for cellular signalling, for example by inositol-1,4,5 tris-phosphate-activated ER channels. Lowering cytosolic Ca2+ requires an active process whereby Ca2+ is removed from the cytosol against steep gradients. We hypothesised that the decreased cytosolic Ca2+ was caused by AS oligomers activating SERCA. To explore this hypothesis, we first conducted a co-immunoprecipitation experiment to test whether toxic AS oligomers could bind SERCA. The AS oligomers used as bait were purified by gel filtration 19, 20 and share aggregate-specific epitopes with AS species in pathological human and mouse brain tissue and insoluble AS filaments 16, 17, 21. Freshly eluted from the gel filtration column, the oligomers reveal heterogeneous morphologies in negative stain transmission electron microscopy. Here, an underlying structure of ribbons with a width of 13 nm form spirals with a diameter of around 20 nm which further displayed a tendency to form closed and open circular structures with a diameter of around 40 nm (Fig 2A). As sources for SERCA we used detergent extracts of (i) SERCA2b-rich membrane fractions from mice brains of C57BL/6 OlaHsd (ASdel) mice, which do not express AS, to avoid interference from endogenous mouse AS (Fig 2B), and (ii) SERCA1a-rich rabbit muscle microsomes (Fig EV2A). The extracts were supplemented with purified recombinant human monomeric and oligomeric AS (or PBS as negative control) followed by co-immunoprecipitation using anti-AS IgG conjugated to Sepharose beads. SERCA displayed preferential binding to oligomeric AS with negligible binding to monomeric AS irrespective of its source being muscle or brain (Figs 2B and EV2A). During the pumping cycle, SERCA exhibits two grossly different conformations, namely a Ca2+-bound E1 state and a low Ca2+-affinity E2 state. Using 5 μM Ca2+ or 1 μM of the SERCA inhibitor, thapsigargin, it was possible to capture SERCA in the E1 and E2 conformations, respectively 22. Co-immunoprecipitation of the conformation-trapped SERCA, and AS revealed that AS oligomers bind preferentially to the Ca2+-bound E1 conformation, although binding to E2 is not completely abolished (Fig 2C and D). The higher binding to the Ca2+-bound E1 state is unlikely to be due to Ca2+ interactions with AS because this binding is of low affinity with a Kd around 0.5 mM 23. This indicates that AS oligomer interaction with SERCA displays a high degree of structural specificity as physiological changes in the SERCA structure are able to modulate the binding strength. Figure 2. The endoplasmic reticulum calcium ATPase, SERCA, is an AS oligomer-interacting protein Transmission electron microscopy of freshly isolated oligomers. Arrowheads appoint heterogeneous population of twisted ribbons with a maximum width of 15 nm. 100 nm scale bar is presented. Purified AS oligomers (O) and monomers (M) were incubated with detergent extract of ASdel mouse brain before being subjected to co-immunoprecipitation (IP) using ASY-1 (anti-AS) and non-immune rabbit IgG (NI IgG) coupled to sepharose. PBS was used as additional negative control. 2% input of each sample was used as input control. The co-IP samples were analysed by immunoblotting anti-SERCA (J15.5) and rabbit polyclonal anti-AS (ASY-1). One representative immunoblot of three independent experiments is shown. The Ca2+-bound E1-state of SERCA was stabilised by 5 μM free Ca2+, and the Ca2+-free E2-state was stabilised by 1 μM thapsigargin. A representative blot of three replicates is shown. Quantification of immunoprecipitation experiments in (C). Bars represent geometric mean ± 95% CI of SERCA signal relative to the AS signal. N = 3 (Wilcoxon signed rank test, *P = 0.0382). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Aggregated AS interacts with SERCA.The interaction is inhibited by aggregate inhibitor, ASI-1D, when analyzed by proximity ligation assay. The inhibition of interaction is not due to ASI-1D blocking the anti-AS antibody quenching the proximity ligation signal. Aggregated AS interacts with SERCA 1A. Purified AS oligomers (O) and monomers (M) were incubated with detergent extract of SERCA1a from sarcoplasmic reticuli isolated from rabbit muscle before being subjected to co-immunoprecipitation (co-IP) using anti-AS (AS) and non-immune rabbit IgG (NI) coupled to sepharose. PBS was used as additional negative control. 2% input of each sample was used as input control. The co-IP samples were analysed by immunoblotting using rabbit polyclonal anti-SERCA (J15.5) and rabbit polyclonal anti-AS (ASY-1). A representative blot of three replicates is shown. ELISA shows that the aggregation inhibitor ASI-1D does not bind the primary anti-AS antibody and thereby quench the proximity ligation assay. The aggregation inhibitor ASI-1D was able to completely abolish the signal from the proximity ligation assay (Fig 3B); therefore, we tested if the binding of the Syn211 antibody to AS can be inhibited by the aggregate inhibitor ASI-1D. An ELISA assay with 0.7 nM biotinylated AS was developed to test whether increasing amounts of ASI-1D can compete with biotinylated AS bound to Syn211. Increasing amounts of ASI-1D cannot compete against the binding of biotinylated AS to Syn211 even at a concentration of 20 μM. As control for the competition, increasing amounts of non-biotinylated AS were tested, and a concentration of 7 nM AS resulted in ˜20% less bound biotinylated AS, and 35 nM AS reduced the biotinylated AS to ˜30%. This shows that the effect of the aggregate inhibitor in our PLA assay is not caused by inhibition of the binding between Syn211/AS by ASI-1D. Bars represent mean absorbance ± SD (Kruskal–Wallis one-way rank test with Dunn's post hoc test, *P = 0.001). N =
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