Local administration of glucocorticoids decreases synovial citrullination in rheumatoid arthritis

Dimitrios Makrygiannakis
Shankar Revu
Marianne Engström
Erik Af Klint
Anthony P Nicholas
Ger Jm Pruijn
Anca I Catrina
Local administration of glucocorticoids decreases synovial citrullination in rheumatoid arthritis
R E S E A R C H A R T I C L E Open Access
Introduction: Protein citrullination is present in the rheumatoid synovium, presumably contributing to the perpetuation of chronic inflammation, in the presence of specific autoimmunity. As a result, the present study examined the possibility that effective antirheumatic treatment will decrease the level of synovial citrullination.Methods: Synovial biopsies were obtained from 11 rheumatoid arthritis (RA) patients before and after 8 weeks of treatment with 20 mg methotrexate weekly, 15 RA patients before and 2 weeks after an intraarticular glucocorticoid injection, and eight healthy volunteers. Synovial inflammation was assessed with double-blind semiquantitative analysis of lining thickness, cell infiltration, and vascularity by using a 4-point scale. Expression of citrullinated proteins (CPs) with the monoclonal antibody F95 and peptidylarginine deiminase (PAD) 2 and 4 was assessed immunohistochemically with double-blind semiquantitative analysis. In vitro synovial fluid (SF), peripheral blood (PB), mononuclear cells (MCs), and synovial explants obtained from RA patients were incubated with dexamethasone and analyzed with immunohistochemistry for expression of CP as well as PAD2 and PAD4 enzymes. Results: The presence of synovial CP was almost exclusive in RA compared with healthy synovium and correlated with the degree of local inflammation. Treatment with glucocorticoids but not methotrexate alters expression of synovial CP and PAD enzymes, in parallel with a decrease of synovial inflammation. Ex vivo and in vitro studies suggest also a direct effect of glucocorticoids on citrullination, as demonstrated by the decrease in the level of citrullination and PAD expression after incubation of SFMC and synovial explants with dexamethasone. Conclusion: Synovial citrullination and PAD expression are dependent on local inflammation and targeted by glucocorticoids.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the presence of highly specific anti-citrullinated protein antibodies (ACPAs) [1]. These antibodies recognize several different proteins that are citrullinated. Citrullination is the conversion of peptidylarginine to peptidylcitrulline through a calcium-dependent process catalyzed by the peptidylarginine deiminase (PAD) enzymes. Five PAD isotypes have been described in humans (PAD1, PAD2, PAD3, PAD4, and PAD6), which are expressed in a variety of tissues, but only PAD2 and PAD4 have been found to be expressed in inflamed synovial tissue of RA and other inflammatory arthritides [2].

Despite the high specificity of ACPA in RA in comparison to other arthritides and other inflammatory diseases [3], the presence of CP is not restricted to RA synovial tissue [4,5], but rather associated with inflammation in general [6]. Synovial citrullination appears therefore not to be essential for the predisease phase of induction of specific anti-citrulline immunity. Conversely, protein citrullination enhances the HLA binding capacity of synovial-derived proteins and promotes NF-B and tumor necrosis factor (TNF) production in the presence ACPA [7]. This suggests that local synovial citrullination might be essential in a later phase of the disease process, contributing to occurrence and perpetuation of chronic synovitis in the presence of specific anti-citrulline antibodies. It is therefore conceivable that downregulation of synovial citrullination by any means will contribute to the resolution of local inflammation. We hypothesize that effective antirheumatic treatment with either antiinflammatory, intraarticular glucocorticoids (GCs), or a disease-modifying antirheumatic drug, such as methotrexate (MTX) will decrease synovial citrullination in vivo. As a result, the present study aimed to investigate any direct effect of these drugs on protein citrullination.

Materials and methods

Patients Twenty-six patients meeting the 1987 American College of Rheumatology criteria for RA [8] were recruited for this study. In a first group, 11 patients (six women and five men; median age, 56 years; range, 33 to 78 years) with newly diagnosed RA (symptom duration less than 1 year) previously disease-modifying antirheumatic drug (DMARD) naïve were started on MTX, 10 mg weekly, and reached a stable dose of 20 mg after 2 weeks, increasing the dose with 5 mg every week. Synovial biopsy samples were obtained by arthroscopy from all patients before and after a median of 8 weeks of treatment. Clinical evaluation of the therapeutic response according to EULAR response criteria was performed a median of 3 months after methotrexate initiation. In a second group, 15 RA patients (11 women and four men; median age, 63 years; range, 34 to 83 years) with active knee arthritis independent of disease duration received an intraarticular injection of 40 mg of triamcinolone hexacetonide, and synovial biopsy samples were obtained with arthroscopy before and after 2 weeks after intraarticular treatment. In this second group, associated DMARD treatments were stable for at least 2 weeks before initiation of treatment and throughout the entire study period. Clinical evaluation of the therapeutic response was performed with macroscopic scoring of the level of inflammation during arthroscopy. Nonsteroidal antiinflammatory drugs and per-oral prednisolone to a maximum dose of 10 mg daily were permitted in both groups. Additionally, synovial biopsy samples were obtained with arthroscopy in eight healthy volunteers. All procedures were approved by the Northern Stockholm Ethical Review Board, and informed consent was obtained from all the participants in the study.

Synovial biopsies handling

Synovial biopsy samples were snap-frozen during arthroscopy in dry-ice cooled isopentane. Serial cryostat sections (7 μm) were fixed for 20 minutes with 2% (vol/vol) formaldehyde and stored at -70°C.

Cell culturing

Paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from RA patients (n = 6) were cultured as duplicates in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin (Gibco, Carlsbad, CA, USA) and incubated at 37°C with 5% CO 2 in a humidified atmosphere. Cells were grown at a density of 1 × 10 6 per ml in polypropylene tubes and incubated with dexamethasone for 24 hours at a final concentration of 1 and 100 μM DXM. After treatment, cells were washed, resuspended in PBS, and seeded on glass slides (Thermo Scientific diagnostic microscopic glass slides, Braunschweig, Germany). Adherent cells were fixed for 20 minutes at 4°C with 2% (vol/vol) formaldehyde (Merck, Darmstadt, Germany).

Synovial explants

Synovial tissue pieces obtained from an orthopedic hipreplacement surgery in an RA patient were selected based on maximal macroscopic score of inflammation, dissected and seeded on 24-well plates in RPMI, supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin (Gibco), and incubated at 37°C with 5% CO 2 in a humidified atmosphere for 24 hours with 100 μM DXM. Tissue pieces were then transferred to six-well plates and cultured for additional 5 days with fresh prepared serum supplemented with the same concentrations of the drugs. At the end of the incubation, medium was removed, and tissue pieces were cryosectioned, fixed for 20 minutes at 4°C with 2% (vol/vol) formaldehyde (Merck), and stored at -80°C.

Immunohistochemistry and histologic evaluation

Presence of citrullinated proteins (CPs) was detected by using a mouse IgM monoclonal antibody (F95), which was raised against a decacitrullinated peptide linked to the carrier protein, keyhole limpet hemocyanin [9][10][11]. For detection of PAD enzyme expression, we used two PAD4 (SN823 and SG1467) [12,13] and one PAD2 (SN665) [12] rabbit polyclonal antibodies and one additional rabbit polyclonal antibody (ROI002) (CosmoBio, Tokyo, Japan) to identify PAD2 expression [14]. Appropriate negative controls were used for each antibody.

Two independent observers (DM and AIC), who were unaware of the sample's identity, scored the presence of CP and PAD enzymes in synovial tissue by using a 4point scale (0, no staining; 1, low amounts of staining; 2, moderate amounts of staining; 3, high amounts of staining). In parallel, histologic scoring of the degree of inflammation was performed, by using a 4-point scale, by double-blind semiquantitative analysis (DM and SR) of the lining thickness, infiltration level, and vascularity in serial hematoxylin eosin staining. In every instance, the final scores represent the mean of the two observations. The presence of CP and PAD enzymes in SFMCs and PBMCs was assessed with manual counting of positive cells and expressed as a percentage of positive cells in the total number of the cells.

Statistical analysis

Statistical analysis was performed by using the Wilcoxon test for comparison of paired samples, the Mann-Whitney test for comparison of independent samples, and the Spearmen rank correlation test. All comparisons were planned, and therefore, no Bonferroni correction was applied. Differences between proportions were analyzed with the Fisher Exact test. In vitro experiments results were analyzed by using one-way (ANOVA) analysis of variance, as appropriate. P values less than 0.05 were considered statistically significant.

Results

Intracellular but not extracellular presence of citrullinated peptides is RA specific

It was previously suggested that the intracellular pattern of F95 staining is a specific trait of RA as compared with healthy synovium [11]. Further to investigate this, we analyzed the immunohistochemical expression of citrullinated peptides (as detected by F95 antibody) and the PAD2 and PAD4 enzymes in baseline RA samples obtained before initiation of treatment, as compared with healthy synovial biopsies. Intracellular F95 staining was readily detected in RA samples (24 of 28, 85.7%) and absent in healthy synovium (none of eight; P < 0.05). Extracellular F95 staining was present in RA (24 of 28, 85.7%) and to a lesser extent in healthy synovial tissue (one of eight, 12.5%; P < 0.05). PAD2 and PAD4 expression was detected in all baseline RA samples (100%) and a vast majority of the healthy synovial biopsies (seven of eight, 87.5% positive for ROI002 antibody; six of eight, 75% positive for SN665 antibody; all eight, 100% positive for SG1467 antibody, and seven of eight, 87.5% positive for SN8233 antibody) and with a general higher expression of all investigated molecules in RA as compared with healthy synovium (Figure 1; P < 0.05 for all investigated antibodies).

Synovial citrullination and PAD expression correlate with the degree of inflammation

To investigate further the relation between inflammation and citrullination, we analyzed the correlation between the presence of total and intracellular CP, as well as PAD enzymes, and the degree of local inflammation in baseline samples obtained from all RA patients and healthy individuals (n = 34). As expected, most of the investigated markers correlated with the degree of lining thickness, cell infiltration, and vascularity (Table 1).

Intraarticular GC, but not MTX, decreases synovial citrullination and PAD4 expression

The observed correlation between inflammation and citrullination prompted us to investigate the effect of antirheumatic treatment on synovial citrullination and PAD expression.

RA synovium

Healthy synovium Local administration of GC significantly decreased both total and intracellular expression of CP from a median score of 2 (range, 0 to 3) to a median score of 1 (range, 0 to 2), P < 0.05. PAD4 expression also decreased after treatment from a median score of 2 (range, 1 to 3) to a median score of 1 (range, 0 to 2.5), P < 0.05, when evaluated with the SG1467 antibody and to a lesser extent when evaluated with the SN823 antibody (P = 0.06), whereas no changes were observed for PAD2 expression after intraarticular administration of GCs ( Figure 2). The GC effect on citrullination and PAD expression was paralleled by a significant decrease (P < 0.05) in the lining thickness (from a median score of 1, range, 0 to 2, to a median score of 0.5, range, 0 to 1) and cell infiltration (from a median score of 3, range, 2 to 3, to a median score of 2, range, 1 to 3), but not vascularity. All patients were good clinical responders, as evaluated with macroscopic investigation of the joint inflammation at the time of arthroscopy and retrospective scoring of photo records.

In contrast, methotrexate treatment had no effect on either synovial inflammation or local expression of CP and PAD (Figure 3), despite a good clinical response in nine of 11 treated patients with a significant decrease in the DAS28 score from a mean ± SEM of 5.5 ± 0.3 to a mean ± SEM of 3.4 ± 0.4 after 3 months of treatment. Interestingly, the same was true when only EULAR responders (n = 9) were included in the analysis.

In vitro GCs have a direct effect on cellular expression of CP, potentially through a PAD-dependent mechanism Apart from their inflammation-damping effect, GCs may also directly target the process of citrullination. To investigate this, we tested the effect of DXM on CP and PAD enzymes expression in vitro in SFMC and PBMC paired samples of RA individuals. Confirming our in vivo results, DXM decreased the expression of CP, PAD4, but also PAD2, in SFMC ( Figure 4) but not PBMC (data not shown). The effect of DXM is dose dependent and present at doses as low as 1 μM ( Figure 5). No such effects were observed for methotrexate when tested in the same systems (Additional file 1).

GC effect can be reproduced ex vivo in synovial explants biopsies

SFMCs are in vitro surrogate replacements of the synovial biopsy complex milieu, although lacking important characteristics, such as the local complex intracellular interplay. To circumvent this caveat, we established explants from a synovial biopsy obtained from an RA patient at open surgery for hip replacement and tested the effect of GCs to confirm our results further. DXM treatment of the explants decreased the expression of CP and PAD4 but not PAD2, whereas parallel histologic investigation did not reveal major changes in the histologic composition of the samples. Once again, no such effects were observed for methotrexate (Additional file 2).

Discussion

Protein modification through posttranslational citrullination in the rheumatoid joint is thought to play an important role in perpetuation of local chronic inflammation in the presence of specific anticitrulline immunity. This is the first report showing that synovial citrullination is actively modulated by antirheumatic treatment.

Previous studies characterized synovial expression of citrullination, showing a general increase in the level of citrullination in the presence of active inflammation [2,[4][5][6]11,15]. These early reports used one particular method for detection of citrullination, consisting of the chemical modification of the citrullinated tissue proteins to allow their recognition by an antibody raised against similarly modified citrullinated proteins [16]. However, this technique is no longer available, and in our hands, the only other antibody performing well in immunohistochemistry is F95 [10]. As with the modified anti-citrulline antibody, F95 is supposed to recognize a large array of citrullinated molecules independent of the amino acid context. We previously demonstrated that this antibody is able to recognize specifically at least one RA-relevant citrullinated antigen, citrullinated fibrinogen [17]. However, a somewhat more restricted pattern of citrullination, as compared with the modified anti-citrulline antibodies, has been observed both in synovial biopsies [11] and in other tissues, such as lungs of smoking healthy individuals [18]. By using this new antibody, De Rycke et al. [11] were able to describe an RA-specific intracellular F95 staining. We confirm this finding by the virtually absence of F95positive cells in synovial biopsies of healthy individuals.

Only few reports of PAD synovial expression are currently available [2,11,19]. The most thorough investigation of synovial PAD expression was performed by Foulquier et al. [2], showing correlation between expression of both PAD2 and PAD4 expression with the degree of synovial inflammation. We confirm these findings and identify the same correlation for the CP expression.  Hematoxylin-eosin (HTX-EOS) staining illustrates the histologic pattern of frozen synovial biopsy sections of an RA patient before (a) and after (b) intraarticular GC treatment. Brown diaminobenzidine immunoperoxidase staining shows a significant decrease in the expression of citrullinated proteins, as detected by F95 antibody (c, before treatment; d, after treatment), no changes in PAD2 expression as detected by either ROI001 (f, before treatment; g, after treatment) antibody or SN665 antibody (i, before treatment; j, after treatment) and a significant decrease in PAD4 expression, as detected by SG1467 antibody (o, before treatment; p, after treatment) but not by SN823 antibody (l, before treatment; m, after treatment). Original magnification, ×100; insets show close-up details from the same samples. Graphs show results of semiquantitative analysis on a 4-grade scale for citrullinated proteins (e), PAD2 as detected with either ROI001 (h) or SN665 (k) antibody, and PAD4 as detected with either SN823 (n) or SG1467 (q) antibody. Values represent the median, and whiskers, the range (*P < 0.05).  Brown diaminobenzidine immunoperoxidase staining shows no changes in expression of citrullinated proteins as detected by F95 antibody (c, before treatment; d, after treatment), PAD2 as detected with either ROI001 antibody (f, before treatment; g, after treatment), or SN665 antibody (i, before treatment; j, after treatment) and PAD4 as detected with either SN823 antibody (l, before treatment; m, after treatment), or SG1467 antibody (o, before treatment; p, after treatment). Original magnification ×100, whereas insets show close-up details from the same samples. Graphs show results of semiquantitative analysis on a 4-grade scale for citrullinated proteins (e), PAD2 as detected with either ROI001 (h) or SN665 (k) antibody, and PAD4 as detected with either SN823 (n) or SG1467 (q) antibody. Values represent the median, and whiskers, the range (*P < 0.05).

HTX-EOS

The novel finding of the current study is the reduction in citrullination and PAD expression induced by intraarticular GC, which was not observed after MTX treatment. One potential explanation for this difference is the different time points to obtain the follow-up biopsy, 2 weeks after treatment initiation with GCs, as compared with 8 weeks for MTX. However, the two different time points were chosen in accordance with the clinical expectation of maximal effect of the administered drug, which would theoretically increase the chance to observe a change, not only for GCs, but also for MTX. We were able also to demonstrate a direct effect of GCs independent of inflammation in our synovial explants. This is further supported by the results of the in vitro experiments, in which we observed a dosedependent effect of GCs despite obvious difficulties in standardization of a quantitative analysis by using immunohistochemistry on cells. Interestingly, although GCs decreased citrullination and PAD4 expression in SFMCs, no such effect was observed in PBMCs. This could be due to the lower baseline levels and, as a consequence, to the lower sensitivity to detect changes of expression of the investigated molecules in PBMCs as compared with SFMCs. Conversely, it could be due to different regulatory mechanisms and cellular activation states of PBMCs, as compared with SFMCs, as previously suggested [20]. Despite major advances in understanding the central role of citrullination and anticitrulline immunity in RA pathogenesis, we face a striking lack of knowledge regarding regulatory factors responsible for induction, perpetuation, and/or amelioration of the process of citrullination, both locally in the joint and even more generally in other tissues where citrullination occurs either under physiologic (skin) [21] or pathologic conditions (lungs of smokers) [18,22]. It is generally accepted that citrullination accompanies inflammation, and it has been suggested that induction of citrullination by inflammatory stimuli such as TNF occurs after PAD4 activation and induction of signaling pathways dependent on NF-B [23]. Interestingly, the antiinflammatory effects of GCs are at least partially dependent on NF-B, as demonstrated by their lack of effect in animal models of acute inflammation in NF-B knockout mice [24]. In contrast, MTX appears to have limited effects on NF-B activation, as demonstrated by high levels of activation in PBMCs of MTX-treated RA patients that can be reversed by anti-TNF agents [25]. These findings suggest that GCs might affect citrullination through PAD4 downregulation through an NF-Bdependent mechanism.

Conclusion

The inflamed RA synovium is characterized by high expression of intracellular CP and PAD4 enzyme that is reversed through GCs, but not MTX treatment. Further investigation of the exact mechanism of action and comparison with other antirheumatic drugs is warranted.

Additional material

Additional file 1: Methotrexate (MTX) has no effect on expression of citrullinated proteins and PAD4 and PAD2 in SFMCs. Brown diaminobenzidine immunoperoxidase staining detects citrullinated proteins, as detected with F95 antibody, PAD2 as detected with ROI001 antibody, and PAD4 as detected with SN823 antibody. Original magnification, ×200. Graphs show results of manual counting in six different SFMC samples analyzed in duplicate, and results are expressed as fold decrease of the number of positive cells in the treated samples as compared with controls.

Additional file 2: MTX has no effect on expression of citrullinated proteins and PAD4 expression in an RA synovial explant. Brown diaminobenzidine immunoperoxidase staining shows citrullinated proteins as detected with F95 antibody, PAD2 as detected with ROI001 antibody, and PAD4 as detected with SN823 antibody. Original magnification, ×250.

Figure 1
1Representative picture showing that expression of intracellular and total citrullinated protein (CP), as well as PAD2 and PAD4 enzymes, is higher in rheumatoid arthritis (RA; upper panel) as compared with healthy (lower panel) synovial tissue. Frozen synovial biopsy sections show diaminobenzidine (brown) immunoperoxidase staining (hematoxylin counterstained) for citrullinated proteins and peptidylarginine deiminase (PAD) enzymes detected with the F95 antibody, anti-PAD2 (with two different antibodies, ROI002 and SN665), and PAD4 (with two different antibodies, SN823 and SG1467) antibodies (original magnification, ×100). Insets show close-up details from the same samples.

Figure 2
2Values are expressed as Spearman rank correlation coefficients (P values). CP, citrullinated protein; NS, not significant. Intraarticular glucocorticoid (GC) treatment of rheumatoid arthritis (RA) patients (n = 15) decreases synovial expression of F95 and peptidylarginine deiminase 4 (PAD4) but not PAD2.

Figure 3
3Methotrexate (MTX) treatment of rheumatoid arthritis (RA) patients (n = 11) does not modulate synovial expression of either citrullinated proteins or PAD2 and PAD4 enzymes. Hematoxylin-eosin (HTX-EOS) staining illustrates the histologic pattern of frozen synovial biopsy sections of an RA patient before (a) and after (b) MTX treatment.

Figure 4
4Dexamethasone (DXM) decreases the expression of citrullinated proteins and PAD4 and PAD2 in synovial fluid mononuclear cells (SFMCs), in a dose-dependent manner. Brown diaminobenzidine immunoperoxidase staining detects citrullinated proteins, as detected with F95 antibody, PAD2 as detected with ROI001 antibody, and PAD4 as detected with SN823 antibody. Original magnification, ×100. Graphs show results of manual counting in six different SFMC samples analyzed in duplicate, and results are expressed as fold decrease of the number of positive cells in the treated samples as compared with controls.

Figure 5
5Abbreviations ACPA: anti-citrullinated protein antibodies; CP: citrullinated protein; DMARD: disease-modifying antirheumatic drug; DXM: dexamethasone; FCS: fetal calf serum; GC: glucocorticoid; MC: mononuclear cell; MTX: methotrexate; PAD: peptidylarginine deiminase; PB: peripheral blood; RA: rheumatoid arthritis; SF: synovial fluid. Dexamethasone (DXM) decreases the expression of citrullinated proteins and PAD4 expression in a rheumatoid arthritis (RA) synovial explant. Brown diaminobenzidine immunoperoxidase staining shows citrullinated proteins as detected with F95 antibody, PAD2 as detected with ROI001 antibody, and PAD4 as detected with SN823 antibody. Original magnification, ×250.

Table 1
1Correlations between CP, PAD2, and PAD4 expression and synovial inflammation
Makrygiannakis et al. Arthritis Research & Therapy 2012, 14:R20 http://arthritis-research.com/content/14/1/R20
Makrygiannakis et al. Arthritis Research & Therapy 2012, 14:R20 http://arthritis-research.com/content/14/1/R20 Page 6 of 9
AcknowledgementsThis work was supported in part by research funding from the European Community FP6 funding project AutoCure, FP7 funding project Gums and joints, Innovative Medicine Initiative Be the CURE, the Swedish Research Council, and through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Instituet.Authors' contributions DM, AIC, and SR participated in study design, collection and interpretation of the data, and manuscript writing. ME and EaK participated in the collection of data. APN and GP participated in data collection, interpretation of the data, and manuscript writing. DM and SR contributed equally to this work. All authors read approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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The nuclear envelope localization of DYT1 dystonia torsinA-ΔE requires the SUN1 LINC complex component

Michael T Jungwirth
Dhivya Kumar
Danielle Y Jeong
Rose E Goodchild
The nuclear envelope localization of DYT1 dystonia torsinA-ΔE requires the SUN1 LINC complex component
R E S E A R C H A R T I C L E Open Access
Background: DYT1 dystonia is an autosomal dominant neurological condition caused by a mutation that removes a single glutamic acid residue (ΔE) from the torsinA (torA) AAA+ protein. TorA appears to possess a nuclear envelope (NE) localized activity that requires Lamina-Associated-Polypeptide 1 (LAP1), which is an inner nuclear membrane localized torA-binding partner. Although hypoactive, the DYT1 dystonia torA-ΔE isoform often concentrates in the NE, suggesting that torA-ΔE also interacts with an NE-localized binding partner. Results: We confirm that NE-localized torA-ΔE does not co-immunoprecipitate with LAP1, and find that torA-ΔE continues to concentrate in the NE of cells that lack LAP1. Instead, we find that variability in torA-ΔE localization correlates with the presence of the SUN-domain and Nesprin proteins that assemble into the LINC complex. We also find that siRNA depletion of SUN1, but not other LINC complex components, removes torA-ΔE from the NE. In contrast, the LAP1-dependent NE-accumulation of an ATP-locked torA mutant is unaffected by loss of LINC complex proteins. This SUN1 dependent torA-ΔE localization requires the torA membrane association domain, as well as a putative substrate-interaction residue, Y147, neither of which are required for torA interaction with LAP1. We also find that mutation of these motifs, or depletion of SUN1, decreases the amount of torA-WT that colocalizes with NE markers, indicating that each also underlies a normal NE-localized torA binding interaction.Conclusions: These data suggest that the disease causing ΔE mutation promotes an association between torA and SUN1 that is distinct to the interaction between LAP1 and ATP-bound torA. This evidence for two NE-localized binding partners suggests that torA may act on multiple substrates and/or possesses regulatory co-factor partners. In addition, finding that the DYT1 mutation causes abnormal association with SUN1 implicates LINC complex dysfunction in DYT1 dystonia pathogenesis, and suggests a gain-of-function activity contributes to this dominantly inherited disease.

Background DYT1 dystonia is a neurological disease characterized by prolonged, involuntary movements that develop in childhood or early adolescence, and occur in the absence of CNS pathology [1,2]. The disease is caused by an inframe, loss-of-function mutation that removes a glutamic acid residue (ΔE) from torA [3,4]. TorA is a member of the AAA+ ATPase family (ATPases Associated with a variety of cellular Activities) that typically couple the energy released by ATP hydrolysis to conformational changes in binding partners. The structural changes induced by AAA+ proteins vary. However, in most cases, an oligomeric ring arrangement of AAA+ enzyme subunits pulls the binding-partner substrate into the central pore and, by doing so, 'stretches' or removes secondary structure from the substrate. This action often destabilizes an otherwise energetically favorable binding interaction, such as the presence of substrate in a protein complex, aggregate, or association with a lipid bilayer. There are many hundreds of AAA+ enzymes and substrates, and AAA+ enzymes are used in processes as diverse as DNA replication, membrane fusion, protein degradation and cytoskeletal movement [5][6][7].

Multiple studies and research groups have found that torA is an endoplasmic reticulum (ER) resident protein [8][9][10]. However, despite localization throughout the ERsystem, torA loss specifically affects the NE subdomain and this suggests that torA AAA+ activity is targeted to a NE localized protein [4,11]. It is well established that torA interacts with the inner nuclear membrane protein, lamina-associated-polypeptide-1 (LAP1; TOR1AIP1) [12][13][14][15]. The importance of LAP1 is further underscored by the recent finding that LAP1 loss causes similar NE abnormalities to those seen in torA null cells [14]. In addition, the interaction between torA and LAP1 is stabilized by AAA+ domain mutations that typically inhibit ATP hydrolysis, such as the WalkerB box E171Q mutation in human torA [12,13,15]. Since the majority of AAA+ proteins interact with substrate in their ATP bound form, this stabilization suggests that LAP1 is a torA substrate. To date, however, the cellular functions of LAP1 remain unknown, no other luminal binding partners are identified, and LAP1 levels and subcellular localization appear unaffected by torA loss [4] -a surprising state of affairs for the predicted substrate of a physiologically important AAA+ protein. Other torA binding partners are also described, including the Nesprin proteins that are components of the LINC complex that couples the nuclear interior to cytoskeletal networks [16]. However, the relationship between these reported interacting partners, and the biochemical [12,13,15], genetic [14] and cell biologically [12,13] verified association between torA and LAP1 remains unclear.

Genetic analysis has demonstrated that the diseaseassociated torA-ΔE protein is expressed in DYT1 dystonia, but is hypoactive or inactive [4,10]. Consistent with these findings, recent studies demonstrated that ΔE appears to inhibit torA interaction with LAP1 and the homologous LULL1 membrane protein [13,15]. However, torA-ΔE can also concentrate in the NE [10,17], which suggests that ΔE stabilizes, rather than inhibits, interaction with an NE binding partner. The possibility that torA-ΔE displays enhanced NE-localized binding also argues against DYT1 dystonia being caused by a pure loss-of-function mutation. Furthermore, a gain-of-function activity is also consistent with the dominant nature of disease inheritance and that the torA-ΔE generating mutation is the sole causative mutation. Here we further examined torA-ΔE behavior in order to determine whether dysfunction of the mutant disease protein might contribute to DYT1 dystonia pathogenesis. We describe an association between torA-ΔE and the SUN1 inner nuclear membrane protein, and present data suggesting that SUN1 is also a normal torA binding partner. This abnormal ability of torA-ΔE provides more evidence for a gain-of-function action in DYT1 dystonia, and implicates the neurodevelopmentally important LINC complex dysfunction in disease pathogenesis.

Results

The majority of NE membrane proteins are localized and immobilized in this ER subdomain by binding to the nuclear lamina [18,19]. Although torA is a luminal protein, torA-E171Q and torA-ΔE concentrate in the NE of several cell lines, and each diffuses more slowly than ER-localized torA [12], which suggests that both isoforms interact with a lamina-associated NE membrane protein, such as LAP1. We examined the LAP1-binding of human torA-ΔE and torA-E171Q using mouse NIH-3T3 cells where both isoforms similarly localize in the NE ( Figure 1A), whereas this is previously assessed using cell lines where torA-ΔE is predominantly ER-localized [15,20]. However, we also find that LAP1 specifically co-immunoprecipitates with (GFP)torA-E171Q, but not (GFP)torA-ΔE ( Figure 1B, top row), despite similar expression and capture of both GFPtagged torsinA isoforms ( Figure 1B, bottom row). We also compared (GFP)torA-E171Q and (GFP)torA-ΔE binding to the ER-localized torA binding partner, LULL1. Again, we find no detectable co-immunoprecipitation of LULL1 with (GFP)torA-ΔE, although LULL1 is co-captured by (GFP)torA-E171Q immunoprecipitation ( Figure 1B, middle row). Thus, it appears that the E171Q and ΔE mutations differentially affect binding to the two verified torA binding partners, even in cell systems where both mutations cause torA to accumulate in the NE.

Co-immunoprecipitation of LAP1 with torA requires that binding is maintained after detergent solubilization removes both proteins from the ER membrane. It is therefore possible that ΔE inhibits this assessment of torA binding to LAP1, while interaction in vivo continues to underlie torA-ΔE localization in the NE, and this could be exacerbated because we are examining human torA in a mouse cell line. Consequently, we next examined whether LAP1 is important for the in vivo localization of torA-ΔE. To semi-quantitatively assess protein localization in vivo, we measured the relative amount of NE and ER-localized protein in individual cells. This method finds ratios of NE/ ER localized fluorescent signal above one for the inner nuclear membrane proteins of LAP1, LAP2 and emerin (the mean ratio ± standard error (s.e.m.) of NE/ER localized LAP1 fluorescence is 2.86 ± 0.05 (n = 25), LAP2 is 1.59 ± 0.05 (n = 10), and emerin is 1.63 ± 0.08 (n = 23)). In contrast, ER-resident proteins that do not accumulate in the NE display relative NE/ER fluorescence ratios below one (mean ± s.e.m. ratio of NE/ER calreticulin fluorescence is 0.94 ± 0.04, n = 12, and protein disulphide isomerase (PDI) is 0.79 ± 0.19, n = 25).

We subsequently co-transfected NIH-3T3 cells with (GFP)torA-WT, (GFP)torA-ΔE, or (GFP)torA-E171Q, together with scrambled control or LAP1 siRNA duplexes. In agreement with previous reports where LAP1 was required for the NE accumulation of (GFP) torA-E171Q [14,21], the semi-quantitative analysis detected that LAP1 depletion significantly reduces the mean ratio of NE/ER localized (GFP)torA-E171Q fluorescence ( Figure 1C). However, in contrast, LAP1 knockdown did not affect the ratio of NE/ER (GFP)torA-ΔE fluorescent signal ( Figure 1C). Qualitative analysis similarly highlights that (GFP)torA-ΔE is strongly concentrated in the NE of LAP1 depleted cells ( Figure 1D; central panels), while the absence of LAP1 inhibits the NE-accumulation of (GFP)torA-E171Q, which is instead predominantly ER-localized ( Figure 1D; right column). Although torA-WT is previously shown to biochemically associate with LAP1 [13,15], and LAP1 overexpression increases the amount of NE-localized (GFP)torA-WT [12], we failed to detect LAP1-induced change in (GFP) torA-WT localization. However, this may be due to the insensitivity of our measurements, rather than absence of in vivo interaction between torA-WT and LAP1.  [14,43] and anti-LULL1 (middle row) detects 75 kD LULL1. Right panels show levels of LAP1, LULL1, or GFP that immunoprecipitate with anti-GFP in buffer containing 2 mM ATP and 0.5 mM ADP. Note: mild elution conditions were used to prevent release of captured anti-GFP IgG molecules. (C) LAP1 depletion specifically affects (GFP)torA-E171Q localization. Columns show the mean and s.e.m. for ratios of NE/ER localized GFP fluorescence from n > 25 NIH-3T3 cells co-transfected with scrambled or LAP1 siRNA duplexes and (GFP)torA isoforms. *** symbol indicates that LAP1 depletion significantly reduces the (GFP)torA-E171Q NE/ER fluorescence ratio (p < 0.001; Two-Way ANOVA with Bonferroni post-hoc analysis). (D) (GFP)torA-ΔE remains NE-localized in LAP1 depleted cells, while (GFP)torA-E171Q redistributes into the ER. Images show anti-GFP labeling of NIH-3T3 cells co-transfected with (GFP)torA isoforms and control (upper row) or LAP1 siRNA duplexes (lower row). Scale bars show 10 μm.

LINC complex components are required for torA-ΔE localization in the NE

This lack of interaction between torA-ΔE and LAP1 reveals that a distinct mechanism underlies the NE-accumulation of the disease-associated torA isoform. Furthermore, although (GFP)torA-ΔE concentrates in the NE of the majority of NIH-3T3 and BHK21 cells, we consistently observe a minority of cells where (GFP)torA-ΔE is diffusely distributed through the ER ( Figure 1A, where the * symbol highlights a cell with ER-localized (GFP) torA-ΔE), and this variability does not correlate with levels of LAP1 (not shown). We hypothesized that this inconsistency in torA-ΔE localization might reflect the variable presence of a NE-localized torA-ΔE binding partner and we proceeded to examine the subcellular localization of several NE membrane proteins. We focused on the LINC complex proteins that couple the nucleus with cytoskeletal networks, as these have previously been associated with torA [16,20]. The LINC complex is formed in the NE lumen between inner nuclear membrane SUN-domain proteins and outer nuclear membrane Nesprins, and we find that NIH-3T3 cells express both of the functionally homologous SUN1 and SUN2 proteins [22,23]. We also detect NE-localized Nesprin2 in NIH-3T3 cells, although Nesprin1 is absent [24] and anti-Nesprin3 signal appears Golgi-localized (Additional File 1, Figure S1), thus indicating that Nesprin2 is the predominant Nesprin protein of NIH-3T3 cells.

While the majority of NIH-3T3 cells possess NE-localized anti-SUN1, anti-SUN2 and anti-Nesprin2 immunoreactivity, we also observe some interphase cells with minimal amounts of NE-localized fluorescent signal, or punctate anti-LINC component immunoreactivity outside of the NE (Figure 2A shows SUN1 localization). In contrast, the inner nuclear membrane proteins of emerin, LAP1 and LAP2 are consistently NE-localized in interphase cells (not shown), suggesting that this variability is LINC complex specific. We consequently examined whether (GFP)torA-ΔE localization correlates with the variability in LINC complex protein localization and found that cells with NE-localized anti-SUN1, anti-SUN2 or anti-Nesprin2 labeling also display NE-localized (GFP) torA-ΔE ( Figure 2B-D). In contrast, (GFP)torA-ΔE is diffusely distributed through the ER system in cells that lack NE-localized LINC complex components, or when these were present at low levels ( Figure 2B-D, cells highlighted by * symbols). Furthermore, we often also observed that (GFP)torA-ΔE co-localized with SUN1-immunoreactive puncta outside the NE ( Figure 2B; white arrow head). We subsequently confirmed this qualitative association between (GFP)torA-ΔE and LINC complex localization by examining the ratio of NE/ER localized anti-SUN1, anti-SUN2 and anti-Nesprin2 fluorescence in cells with either NE-or ER-localized (GFP)torA-ΔE. This analysis found that the ratios of NE/ER localized LINC complex components were significantly higher for cells with NElocalized (GFP)torA-ΔE ( Figure 2E, black columns), compared with cells where (GFP)torA-ΔE is predominantly present in the main ER ( Figure 2E, grey columns). In contrast, we found no correlation between torA-ΔE localization and the relative NE/ER levels of LAP1, emerin or LAP2 ( Figure 2E).

We next examined whether any LINC complex components are required for torA-ΔE to localize in the NE. We co-transfected NIH-3T3 cells with (GFP)torA isoforms alongside scrambled control siRNA duplexes, or SUN1, SUN2, or Nesprin2 siRNA duplexes that were verified to reduce expression of each LINC complex component (Additional File 1, Figure S1B -D). This revealed that SUN1 is required for (GFP)torA-ΔE to concentrate in NE, and we find ER-localized (GFP) torA-ΔE, or a punctate distribution of (GFP)torA-ΔE, in cells lacking SUN1 ( Figure 3A middle row). As expected, this change in subcellular localization is reflected by a significant decrease in relative NE/ER (GFP)torA-ΔE fluorescence between control and SUN1 depleted cells ( Figure 3B). In contrast, (GFP)torA-E171Q continues to concentrate in the NE of SUN1 siRNA treated cells ( Figure 3A, bottom row) and SUN1 siRNA does not appear to alter the relative NE/ ER localization of (GFP)torA-E171Q ( Figure 3B). This suggests that SUN1 loss specifically affects torA-ΔE without perturbing all NE-localized interactions. We do not observe gross differences in the localization of any torA isoform after SUN2 or Nesprin2 depletion (not shown). We also determined that the relative NE/ ER fluorescence of both (GFP)torA-ΔE and (GFP)torA-E171Q remains high in SUN2 siRNA transfected cells ( Figure 3C). Surprisingly, this semi-quantitative analysis identified that Nesprin2 depletion increases the ratio of NE/ER localized (GFP)torA-ΔE ( Figure 3D). Thus, although Nesprins are identified as torA-binding proteins [16], it appears that removing Nesprin2 may increase the number of NE-localized torA-ΔE binding sites. TorA-WT and torA-ΔE localization in the NE requires motifs that are not involved in torA-E171Q interaction with LAP1

These observations suggest that an association exists between torA-ΔE and the SUN1 LINC complex component, and therefore that SUN1, rather than LAP1, may be an NE partner of torA-ΔE. We subsequently examined whether these two putative torA binding interactions require distinct torA domains or residues. We first investigated the importance of the torA membrane association domain that immediately follows the cleaved ER targeting sequence [20,25] (Figure 4A). We transfected NIH-3T3 cells with either full-length (GFP)torA isoforms, or torA isoforms that lacked residues 22 to 40 (Δ22-40) that includes the membrane-association domain. We found that (GFP)torA-E171Q lacking residues 22-40 continues to accumulate in the NE ( Figure 4B middle row), indicating that this domain is not required for LAP1 binding, as is consistent with previous study of the torA -LULL1 interaction [20]. In contrast, Δ22-40 inhibits the NE-concentration of (GFP)torA-ΔE, and we fail to observe any cells with NE-localized (GFP)torA-ΔE when residues 22-40 are removed ( Figure 4B). We also observed that Δ22-40 appeared to decrease the amount of NElocalized torA-WT ( Figure 4B top row). We therefore semi-quantitatively examined (GFP)torA-WT localization, which revealed that introduction of Δ22-40 caused a significant (p < 0.001, Students Two-Tailed T-Test) decrease in the relative amount of NE/ER localized (GFP) torA-WT fluorescence from a mean ± s.e.m. of 1.15 ± 0.03 to 0.98 ± 0.03 (n = 25). Thus, the membrane association domain of torA is both essential for the SUN1dependent NE accumulation of torA-ΔE, and appears to play a role in a torA-WT localization, despite not affecting torA interaction with LAP1 or LULL1.

We also explored whether other torA motifs are important for torA localization in the NE. AAA+ enzymes typically alter substrate conformation using aromatic aminoacid residues that line the pore of the assembled AAA+ ring structure. Work with model AAA+ proteins has suggested that these residues interact with substrate and undergo 'lever-like' movements to pull substrates into the central pore [26][27][28]. We identified a torA tyrosine residue (Y147 in human torA) that lies in the approximate position of other AAA+ substrate-binding tyrosine residues. Furthermore, this residue is conserved between all vertebrate and invertebrate torA homologs (torsin family proteins [21]), suggesting a functional importance in torsin enzyme activity. With the hypothesis that this residue may be specifically important for torA binding with substrate, we compared how mutation (Y147A) affects (GFP)torA-ΔE localization in the NE compared with the LAP1-dependent NE-localization of (GFP)torA-E171Q. As with residues 22-40, we observed that the Y147A mutation specifically inhibits the NE-localization of (GFP)torA-ΔE, while (GFP)torA-E171Q continues to accumulate in the NE ( Figure 4B, right column). We again also observed an apparent loss of NE-localized (GFP)torA-WT fluorescent signal and therefore semiquantitatively examined how Y147A affects (GFP)torA-WT localization. This revealed that the Y147A mutation significantly reduces (p < 0.05, Students Two-Tailed T-Test) the ratio of NE/ER (GFP)torA-WT fluorescence from a mean ± s.e.m. of 0.98 ± 0.02, to 0.90 ± 0.02 (n = 45). Thus, the Y147 residue is required for both the NE- concentration of torA-ΔE, as well as for torA-WT to localize in the NE.

SUN1 depletion removes torA-WT from the NE

The importance of 22-40 and Y147 for WT and torA-ΔE localization in the NE suggests that these torA isoforms form a similar, LAP1-independent, binding interaction. Consistent with this hypothesis, we often observed that SUN1 depletion reduced the small amount of NE-localized (GFP)torA-WT fluorescence present in most cells [10,29] (Figure 5). Our initial analysis found that SUN1 siRNA did not significantly decrease the ratio of NE/ER localized (GFP)torA-WT fluorescence. However, since a proportion of control treated cells also lack NE-localized SUN1 (Figure 2A shows variability in SUN1 levels), we hypothesized that our approach may have yielded a falsenegative result. Consequently, we reassessed the ratio of NE/ER localized (GFP)torA-WT fluorescence in individual control siRNA transfected cells that we verified possessed SUN1, and compared this with cells that were SUN1 siRNA treated and verified to lack SUN1. This analysis found that the control ratio of NE/ER (GFP) torA-WT fluorescence in SUN1-containing cells (mean ± s.e.m. of 1.03 ± 0.02 (n = 34)) is significantly reduced (p < 0.0001, Students Two Tailed T-Test) to 0.89 ± 0.02 (n = 41) when SUN1 siRNA transfection depletes cellular SUN1. Thus, it appears that some torA-WT is normally SUN1-associated in vivo, which suggests SUN1 is a normal torA partner, as well as the protein responsible for the abnormal NE accumulation of disease-associated torA-ΔE.

Discussion

LAP1 has been consistently identified as a NE-localized binding partner of torA [12,14,15,20], and it appears that LAP1 is required for NE-localized torA activity [14]. However, we find that the NE localization of both torA-WT and torA-ΔE require torA-motifs that are unimportant for the LAP1 interaction. Furthermore, we show that SUN1 depletion removes torA-WT and torA-ΔE from the NE, while LAP1 is not required for torA-ΔE to concentrate in the NE. Therefore, although LAP1 is the confirmed binding partner for ATP-bound torA, LAP1 is neither the sole NE-localized torA interacting protein nor the partner of disease-associated torA-ΔE.

The multiple differences between torA-E171Q and torA-ΔE reveal that distinct mechanisms underlie their superficially similar ability to concentrate in the NE lumen [13,15]. While it appears that the E171Q mutation traps torA in an ATP-bound state, the mechanism by which ΔE affects torA remains unclear. A previous genetic analysis found that torA-ΔE cannot compensate for torA loss, therefore revealing that this isoform is hypoactive or inactive [4]. Biochemical analysis has also demonstrated that torA-ΔE may fail to bind ATP, or perhaps fail to undergo a structural rearrangement on ATP-binding [15], either of which may explain why torA-ΔE cannot form the ATPdependent interaction with LAP1 or LULL1 [13,15]. Despite these inhibitory effects, our data now highlight that ΔE does not generate a 'dead' torA isoform that is incapable of all binding interactions. Instead, the abnormal association between torA-ΔE and SUN1 suggests that ΔE 'traps' torA in a specific conformation that is distinct to the ATP-bound state. This finding also provides further evidence that the DYT1 ΔE mutation has a gain-offunction activity, in addition to producing the previously characterized impairment of torA function.

Our data indicate that torA associates with the SUN1 LINC complex component, and that the DYT1 mutation abnormally promotes or stabilizes this interaction. SUN1 is an inner nuclear membrane component of the LINC complex that couples the nuclear interior to cytoskeletal networks. SUN1 has a nucleoplasmic domain that mediates interaction with lamins, an extended membrane spanning hydrophobic region, and an approximately 50 kD luminal domain that appears to mediate both SUN-protein multimerization and interaction with KASH-domains of outer nuclear membrane Nesprin proteins [30][31][32][33]. There is loss-of-function evidence for functional redundancy between SUN1 and the similarly widely expressed and homologous SUN2 [22,23]. It is therefore surprising to find that SUN1 is specifically required for torA localization in the NE. We cannot rule out that SUN1 is selectively required because NIH-3T3 cells predominantly rely on SUN1, or that SUN1 siRNA more efficiently depletes SUN1 from the NE. However, there are reported differences in SUN1 and SUN2 characteristics [20,22,24,34], and our finding that SUN1 is selectively important for torA localization provides further evidence that these proteins have distinct, as well as overlapping cellular roles.

We do not resolve whether torA interacts directly or indirectly with SUN1. However, there is evidence against the possibility that SUN1 loss causes general NE disruption that removes torA by a highly indirect mechanism. Several previous studies have shown that NE structure and LINC complex function are largely normal in the absence of SUN1, and that combined SUN1 and SUN2 loss is required to perturb NE morphology [23,30,35,36]. Consistent with these findings, we observe that the NE-localization of LAP1-associated torA-E171Q is undisturbed by SUN1 loss, and that other inner nuclear membrane proteins are normally localized in cells lacking SUN1 (not shown). Furthermore, we also find that torA-ΔE colocalizes with SUN1 in puncta that lie outside of the NE. Thus, while it is possible that torA indirectly associates with SUN1, any such indirect interaction would be mediated by a protein or proteins that are also tightly coupled to SUN1. Unfortunately, we were unable to biochemically verify that a direct or indirect interaction exists between torA and SUN1. Like many inner nuclear membrane proteins, SUN1 solubilization requires that ionic detergents disrupt binding to the nuclear lamina and chromatin [31]. This treatment necessarily also impairs luminal interactions and a failure to detect biochemical association does not preclude that SUN1 and torA interact in vivo. Furthermore, despite our negative findings with anti-GFP immunoprecipitation of (GFP)torA isoforms, one previous report described that nesprin KASH-domains co-immunoprecipitate with anti-torA antibodies [16], which provides general support for an interaction between torA and a LINC complex component. However, our finding that Nesprin2 depletion increases, rather than decreases, the amount of NElocalized torA-ΔE, indicates that this previous result reflects an indirect, rather than direct, interaction between torA and nesprins. It is unclear why Nesprin2 loss increases torA-ΔE localization in the NE. However, an increased number of torA-ΔE binding sites, perhaps caused by compensatory upregulation of other LINC components, could account for both our observations and the previous association between torA and the nesprin KASH-domain.

Our findings support a model where torA interacts with at least two different NE proteins [16]. The existence of additional NE-localized partners suggests that torA either operates on multiple substrates and/or that some identified torA interacting partners have a regulatory function and are not subject to torA AAA+ activity. Finding that the SUN1-dependent localization requires a putative substrate interaction residue [26,28], Y147, suggests that SUN1 or the LINC complex is a substrate affected by torA AAA+ activity. Surprisingly, this also suggests that Y147A destabilizes an interaction that is distinct to the ATP-bound torA state that associates with LAP1 and LULL1. This insensitivity of LAP1 and LULL1 binding raises the possibility that these proteins are not torA substrates. Furthermore, these data also suggest that torA could operate using an atypical biochemical AAA+ mechanism; a hypothesis that is supported by the presence of a non-canonical nucleotide binding motif in the torA AAA+ domain that appears to convey preferential binding to ADP, rather than ATP [37].

The possibility that torA activity modifies the LINC complex is also supported by two previous reports. In one study, torA loss appeared to remove the Nesprin3 LINC complex component from the NE, suggesting that torA normally maintains intact LINC complexes [16]. In contrast, a separate study made the reverse observation, and found that overexpression of the LULL1 torA-binding partner appeared to induce a NE-localized torA activity that removed SUN2 and Nesprin2 [20]. This LULL1activated torA was strongly concentrated in the NE of cells that lacked SUN2 and Nesprin2, which supports our finding that SUN1 is important for the NE retention of torA. Furthermore, since AAA+ activity often disassembles otherwise stable protein complexes [6,38], this data is also consistent with torA dissociating SUN1 binding interactions to release SUN1-associated proteins from the NE lumen. The relationship between the LULL1-overexpression paradigm and physiological torA function is unclear, and there are some AAA+ proteins that promote protein complex formation or remodeling, rather than disassembly [39]. Therefore, a difference between the actions of physiological levels of torA activity, versus overactive torA enzymes, could explain the discrepancy between these two studies.

While our study emphases a relationship between torA and the LINC complex, it is also clear that LAP1 has a central role in torA function and, notably, LAP1 and torA gene knock-out result in the same NE membrane abnormalities [14]. Although this association does not resolve whether LAP1 activity is upstream (an essential torA regulator/co-factor) or downstream (a substrate) of torA activity, it is clear that LAP1 is the partner of the ATP-bound form of torA that is typically the substrate-associated state of a AAA+ protein [13,15].

There are examples of promiscuous AAA+ enzymes that operate on several distinct substrate proteins, and it is possible that torA is a multi-functional AAA+ protein that operates on both LAP1 and the LINC complex. Furthermore, since adapter proteins often regulate the substrate selection of multifunctional AAA+ proteins [40], the importance of Y147, residues 22-40, and the effect of ΔE, could be explained if these motifs affect torA interaction with an adapter that promotes the LINC complex association over LAP1 binding.

Our study is performed using cells that also express torA. It is therefore possible that these endogenous torA-WT subunits play a role in the association between (GFP)torA-ΔE and SUN1. Nevertheless, mixed torA-ΔE and torA-WT expression also occurs in DYT1 dystonia, and it is demonstrated that torA abnormally concentrates in the NE of DYT1 dystonia cells, as well as neurons ectopically expressing torA-ΔE [10,29]. Thus, our findings suggest that the LINC complex of DYT1 dystonia neurons is abnormally associated with torA-ΔE. There are several mechanisms by which an abnormal association between torA-ΔE and SUN1 could negatively impact cell function. Firstly, torA-ΔE occupation of binding sites could prevent functional torA enzymes from accessing the LINC complex, and therefore inhibit torA activity in the event that this normally modifies the LINC complex. Secondly, there is evidence that torA-WT and torA-ΔE co-oligomerize, and that the abnormal SUN1-association of torA-ΔE is conferred to coexpressed torA-WT [10]. This could result in torA-WT sequestration away from other molecular targets of torA AAA+ activity. A third possibility is that abnormal association with torA-ΔE directly impacts LINC complex assembly or function, independent of torA-WT activity. These mechanisms are not mutually exclusive, and it is possible that a combination of such loss-of-function and/or gain-of-function torA-ΔE actions underlie why DYT1 dystonia is dominant and the torA-ΔE generating mutation is the only identified cause of this disease.

Our study did not identify grossly abnormal LINC complex component localization in cells transfected to express (GFP)torA-ΔE. However, our assessment did not examine whether physiological torA-ΔE expression impacts LINC complex components as these are utilized during particular cellular behaviors, such as nuclear movement [23,24]. It is unlikely that torA-ΔE expression completely ablates LINC complex activity, as genetic deletion of the complex in mice results in severe neurodevelopmental abnormalities [23], compared with undetectable or limited neuropathology in human DYT1 dystonia patients and heterozygous torA-ΔE expressing mice [41]. However, given the importance of the LINC complex for nervous system development [23], it is conceivable that torA-ΔE driven abnormalities in LINC complex mediated events impact neuronal development to generate the circuit abnormalities that are thought to underlie the debilitating twisting movements of DYT1 dystonia [42].

Conclusions

We have demonstrated an association between the DYT1 dystonia protein, torA-ΔE, and the SUN1 component of the LINC complex. We also find evidence that SUN1 is a normal torA partner, which therefore suggests that the ΔE mutation stabilizes a normally transient torA binding interaction. These findings raise the possibility that torA-ΔE may impact the LINC complex, and therefore implicate LINC complex dysfunction in DYT1 dystonia.

Methods

Cell culturing and transfection

We obtained NIH-3T3 and BHK21 cells from ATCC and cultured these under recommended conditions. Transfections were performed for 3 hours with cells plated onto collagen-coated coverglasses in 24 well plates using Plus Reagent with either Lipofectamine or Lipofectamine LTX. siRNA transfection of NIH-3T3 cells was performed using 25 pmol dsRNA and RNAiMAX reagent. Dual plasmid and RNA transfection of~30% confluent NIH-3T3 cells was performed using 200 ng DNA, 25 pmol dsRNA and Lipofectamine 2000. All transfection reagents were purchased from Invitrogen and used according to the manufacturers recommendations except as noted above. We generated (GFP)torA stably expressing NIH-3T3 lines by placing plasmid transfected cells under selection with 1 mg/ml G418 (Fisher Scientific) until patches of cells were visible. Individual colonies were collected by trypsinization, expanded in the continued presence of G418, and assayed for torA expression. All other transfections were performed at least twice and coverslip cultured cells were fixed with formaldehyde at 24 hours post-transfection (plasmid transfections) or between 36 and 48 hours post-transfection (siRNA transfections).

Plasmid generation and siRNA sequences

The methods and production of torA expression constructs are previously described [10,12]. We generated torA cDNA constructs that lacked residues 22-40 by first using QuikChange PCR mutagenesis to introduce Nhe1 sites between residues 21 and 22, and between 40 and 41, of human torA. We then replaced the sequence between the Nhe1 sites with GFP from pEGFP1 (Clontech). QuikChange was also used to introduce the Y147A mutation. All torA cDNA sequences were fully sequenced following PCR based mutagenesis.

siRNA duplexes were custom synthesized by Invitrogen. Mouse Tor1aip1 (LAP1) siRNA duplex sequence begins at basepair 956 in NM_144791 and is previously described [21].

Fluorescent imaging, quantification and statistical analysis

Immunofluorescent labeling of coverslip cultured cells is previously described [21]. Quantification of the relative amounts of NE and ER localized (GFP)torA fluorescence was performed on transfected cells co-labeled with antibodies against GFP and the ER marker Protein Disulphide Isomerase (PDI). We also either colabeled cells, or processed duplicate sets of transfected cells, with antibodies that detect siRNA-targeted proteins to assess the efficiency of target protein knock-down. All images of target proteins were removed prior to assessment of GFP localization to ensure that quantification of (GFP)torA NE/ER fluorescence ratios was conducted blind to both the siRNA treatment and torA isoform.

Labeled samples were examined using a Nikon Eclipse Ti inverted microscope and imaged with Nikon Digital Sight DS-QiMc camera and NIS-elements BR3.0 software. Images of GFP positive cells were captured so that the focal plane visualized the NE. Exposure times were 100 milliseconds or greater and images with saturated signal were excluded to avoid analysis of highly expressing cells. A one-pixel wide measuring line was positioned to bisect the NE and a portion of PDI-labeled ER. The peak of NE localized pixel intensity was used as measure of NE fluorescence. We reduced the possibility of biasing ER fluorescence measurements by consistently determining GFP signal in a region of PDI-positive perinuclear ER. Thus, ER localized GFP signal was calculated as the average intensity of pixels between 0.5 μm to 1 μm distance from the NE. We recorded a 'Background' fluorescence measurement from an untransfected cell within the field and subtracted this value from the NE and ER fluorescence measurements. The intensity of anti-LAP1, SUN1, SUN2, Nesprin2, LAP2, and emerin NE and ER immunofluorescent signals were similarly collected, with the exception that quantification was performed from cells containing either NE-or ER-localized (GFP)torA-ΔE, and background corrections were made from a section of image that lacked adherent cells. A similar procedure was used to determine the ratio of NE/ER localized (GFP)torA-WT fluorescence from verified SUN1 positive or negative cells. In all cases, we used GraphPad prism to analyze the ratios of NE/ER localized fluorescence collected from individual cells, including calculation of mean, s.e.m., Two-Way ANOVA, T-Test, and post-hoc analyses. All graphs show the mean and standard error (s.e.m.) of these values.

Immunoprecipitations and Western blotting

Confluent dishes of control or (GFP)torA expressing NIH-3T3 cells were collected, pelleted and solubilized in buffer containing 50 mM imidazole-HCl (pH 7.5), 5% glycerol, 0.5% digitonin, 4 mM MgCl 2 , 50 mM NaCl, 1X protease inhibitor cocktail, 2 mM PMSF and nucleotides as indicated. We removed insoluble material by centrifugation at 20, 000 × g and then incubated lysates for 2.5 hour at 4°C with anti-GFP conjugated AminoLink resin (Pierce). We subsequently washed the agarose resin twice with solubilization buffer and eluted captured proteins by 24°C incubation with non-reducing 2X Laemmli buffer. Elutes and lysates were analyzed using standard SDS-PAGE and Western blotting procedures.

Antibodies

Rabbit anti-GFP and anti-LAP1 antibodies are previously described [21]. We used rabbit polyclonal anti-SUN1 (AbCam), anti-SUN2 (UNC84B) from Sigma and an anti-SUN2 from AbCam. We also used previously described procedures [21] to generate rabbit serum containing polyclonal antibodies against the recombinant luminal domain of SUN1 [30] that we expressed and purified from BL21-RIPL E. coli (Stratagene). Anti-Nesprin1 (Abcam), anti-Nesprin2 (Santa Cruz), anti-Nesprin3 (Abcam), anti-emerin (Santa Cruz), anti-LAP2 (Sigma),

Figure 1
1TorA-ΔE does not interact with the NE-localized torA-E171Q binding partner, LAP1. (A) (GFP)torA-ΔE and (GFP)torA-E171Q both concentrate in the NE of NIH-3T3 cells. The * symbol highlights a cell with ER-localized (GFP)torA-ΔE. Scale bars show 10 μm. (B) LAP1 and LULL1 co-immunoprecipitate with (GFP)torA-E171Q and not (GFP)torA-ΔE. Left panels show levels of LAP1, LULL1 and (GFP)torA in 5% of lysate input collected from control, (GFP)torA-E171Q or (GFP)torA-ΔE stably expressing NIH-3T3 cells. Anti-LAP1 (top row) detects the alternatively spliced LAP1 isoforms

Figure 2
2TorA-ΔE co-localizes with LINC complex components. A) SUN1 localization in the NE varies between individual NIH-3T3 cells. Panels show anti-SUN1 (red), DAPI labeling of DNA, anti-PDI labeling of the ER (green) in sub-confluent, unsynchronized NIH-3T3 cells. Scale bars show 10 μm. (B -D) Positive correlation between the localization of LINC complex components and transfected (GFP) torA-ΔE. Left panels (red) show (B) anti-SUN1, (C) anti-SUN2, and (D) anti-Nesprin2 labeling of NIH-3T3 cells. Middle panels (green) show the subcellular localization of transfected (GFP)torA-ΔE. * symbols highlight that cells with ER-localized (GFP)torA-ΔE also lack NElocalized LINC proteins. White arrowheads highlight that transfected (GFP)torA-ΔE also colocalizes with SUN1 outside the NE. (E) Ratios of NE/ER localized anti-SUN1, SUN2 and Nesprin2 fluorescence are higher in cells with NE-localized (GFP)torA-ΔE. Columns show the mean and s.e.m. of the relative NE/ER localized anti-SUN1, SUN2, Nesprin2, LAP1, emerin and LAP2 fluorescence from n > 15 cells. Black columns show the ratios of NE/ER fluorescence from cells with predominantly NE-localized (GFP)torA-ΔE, while grey columns show ratios from cells with ER-localized (GFP)torA-ΔE. ***, ** indicate that the mean ratio of NE/ER fluorescence is significantly (p < 0.001, 0.01) different between cells with NE-or ER-localized (GFP)torA-ΔE (Two-Way ANOVA with Bonferroni post-hoc analysis).

Figure 3
3TorA-ΔE localization in the NE requires SUN1. (A) SUN1 depletion selectively inhibits the NE-localization of (GFP)torA-ΔE. Images show anti-GFP labeling of NIH-3T3 cells co-transfected with (GFP)torA isoforms and control (left panels) or SUN1 siRNA duplexes (right panels). Scale bar shows 10 μm. (B -D) Only SUN1 siRNA reduces the ratio of NE/ER localized (GFP)torA-ΔE fluorescence. Columns show the mean and s.e.m. of the relative NE/ER GFP fluorescence from n > 25 NIH-3T3 cells transfected with (GFP)torA isoforms alongside (B) SUN1, (C) SUN2, or (D) Nesprin2 siRNA duplexes, or a sequence matched scrambled control siRNA duplex. The ** and *** symbols indicate a significant (p < 0.01, < 0.001, respectively) difference between control and siRNA treated cells. (Two-Way ANOVA with Bonferroni post-hoc analysis).

Figure 4
4A putative membrane association domain and porelining residue are required for torA-ΔE localization in the NE.(A) Schematic of torA structure showing the location of aminoterminal ER targeting sequence, putative membrane association domain, together with the Y147, E171 and E302/3 (mutated by ΔE) residues. (B) Images show anti-GFP labeling of BHK21 cells expressing (left panels) "control" (GFP)torA-WT, (GFP)torA-ΔE or (GFP) torA-E171Q proteins, or (central column) these torA isoforms lacking residues 22 -40 ("Δ22-40"), or (right column) torA isoforms containing the Y147A mutation ("+ Y147A"). Scale bars show 10 μm.

Figure 5
5SUN1 depletion also reduces NE-localized torA-WT. The (GFP)torA-WT normally present in the NE of NIH-3T3 cells is reduced by SUN1 siRNA treatment. Images show anti-GFP labeling of NIH-3T3 cells expressing (GFP)torA-WT (green) and transfected with control (left) or SUN1 siRNA duplexes (right). Anti-LaminA/C labeling (red) highlights the position of the NE and lower panels show the merge (yellow) of (GFP)torA-WT and NE-marker signals, with less yellow signal present in the cell lacking SUN1. Scale bars show 10 μm.

Mouse Unc84a (SUN1) siRNA duplex sequence begins at basepair 261 in NM_024451: (GCU AUU GAU UCG CAC AUU A; UAA UGU GCG AAU CAA UAG C) and is matched with control duplex (GCU UAG UCG CUA CAU AUU A; UAA UAU GUA GCG ACU AAG C). Mouse Unc84b (SUN2) begins at basepair 2142 in NM_194342: (GCA GGA AGG GAC ACU UCU U; AAG AAG UGU CCC UUC CUG C) and is matched with control duplex (GCA GGA AAC AGU UCG GCU U; AAG CCG AAC UGU UUC CUG C). Mouse Syne2 (Nesprin2) begins at basepair 21173 in NM_001005510 and contains sequence from the transmembrane and KASH domain encoding terminal exon of nesprin2: (AUA UAG GUC AUU UAC GUG C; GCA CGU AAA UGA CCU AUA U) and is used with control duplex (GCA AAU GUA CCA AUC GUA UTT; AUA CGA UUG GUA CAU UUG CTT). Mouse 4831426I19Rik (Nesprin3) siRNA duplex sequence begins at basepair 2732 of NM_172500, which is within the terminal exon that contains the KASH domain sequence: (GCU GUU GCU CCU GCU CUU UTT; AAA GAG CAG GAG CAA CAG CTT) and this is matched with control duplex (GCU UCG UGU CCC UCU GUU UTT; AAA CAG AGG GAC ACG AAG CTT).
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AcknowledgementsWe thank Mary Lynn Dear, Paolo Vignali, Erica Ellis, Rex Siu, and Dang Xiong for experimental assistance. We also appreciate helpful advice from Dan Roberts and Barry Bruce. A Dystonia Medical Research Foundation award to REG provided some of the funding for this work.Authors' contributions MTJ, DK performed experiments, DYJ performed image analysis, REG designed experiments, analyzed data, prepared data images and wrote the manuscript. All authors read and approved the final manuscript.chicken anti-GFP (AbCam), goat anti-laminA/C (Santa Cruz) and anti-PDI (Assay Designs), are other commercially available antibodies used in this study. Immunofluorescent labeling of cells utilized anti-mouse, chicken or rabbit secondary antibodies conjugated to DyLight 488, FITC or Rhodamine RedX (Jackson Immunoreagents), or AlexaFluor350 conjugated donkey anti-goat (Invitrogen). Primary antibodies used in Western blotting were detected using peroxidase conjugated goat anti-rabbit secondary antibody (Pierce) or an anti-rabbit lightchain specific secondary antibody (Jackson Immunolabs).Additional materialAdditional file 1:Figure S1. LINC complex components are depleted by siRNA transfection of NIH-3T3 cells.
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The nuclear envelope localization of DYT1 dystonia torsinA-ΔE requires the SUN1 LINC complex component. Jungwirth, BMC Cell Biology. 1224Cite this article asCite this article as: Jungwirth et al.: The nuclear envelope localization of DYT1 dystonia torsinA-ΔE requires the SUN1 LINC complex component. BMC Cell Biology 2011 12:24.