ALS / FTD ‐associated FUS activates GSK ‐3β to disrupt the VAPB – PTPIP 51 interaction and ER –mitochondria associations
2016; Springer Nature; Volume: 17; Issue: 9 Linguagem: Inglês
10.15252/embr.201541726
ISSN1469-3178
AutoresRadu Stoica, Sébastien Paillusson, Patricia Gómez‐Suaga, Jacqueline C. Mitchell, Dawn H. W. Lau, Emma H. Gray, Rosa M. Sancho, Gema Vizcay‐Barrena, Kurt J. De Vos, Christopher E. Shaw, Diane P. Hanger, Wendy Noble, Christopher C.J. Miller,
Tópico(s)Amyotrophic Lateral Sclerosis Research
ResumoArticle14 July 2016Open Access Transparent process ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB–PTPIP51 interaction and ER–mitochondria associations Radu Stoica Radu Stoica Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Sébastien Paillusson Sébastien Paillusson Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Patricia Gomez-Suaga Patricia Gomez-Suaga Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Jacqueline C Mitchell Jacqueline C Mitchell Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Dawn HW Lau Dawn HW Lau Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Emma H Gray Emma H Gray Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Rosa M Sancho Rosa M Sancho Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Gema Vizcay-Barrena Gema Vizcay-Barrena Centre for Ultrastructural Imaging, King's College London, London, UK Search for more papers by this author Kurt J De Vos Kurt J De Vos orcid.org/0000-0003-2161-6309 Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Christopher E Shaw Christopher E Shaw Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Diane P Hanger Diane P Hanger Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Wendy Noble Wendy Noble Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Christopher CJ Miller Corresponding Author Christopher CJ Miller orcid.org/0000-0002-5130-1845 Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Radu Stoica Radu Stoica Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Sébastien Paillusson Sébastien Paillusson Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Patricia Gomez-Suaga Patricia Gomez-Suaga Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Jacqueline C Mitchell Jacqueline C Mitchell Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Dawn HW Lau Dawn HW Lau Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Emma H Gray Emma H Gray Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Rosa M Sancho Rosa M Sancho Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Gema Vizcay-Barrena Gema Vizcay-Barrena Centre for Ultrastructural Imaging, King's College London, London, UK Search for more papers by this author Kurt J De Vos Kurt J De Vos orcid.org/0000-0003-2161-6309 Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Christopher E Shaw Christopher E Shaw Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Diane P Hanger Diane P Hanger Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Wendy Noble Wendy Noble Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Christopher CJ Miller Corresponding Author Christopher CJ Miller orcid.org/0000-0002-5130-1845 Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK Search for more papers by this author Author Information Radu Stoica1,‡, Sébastien Paillusson1,‡, Patricia Gomez-Suaga1, Jacqueline C Mitchell1, Dawn HW Lau1, Emma H Gray1,3, Rosa M Sancho1,4, Gema Vizcay-Barrena2, Kurt J De Vos1,5, Christopher E Shaw1, Diane P Hanger1, Wendy Noble1 and Christopher CJ Miller 1 1Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, UK 2Centre for Ultrastructural Imaging, King's College London, London, UK 3Present address: Multiple Sclerosis Society, London, UK 4Present address: Alzheimer's Research UK, Cambridge, UK 5Present address: Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 207 8480393; Fax: +44 207 7080017; E-mail: [email protected] EMBO Reports (2016)17:1326-1342https://doi.org/10.15252/embr.201541726 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 Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca2+ uptake by mitochondria following its release from ER stores, which is a physiological read-out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS-expressing cells; mitochondrial ATP production is linked to Ca2+ levels. Finally, we demonstrate that the FUS-induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase-3β (GSK-3β), a kinase already strongly associated with ALS/FTD. Synopsis This study shows that FUS, by disrupting ER and mitochondria associations, regulates calcium uptake into and ATP production by mitochondria, with implications for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). FTD/ALS-associated fused in sarcoma (FUS) disrupts ER–mitochondria associations and links calcium homoeostasis and mitochondrial ATP production. FUS-induced disruption of ER–mitochondria associations involves activation of GSK-3β and breaking of the VAPB–PTPIP51 ER–mitochondria tethering proteins. Introduction Mitochondria and the endoplasmic reticulum (ER) form tight structural associations such that ~5–20% of the mitochondrial surface is closely apposed (10–30 nm distances) to specialized regions of the ER termed mitochondria-associated ER membranes (MAM) 123. These associations regulate a number of fundamental physiological functions including Ca2+ and phospholipid exchange between the two organelles, energy metabolism and ATP production, mitochondrial biogenesis and trafficking, apoptosis, ER stress responses and autophagy 123. The mechanisms by which MAM become associated with mitochondria are not properly understood but electron microscopy (EM) studies reveal the presence of structures that appear to tether the two organelles 4. Recently, the integral ER protein, vesicle-associated membrane protein-associated protein B (VAPB), was shown to bind to the outer mitochondrial membrane protein, protein tyrosine phosphatase interacting protein 51 (PTPIP51), to form at least some of these tethers 56. Thus, modulating VAPB and/or PTPIP51 expression induces appropriate changes in ER–mitochondria contacts and also Ca2+ exchange between the two organelles, which is a physiological read-out of ER–mitochondria contacts 56. Many of the functions regulated by ER–mitochondria contacts are perturbed in ALS and associated FTD. ALS is the most common form of motor neuron disease, and FTD is the second most common form of presenile dementia after Alzheimer's disease 7. These diseases are now known to be clinically, genetically and pathologically linked 78. Thus, damage to mitochondria, ATP production, Ca2+ homoeostasis, lipid metabolism, axonal transport, autophagy and the ER including activation of the unfolded protein response (UPR) are all features of ALS/FTD, and ER–mitochondria communications impact upon all of these processes 123910. Indeed, recent studies have demonstrated that some ALS/FTD-associated insults disrupt ER–mitochondria contacts and associated functions. These include the expression of Tar DNA-binding protein-43 (TDP-43) and loss of Sigma-1 receptor (Sigma-R1) function 611. Mutations in both TDP-43 and Sigma-R1 cause some familial forms of ALS/FTD and accumulations of TDP-43 are a major pathology of ALS/FTD 1213141516. Defects in fused in sarcoma (FUS) metabolism are strongly implicated in both ALS and FTD. FUS accumulations are a pathological feature in a significant number of ALS/FTD cases, mutations in FUS cause some familial forms of ALS and FTD, and overexpression of wild-type and ALS/FTD-mutant FUS induces aggressive disease in transgenic rodents 71718192021222324252627. FUS is a predominantly nuclear protein where it functions in DNA repair, transcription and splicing but a proportion is also normally present in the cytoplasm 2627. However, the mechanisms by which FUS induces disease are not clear and both gain and loss of function hypotheses have been proposed 2627. Here, we show that the expression of both wild-type and ALS-mutant FUS disrupt ER–mitochondria associations and that this is accompanied by reductions in binding of VAPB to PTPIP51. We also demonstrate that FUS perturbs cellular Ca2+ homoeostasis and mitochondrial ATP production. Damage to mitochondria is strongly linked to ALS 28293031323334. Finally, we show that FUS activates glycogen synthase kinase-3β (GSK-3β) and that GSK-3β is a regulator of ER–mitochondria associations. GSK-3β is already strongly implicated in ALS/FTD 6353637. Thus, our findings reveal a new pathogenic mechanism for FUS involving activation of GSK-3β and disruption to ER–mitochondria associations. Results Wild-type and mutant FUS disrupt ER–mitochondria associations and the VAPB–PTPIP51 interaction To determine the effects of FUS on ER–mitochondria associations, we quantified ER–mitochondria contacts in NSC34 motor neuron cells transfected with either enhanced green fluorescent protein (EGFP) control vector, EGFP-FUS or familial ALS mutants EGFP-FUSR521C or EGFP-FUSR518K. Several previous studies have utilized EGFP-tagged FUS 2438 but to confirm that the EGFP-FUS was functional, we monitored the expression of endogenous FUS 72 h post-transfection. FUS displays an autoregulatory function such that overexpression by transfection reduces endogenous gene expression 38. At this time point, we detected a marked decrease in endogenous FUS expression in both wild-type and mutant EGFP-FUS-transfected cells (Fig EV1). These findings are in agreement with previous studies, which also showed that the EGFP tag does not affect the autoregulatory function of FUS 38. Click here to expand this figure. Figure EV1. Expression of EGFP-FUS reduces the expression of endogenous FUSHEK293 cells were transfected with control EGFP, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and 72 h post-transfection, the samples were probed on immunoblots for FUS (using FUS antibody) and tubulin as a loading control. Download figure Download PowerPoint The EGFP tags were then used to isolate transfected cells using a cell sorter and ER–mitochondria associations quantified by determining the proportion of the mitochondrial surface that was closely apposed (< 30 nm) to ER following analyses by EM. This approach has been used previously 4639. Transfection of FUS did not lead to changes in the expression of the ER–mitochondria tethering proteins VAPB or PTPIP51, or mitofusin-2, which has been proposed as a further ER–mitochondria tether 40 (Fig 1A). Moreover, we detected no change in the numbers of mitochondria or ER profiles in the presence of either wild-type or mutant FUS. However, compared to control cells, the expression of wild-type and mutant FUS all led to significant reductions in ER–mitochondria associations (Fig 1B). Figure 1. Expression of wild-type and ALS/FTD-mutant FUS reduces ER–mitochondria associations in NSC34 cells A. Expression of FUS does not alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in transfected NSC34 cells. Immunoblots of NSC34 cells transfected with EGFP as a control (CTRL), or wild-type or mutant EGFP-FUS. Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated. On the FUS immunoblot, samples were probed with FUS antibody to show endogenous and transfected proteins; tubulin is shown as a loading control. B. Representative electron micrographs of ER–mitochondria associations in NSC34 cells transfected with control EGFP vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K as indicated; arrowheads with loops show regions of association. Scale bar = 200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. N = 27–30 cells and 247–424 mitochondria, error bars are s.e.m.; ***P < 0.001. C, D. siRNA loss of FUS does not affect ER–mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (C) Immunoblots of cells either mock transfected or treated with control (CTRL) or FUS siRNAs; GAPDH is shown as a loading control. (D) Representative electron micrographs of ER–mitochondria associations in control (CTRL) and FUS siRNA-treated cells. Arrowheads with loops show regions of association. Scale bar = 200 nm. Data analysed by unpaired t-test. N = 27–28 cells and 193–202 mitochondria, error bars are s.e.m. Download figure Download PowerPoint We also enquired whether loss of FUS influenced ER–mitochondria associations. To do so, we treated NSC34 cells with control or FUS siRNAs and again monitored ER–mitochondria associations by EM. siRNA knockdown of FUS did not alter the expression of VAPB, PTPIP51 or mitofusin-2, and loss of FUS had no effect on ER–mitochondria associations (Fig 1C and D). To complement the EM studies, we monitored the effects of FUS on ER–mitochondria associations using super resolution structured illumination microscopy (SIM) 41. NSC34 cells were again transfected with EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and then immunostained for protein disulphide isomerase (PDI) and translocase of the outer mitochondrial membrane protein-20 (TOM20) to label ER and mitochondria, respectively. Co-localization analyses of PDI and TOM20 revealed that wild-type and mutant FUS again disrupted ER–mitochondria associations (Fig 2A). Figure 2. Expression of wild-type and mutant FUS reduces ER–mitochondria associations and the VAPB–PTPIP51 interaction in NSC34 cells FU-induced reductions in ER–mitochondria associations can be detected using SIM. NSC34 cells were transfected with either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and immunostained for TOM20 and PDI to label mitochondria (Mito) and ER, respectively; FUS was detected via their EGFP tags. Merge (ZOOM) shows zoomed images of boxed regions, and co-localization shows co-localized pixels. Scale bar = 2 μm. Bar chart shows ER–mitochondria co-localization (Manders coefficient) normalized to control in the different samples. Data were analysed by one-way ANOVA with Tukey's post hoc test. A total of 10–14 cells were analysed per condition from three independent experiments; error bars are s.e.m., **P < 0.01 and ***P < 0.001. ER–mitochondria associations and the VAPB–PTPIP51 interaction are disrupted by wild-type and ALS/FTD-mutant FUS. NSC34 cells were transfected with EGFP control vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies. FUS were detected via their EGFP tags. Scale bar = 10 μm. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey's post hoc test; n = 47–53 cells from five experiments. Error bars are s.e.m.; ***P < 0.001. Overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells. Cells were transfected as indicated with either control empty vector (CTRL), HA-PTPIP51 + CTRL, or HA-PTPIP51 + either EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA-PTPIP51. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals. Data were analysed by one-way ANOVA and Tukey's post hoc test; N = 4. Error bars are s.e.m.; ***P < 0.001. Download figure Download PowerPoint Finally, we used in situ proximity ligation assays 42 to monitor the effects of FUS on ER–mitochondria associations. Here, conventionally fixed cells and tissues are probed with primary antibodies followed by secondary antibodies coupled to specific oligonucleotides. If the distances between antigens are small enough, these oligonucleotides facilitate hybridization and ligation of connector oligonucleotides to form a circular DNA molecule, which then serves as a template for rolling circular amplification. Use of labelled nucleotides enables microscopic detection and quantification of hybridization signals. The distances detected by proximity ligation assays are similar to those detected by resonance energy transfer between fluorophores (i.e. ~10 nm) 42. For these assays, we used antibodies to the ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 since VAPB and PTPIP51 interact directly to tether ER with mitochondria 56. Proximity ligation assays have already been used to quantify ER–mitochondria associations and the VAPB–PTPIP51 interaction 51143. To demonstrate the specificity of the proximity ligation assays, we first performed experiments involving omission of VAPB, PTPIP51 or both VAPB and PTPIP51 primary antibodies. In agreement with previous studies 5, omission of VAPB and/or PTPIP51 antibodies produced very few signals whereas inclusion of these primary antibodies generated robust signals (Fig EV2A). We then monitored how FUS expression affected the VAPB–PTPIP51 interaction. Compared to control EGFP, transfection of either wild-type or mutant FUS did not affect the size of the cells (% size: EGFP 100 ± 5.93, EGFP-FUS 96.86 ± 4.57, EGFP-FUSR521C 102 ± 5.3, EGFP-FUSR518K 112.9 ± 5.9; analysed by one-way ANOVA). However, compared to controls, reduced interactions between VAPB and PTPIP51 were detected in both wild-type and mutant FUS-transfected NSC34 cells (Fig 2B). Thus, in three different assays, wild-type and ALS/FTD-mutant FUS disrupt ER–mitochondria associations. Click here to expand this figure. Figure EV2. Control experiments involving omission of primary antibodies demonstrate the specificity of the VAPB-PTPIP51 proximity ligation assays A, B. Panel (A) shows NSC34 cells; panel (B) shows mice spinal cords. Samples were probed with no primary antibodies (no Abs), VAPB only, PTPIP51 only or VAPB + PTPIP51 antibodies. In (A) samples are counterstained with DAPI to show nuclei. Scale bar = 10 μm (A) and 30 μm (B). Bar charts show proximity signals/cell. Data were analysed by one-way ANOVA and Tukey's post hoc test. N = 16 cells (A) and 12 cells (B), error bars are s.e.m.; ***P < 0.001. Download figure Download PowerPoint As a complement to these cellular studies, we investigated ER–mitochondria associations in spinal cord motor neurons of 10-week-old homozygous FUS transgenic mice and their non-transgenic littermates 19. These transgenic mice express wild-type FUS under control of the commonly used mouse prion gene regulatory elements, which drive expression to a variety of neural cell types including motor neurons 19. The homozygous FUS transgenic mice develop aggressive features of ALS including hindlimb paralysis and death by 12 weeks of age 19. Similar to the findings in NSC34 cells, no changes in the expression of VAPB, PTPIP51 or mitofusin-2 were detected in FUS transgenic mice (Fig 3A). However, EM analyses revealed that compared to non-transgenic control littermates, ER–mitochondria associations were significantly reduced in spinal cord motor neurons of the FUS transgenics (Fig 3B). We also utilized VAPB/PTPIP51 proximity ligation assays to quantify ER–mitochondria contacts in spinal cord motor neurons of the FUS transgenic mice. Again, we first tested the specificity of the assays by performing control experiments in which VAPB, PTPIP51 or both VAPB and PTPIP51 primary antibodies were omitted and these revealed that omission of primary antibodies produced very few signals but their inclusion generated robust signals (Fig EV2B). Compared to control non-transgenic littermates, we detected no differences in motor neuron size in the FUS transgenics (% size: control 100 ± 4.96, FUS 95.65 ± 5.32; analysed by unpaired t-test). However, the VAPB/PTPIP51 proximity ligation assays revealed that in agreement with the EM analyses, ER–mitochondria associations were significantly reduced in spinal cord motor neurons of the FUS transgenics (Fig 3C). Figure 3. Overexpression of FUS reduces ER–mitochondria associations and the VAPB–PTPIP51 interaction in spinal cords of FUS transgenic mice Overexpression of FUS does not affect expression of VAPB, PTPIP51 or mitofusin-2. Immunoblots of spinal cord proteins from three 10-week-old FUS transgenic mice and three age-matched littermates are shown; FUS was detected via its HA tag. Tubulin was used as a loading control. Representative electron micrographs of ER–mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice and their non-transgenic littermates; arrowheads with loops show regions of association. Scale bar = 200 nm. Bar chart shows % of mitochondrial surface closely apposed to ER in the two samples. Data were analysed by unpaired t-test. N = 67–88 cells and 438–749 mitochondria, error bars are s.e.m.; ***P < 0.001. ER–mitochondria associations and the VAPB–PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice. Representative images of proximity ligation signals in 11-week-old FUS and non-transgenic (NTg) littermate mice. Data were analysed by unpaired t-test; N = 40 cells from 3 FUS and 3 non-transgenic littermates (age 10 weeks). Error bars are s.e.m.; ***P < 0.001. Download figure Download PowerPoint Since we detected no FUS-induced changes in expression of VAPB or PTPIP51 in either NSC34 cells or transgenic mice (Figs 1A and C, and 3A), the proximity ligation assays not only confirm that FUS reduces ER–mitochondria associations but also show that this reduction involves breaking of the VAPB–PTPIP51 tethers. To test this further, we performed immunoprecipitation assays to monitor the affect of FUS on binding of VAPB to PTPIP51. To do so, we co-transfected cells with haemagglutinin (HA)-tagged PTPIP51 and either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and monitored the amounts of VAPB bound to immunoprecipitated PTPIP51-HA by immunoblotting of the samples. Consistent with the proximity ligation assays, both wild-type and mutant FUS decreased the amounts of endogenous VAPB bound to immunoprecipitated PTPIP51-HA in these assays (Fig 2C). Thus, the FUS-induced reductions in ER–mitochondria associations involve decreased interactions between the tethering proteins VAPB and PTPIP51. Overexpression of FUS disturbs cellular Ca2+ homoeostasis and mitochondrial ATP production A major function of ER–mitochondria associations is to regulate cellular Ca2+ homoeostasis and in particular, to facilitate Ca2+ exchange between ER and mitochondria following Ca2+ release from ER stores 123440. Thus, disruption of the VAPB–PTPIP51 interaction perturbs cellular Ca2+ homoeostasis 56. Since expression of wild-type or mutant FUS reduced both ER–mitochondria and VAPB–PTPIP51 associations, we monitored the effect of overexpressing FUS on cytosolic and mitochondrial Ca2+ levels after induction of inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ release from ER stores. For these experiments, we used HEK293 cells co-transfected with the M3 muscarinic acetylcholine receptor (M3R) and either control vector or FUS and triggered physiological IP3 receptor-mediated Ca2+ release from ER stores by application of the M3R agonist oxotremorine-M. In line with previous studies on VAPB, PTPIP51 and ER–mitochondria associations, we used HEK293 cells for these experiments since they do not express endogenous M3R and so provide a useful model for monitoring cytosolic and mitochondrial Ca2+ levels after its release from ER specifically in transfected cells 56. Transfection of both wild-type and mutant FUS all induced significant increases in cytosolic and decreases in mitochondrial Ca2+ levels (Fig 4A and B). Such findings are consistent with the observed FUS-induced decreases in ER–mitochondria and VAPB–PTPIP51 interactions. Figure 4. Expression of FUS disrupts cellular Ca2+ homoeostasis and mitochondrial ATP production A, B. FUS disrupts Ca2+ homoeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. Panel (A) shows cytosolic Ca2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolic Ca2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolic Ca2+ levels. Panel (B) shows mitochondrial Ca2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey's post hoc test. (A) N = 49–52 cells from three experiments; (B) N = 50–52 cells from five experiments, error bars are s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. C. FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples. Data were analysed by one-way ANOVA and Tukey's post hoc test. N = 29–54 cells from five experiments, error bars are s.e.m.; *P < 0.05, ***P < 0.001. Download figure Download PowerPoint Damage to mitochondrial ATP production is a prominent feature of ALS 9333444. Ca2+ is required by mitochondria for generating ATP via the tricarboxylic acid cycle 45, and so, the reduced mitochondrial Ca2+ levels seen in FUS-overexpressing cells predict that FUS impairs mitochondrial ATP production. We therefore monitored the effect of FUS-overexpression on mitochondrial ATP production using a FRET r
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