The etiological underpinnings of amyotrophic lateral sclerosis (ALS) are complex and incompletely understood, although contributions to pathogenesis by regulators of proteolytic pathways have become increasingly apparent. Here, we present a novel variant in UBQLN4 that is associated with ALS and show that its expression compromises motor axon morphogenesis in mouse motor neurons and in zebrafish. We further demonstrate that the ALS-associated UBQLN4 variant impairs proteasomal function, and identify the Wnt signaling pathway effector beta-catenin as a UBQLN4 substrate. Inhibition of beta-catenin function rescues the UBQLN4 variant-induced motor axon phenotypes. These findings provide a strong link between the regulation of axonal morphogenesis and a new ALS-associated gene variant mediated by protein degradation pathways.https://doi.org/10.7554/eLife.25453.001
Amyotrophic lateral sclerosis, or ALS for short, is a disease in which parts of the brain and spinal cord progressively degenerate. Specifically, the condition causes the nerve cells that control movement – called motor neurons – to die. As a result, people with ALS lose control of their muscles. The cause of ALS is not known, but evidence suggests that a person’s genetics plays a role in the development of the disease. Learning which genes are involved and what they do within cells may help scientists figure out what goes wrong in patients with ALS and how to treat the condition.
People with ALS often experience an abnormal build up of proteins in their brain and spinal cord. Cells normally rely on molecules working together in the so-called ubiquitin proteasome system to eliminate unwanted proteins. A few mutations linked with ALS, and some other neurodegenerative conditions, have been traced back to genes encoding parts of this protein disposal system, which may help to explain the build up of proteins. However, our understanding of the genetic causes of the disease is far from complete.
Now, Edens et al. report a new mutation in a gene that encodes a protein involved in the ubiquitin proteasome system. The gene, called UBQLN4, had not previously been linked to ALS, but looking at this gene in nearly 700 patients with ALS revealed a mutation in one patient with an inherited form of the disease. This mutation was not found in public databases that contain genetic information from tens of thousands of people without ALS.
To better understand the effect of this newly identified mutation, Edens et al. recreated it in zebrafish embryos and motor neurons from mice. The mutation in UBQLN4 changed the shape of the cells in the spinal cords of the zebrafish and the mouse motor neurons. There was also a build up of excess proteins because the breakdown of proteins by the ubiquitin proteasome system was slowed. Specifically, there was an excess amount of a protein called beta-catenin, which is important for development and activity of the nervous system. Treating the mutant motor neuron cells with a drug called quercetin, which suppresses beta-catenin, reversed the defects seen in the cells. Larger studies are now needed to see how often this mutation occurs in patients with ALS and to determine if other forms of the disease might have a similar cause.https://doi.org/10.7554/eLife.25453.002
Ubiquilins belong to the ubiquitin-like family of proteins and act broadly as key regulators of degradative processes. The best understood function of ubiquilins is in the ubiquitin proteasome system (UPS), where they recognize and bind polyubiquitinated substrate proteins through a C-terminal ubiquitin-associated (UBA) domain and deliver them to the proteasome through an N-terminal ubiquitin-like (UBL) domain (Marín, 2014). Additional roles for ubiquilins in autophagy and ER-associated degradation have also been described (Hjerpe et al., 2016; Lee and Brown, 2012; Lim et al., 2009). The UPS and autophagy are indispensible for multiple aspects of neuronal development (Cecconi and Levine, 2008; Hamilton and Zito, 2013) and are crucial in maintaining homeostasis in the aging nervous system (Groen and Gillingwater, 2015; Rezania and Roos, 2013). Dysfunction of these pathways contributes to neurodegenerative diseases characterized by protein aggregation (Huang et al., 2010; Kim et al., 2009; Wang et al., 2011; Wu et al., 2015). Accordingly, variants in UBQLN1 and UBQLN2 have been linked to neurological disorders. The ubiquilin1 protein, encoded by UBQLN1, has been associated with neurofibrillary tangles in Alzheimer’s disease (AD) and is proposed to contribute to protein aggregates characteristic of AD pathology (El Ayadi et al., 2012; Mah et al., 2000). Single-nucleotide polymorphisms in UBQLN1 were suggested to confer susceptibility to Alzheimer’s disease (Bertram et al., 2005; Kamboh et al., 2006). Mutations in UBQLN2 have been related to familial X-linked ALS/FTD (Deng et al., 2011) and heterogeneous X-linked dominant neurodegeneration (Fahed et al., 2014). Here, we report a novel variant in UBQLN4 that is associated with ALS and demonstrate a mechanism by which wild-type UBQLN4 may regulate motor axon morphogenesis. We also reveal how dysregulation of this mechanism by the ALS-associated variant leads to abnormal motor neuron structure and function through impaired degradation and substrate retention.
Mutations in UBQLN2 have been identified in ALS patients with or without dementia (Deng et al., 2011). A variant, E54D, in UBQLN1 has been reported in a single patient with atypical motor neuron disease consistent with Brown-Vialetto-Van Laere syndrome (González-Pérez et al., 2012). These data suggest a role for ubiquilins in motor neuron diseases. UBQLN4, like UBQLN1 and UBQLN2, is widely expressed and shows substantial homology to human UBQLN2 (Marín, 2014). To test if genetic variants of UBQLN4 are involved in the etiology of ALS, we screened its 11 exons with primers covering coding regions and the exon-intron boundaries. We examined 267 familial ALS index cases and 411 sporadic ALS cases, and identified a variant in a familial ALS case (Figure 1A). This variant, c.269A>C, was located in exon 3, leading to the change of aspartate to alanine, p.D90A at the protein level (Figure 1C). The female patient (III3) with this UBQLN4D90A variant had an age of disease onset at 55 years, with disease duration of two years. Her mother (II3), maternal aunt (II2) and a maternal cousin (III1) also developed ALS and died of respiratory failure two to five years following disease onset. The variant was neither present in our 332 in-house controls, nor in any SNP databases with a total of >15,000 sequenced alleles nor in the Exome Aggregation Consortium (ExAC) database in a total of 60,706 unrelated individuals. The amino acid D90 is adjacent to the UBL domain of UBQLN4 (Figure 1B) and is highly conserved during evolution, suggesting its importance in structural and functional properties of the protein.
To assess the effects of the ALS-associated variant, we expressed wild-type or disease-associated UBQLN4 in cultured mouse spinal motor neurons (Figure 2A, Figure 2—figure supplement 1). UBQLN4D90A-expressing cells showed a significant increase in the total number of neurites as compared to cells expressing wild-type UBQLN4, or non-transfected cells (Figure 2A,C). Importantly, these results were validated in vivo in zebrafish. When mRNAs encoding UBQLN4-WT or UBQLN4D90A were injected into zebrafish embryos, we observed abnormal motor axon branching in UBQLN4D90A but not UBQLN4-WT or uninjected embryos (Figure 2B,D). Both UBQLN4-WT and UBQLN4D90A-injected fish embryos otherwise developed normally and showed neither gross morphological abnormalities nor significant changes in motor axon length, suggesting specificity of the motor axon branching phenotype. In all experiments, expression levels of UBQLN4-WT and UBQLN4D90A were comparable (Figure 2—figure supplement 2A–C). These results indicate that the ALS-associated UBQLN4 variant interferes with normal motor axon morphogenesis in culture and in vivo.
Given the role ubiquilins play in the UPS, we sought to determine if UBQLN4D90A affects proteasome-mediated degradation. We used the UbG76V-GFP fusion protein as a reporter for UPS function (Dantuma et al., 2000). The G76V substitution produces an uncleavable ubiquitin moiety that acts as a proteasome degradation signal, thereby targeting GFP for proteasomal degradation. Rates of UPS-dependent protein turnover can therefore be monitored by GFP protein level. We transfected NSC-34 cells, a motor neuron-derived cell line, with UbG76V-GFP, UbG76V-GFP + UBQLN4-WT, or UbG76V-GFP + UBQLN4D90A and compared GFP levels. UBQLN4D90A expression resulted in reduced protein turnover, as indicated by significantly greater GFP signal compared to UBQLN4-WT or UbG76V-GFP alone (Figure 3A,B). We further confirmed this finding with cycloheximide protein stability assay (Figure 3C,D). Cells transfected with UbG76V-GFP alone or UbG76V-GFP + UBQLN4-WT showed a gradual reduction in GFP levels due to proteasomal degradation in the presence of protein synthesis inhibitor cycloheximide. Cells transfected with UbG76V-GFP + UBQLN4D90A showed much less GFP degradation, indicating impairment of proteasomal function by the ALS-associated UBQLN4D90A variant (Figure 3C,D). Taken together, these results suggest that UBQLN4D90A impairs proteasomal function.
Recently, loss of the ubiquitin-like modifier-activating enzyme 1 (UBA1) was shown to cause motor axon abnormalities similar to what we observed. In the UBA1 study, the phenotypes were shown to result from heightened beta-catenin signaling, caused by loss of Uba1 and dysfunction of ubiquitination pathways (Wishart et al., 2014). Given our finding that UBQLN4D90A results in reduced proteasomal efficiency, we sought to determine if it also affects beta-catenin levels. We first compared beta-catenin levels in UBQLN4-WT, UBQLN4D90A and non-transfected NSC-34 cells (Figure 3E). There was a significant decrease in beta-catenin in UBQLN4-WT-expressing cells compared to non-transfected cells, suggesting a role for UBQLN4 in facilitating beta-catenin degradation. Moreover, we detected a significant increase of beta-catenin in UBQLN4D90A-expressing cells compared to those expressing UBQLN4-WT, suggesting that the UBQLN4D90A mutation disrupts beta-catenin degradation (Figure 3F). Immunostaining in primary mouse spinal motor neurons validated these observations, and further confirmed nuclear beta-catenin accumulation in UBQLN4D90A-expressing cells (Figure 3G,H).
Given our findings that UBQLN4D90A expression results in reduced proteasomal efficiency and nuclear beta-catenin accumulation, we asked if inhibition of beta-catenin could mitigate motor axon morphogenesis defects characteristic of UBQLN4D90A expression. To address this possibility we utilized quercetin, which acts by restricting beta-catenin nuclear localization to reduce beta-catenin-dependent signaling (Park et al., 2005). Immunostaining for beta-catenin in mouse spinal motor neurons transfected with UBQLN4-WT or UBQLN4D90A revealed significantly greater nuclear accumulation of beta-catenin in UBQLN4D90A-expressing cells as compared to UBQLN4-WT-expressing cells (Figure 4A,B). This accumulation was reduced by treatment with 0.1 μM quercetin, confirming the inhibitor’s effectiveness. We next looked at quercetin’s effects on axon morphogenesis in primary neurons and in zebrafish in the context of UBQLN4-WT or UBQLN4D90A expression. Indeed, the total number of neurites in 0.1 μM quercetin-treated UBQLN4D90A-expressing neurons was rescued to wild-type levels (Figure 4C,D). Moreover, the percentage of motor axons with aberrant branching morphology in 50 μM quercetin-treated UBQLN4D90A-expressing zebrafish embryos was significantly reduced as compared to vehicle-treated embryos (Figure 4E and F). Taken together, these findings suggest that motor axon morphogenesis phenotypes characteristic of UBQLN4D90A expression may result from beta-catenin accumulation, and that inhibition of beta-catenin is sufficient to mitigate UBQLN4D90A variant-induced phenotypes.
Here we provide the first evidence of UBQLN4 involvement in ALS, and reveal the association of beta-catenin-dependent signaling with the disease variant-induced phenotypes. Taken with reports of contributions by UBQLN1 and UBQLN2 to neurodegeneration, our findings suggest prominent neuropathological involvement of the UBQLN gene family. In the present study, we identified abnormalities in spinal motor neuron morphogenesis in primary mouse neurons as well as a zebrafish model in vivo. Furthermore, we demonstrated that the novel ALS-associated UBQLN4 variant impaired UPS function, leading to increased beta-catenin in UBQLN4D90A-expressing cells. We also showed that inhibition of beta-catenin was sufficient to mitigate these phenotypes, suggesting a role for beta-catenin signaling in regulating these physiological and pathological functions.
Ubiquilins are characterized by a ubiquitin-like UBL domain and a ubiquitin-associated UBA domain that interact with proteasomes and polyubiquitinated substrates, respectively, to facilitate proteasomal degradation (Ko et al., 2004). Because the D90A mutation lies near the UBL domain, the UBQLN4D90A variant may compromise interaction with proteasomes, consistent with our finding of proteasomal impairment in UBQLN4D90A-expressing neurons. Beta-catenin-dependent signaling has been shown to play a critical role in regulating multiple aspects of neuronal development, including neurite outgrowth (Votin et al., 2005), axon guidance (Avilés and Stoeckli, 2016; Maro et al., 2009), and target innervation (Salinas and Zou, 2008; Wu et al., 2012). Therefore UBQLN4D90A expression, which compromises UPS function and causes beta-catenin accumulation, may affect motor neuron development through dysregulated beta-catenin-dependent signaling. Early neurodevelopmental processes affect mature neuron functions; motor axon morphogenesis is essential for action potential transmission and target innervation. Defects in these processes may therefore confer functional vulnerability at later stages, rendering motor neurons susceptible to degeneration in ALS. This is consistent with reports of aberrant motor axon morphology associated with ALS-related genes including TDP-43 (Kabashi et al., 2010), SOD1 (Clark et al., 2016; Lemmens et al., 2007; Ramesh et al., 2010) and C9orf72 (Burguete et al., 2015; Ciura et al., 2013).
Our observation that UBQLN4D90A expression impairs proteasome function (Figure 3A–D) suggests that the variant acts in a dominant-negative manner. However, our finding that beta-catenin level was still reduced in UBQLN4D90A expressing cells compared to nontransfected cells (Figure 3E,F) suggests that the variant results in a partial loss-of-function. This may be because in the short term UBQLN4D90A functions with reduced efficiency, but still facilitates degradation of substrates. Overtime, reduced UBQLN4 functional efficiency may overload the proteasome pathway; therefore, an initial partial loss of function can eventually lead to a dominant negative gain of function outcome. This notion is consistent with the progressive nature of ALS.
It is notable that our findings link UBQLN4 with beta-catenin in the context of ALS, as recent work suggested a similar role for beta-catenin signaling in the pediatric motor neuron disease Spinal Muscular Atrophy (SMA) (Wishart et al., 2014). Wishart et al. reported similar morphological phenotypes in UBA1-deficient zebrafish motor neurons in which beta-catenin expression was heightened, and showed a dose-dependent rescue effect by quercetin. The degradation of beta-catenin is known to be carried out through the ubiquitin proteasome pathway (Aberle et al., 1997). Disruption of UBQLN4 or UBA1 function, which are both involved in the UPS pathway, could therefore lead to beta-catenin accumulation and aberrant signaling. Given the wide-ranging and critical roles in neuronal development and function played by beta-catenin, its upregulation in ALS and SMA may understandably have dire consequences for the development, function, and survival of affected motor neurons. Collectively these studies suggest heightened beta-catenin activity as a common mechanism between the adult-onset motor neuron disease ALS and the pediatric motor neuron disease SMA.
Taken together, we revealed a novel role for UBQLN4 in regulating motor axon morphogenesis through the UPS. Dysregulation of this function by the ALS-associated UBQLN4D90A variant leads to compromised proteasome function and beta-catenin accumulation, conferring abnormalities in motor axon morphogenesis (Figure 5), and contributing to motor neuron degeneration in ALS. Further exploration of the underlying mechanism may provide new insights for understanding ALS pathogenesis and for therapeutic development.
This study has been approved by the Northwestern University Institutional Review Board. Blood samples were collected after obtaining written informed consent. Eleven sets of primers (Supplementary file 1) were synthesized for PCR amplification of human UBQLN4 exons and Sanger sequencing using the EQ 8000 Genetic Analysis System (Deng et al., 2011). A total of 267 familial ALS index cases and 411 sporadic ALS cases were sequenced. The familial case in which the UBQLN4 variant was identified was compared to numerous control large-scale reference datasets to validate the variant: these included 332 in-house controls, SNP databases with a total of >15,000 sequenced alleles, and the Exome Aggregation Consortium (ExAC) database with a total of 60,706 unrelated individuals (Lek et al., 2016).
This study has been approved by the Institutional Animal Care and Use Committee of the Lurie Children’s Hospital of Chicago. All studies were conducted in accordance with the US Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. Wild-type AB zebrafish (RRID:ZIRC_ZL1) were obtained from the Zebrafish International Resource Center (Eugene, OR) and maintained in standard conditions. Timed-pregnant wild-type CD1 mice (RRID:IMSR_CRL:086) were obtained from Charles River Laboratories (Chicago, IL).
A human UBQLN4 cDNA clone was obtained from GE Dharmacon (Clone ID: 6183942, Accession #BU149502). The coding region was excised and 5’Fse1 and 3’Asc1 sites were inserted via PCR (UBQLN4 FseFwd 5’ GATC GGC CGG CCT ACC ATG GCG GAG CCG AGC GGG GCC GAG 3’; UBQLN4 AscRev 5’ GAT CGG CGC GCC TTA GGA GAG CTG GGA GCC CAG CAG 3’). The product was ligated into a pCS2-Flag vector, and site-directed mutagenesis (Q5 kit, NEB) was performed to convert the 2747 adenine to cytosine (UBQLN4 Q5 Fwd 5’ AAG GCT CAA GcT CCA GCT GCT G 3’; UBQLN4 Q5 Rev 5’ CTG AGG GGT CTT GAT GAC 3’). Both wild-type and mutant constructs were verified by sequencing. UbG76V-GFP was a gift from Nico Dantuma (Addgene plasmid #11941).
Capped mRNAs were generated from linearized UBQLN4 wild-type and mutant constructs via in vitro transcription using the mMessage mMachine SP6 Transcription Kit (Ambion). Zebrafish embryos were injected with capped mRNA (200–300 pg target) at the single-cell stage and grown until 32 hr post-fertilization. Embryos were treated with quercetin (50 μM in 0.25% DMSO in embryo medium) or vehicle control from six hours post-fertilization until fixation.
Primary mouse spinal cord motor neurons were isolated, dissociated, and cultured as described previously (Miller et al., 2015). The NSC-34 motor neuron cell line (RRID:CVCL_D356) was provided by Dr. Neil Cashman (University of British Columbia, Vancouver, Canada; [Cashman et al., 1992]). The NSC-34 line is not included in the International Cell Line Authentication Committee’s Database of Cross-Contaminated or Misidentified Cell Lines, and is negative for mycoplasma. Primary and NSC-34 cells were transfected using Lipofectamine 2000 (Life Technologies) according to manufacturer’s instructions. Cultures were fixed for imaging or lysed for Western blot analysis 48 hr post-transfection. For cycloheximide protein stability assay, cells were treated for 0, 2, 4, or 6 hr with 100 μg/ml cycloheximide following UbG76V-GFP transfection. For quercetin treatment, a working concentration of 0.1 μM was used.
Western blotting was performed as described (Miller et al., 2015) with the following antibodies and dilutions: mouse anti-beta-catenin (BD-Biosciences #610153, RRID:AB_397554; 1:500), goat anti-beta-actin (Santa Cruz #1616, RRID:AB_630836; 1:1000), rabbit anti-GAPDH (Santa Cruz #25778, RRID:AB_10167668; 1:1000), mouse anti-GFP (Affymetrix #14-6674-80, RRID:AB_2572899; 1:300), and mouse anti-Flag M2 (Sigma #F3165, RRID:AB_259529, 1:1000). Whole-mount immunostaining of zebrafish embryos was performed as follows: embryos were fixed overnight in 4% PFA at 4°C. The tissue was permeabilized with proteinase K (10 μg/ml) for 40 min and blocked with 5% BSA and 1% goat serum in PBS for one hour. Embryos were incubated in primary antibody, mouse anti-znp1 (DSHB #znp - 1, RRID:AB_2315626; 1:500), in blocking solution overnight at 4°C. Goat-anti-mouse Cy3 secondary antibody (Jackson ImmunoResearch, 1:250) diluted in blocking solution was applied for one hour at room temperature. Immunostaining of cultured mouse neurons was performed as described (Miller et al., 2015) with the following antibodies and dilutions: mouse anti-DDDDK tag M2 (Abcam #ab45766, RRID:AB_731867; 1:1500), mouse anti-Isl1 (DSHB #40.2D6, RRID:AB_528315; 1:60), and mouse anti-beta-catenin (BD-Biosciences #610153, RRID:AB_397554; 1:300). All images were acquired with the Zeiss 510 Meta Laser Scanning Microscope.
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Anne E WestReviewing Editor; Duke University School of Medicine, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "A Novel ALS-Associated Variant in UBQLN4 Regulates Motor Axon Morphogenesis" for consideration by eLife. Your article has been favorably evaluated by Marianne Bronner (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal his identity: Thomas H Gillingwater (Reviewer #3).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
This manuscript from Edens and colleagues reports on a timely and potentially important set of experiments, detailing a novel variation in the UBQLN4 gene associated with ALS. While this in itself may not be particularly novel (mutations in other ubiquilin genes have been linked to neurodegenerative conditions, including motor neuron disease), the subsequent demonstration of a mechanism of action is exciting and is likely to be of considerable interest to a broad readership. The authors have used a range of in vitro and in vivo approaches to show that the UBQLN4D90A variant; i) modulates motor axon stability/morphology, ii) affects proteasome-mediated degradation, leading to an abnormal accumulation of β-catenin, and iii) modulates motor axon stability via a β-catenin-dependent pathway, with inhibition of β-catenin ameliorating motor neuron defects. The short paper is, in general, well written and the study has been well-designed and performed. Only a few issues need to be addressed.
1) For the in vitro data shown in Figure 2 (and Figure 3F), the authors need to provide evidence that the cells they are quantifying are indeed motor neurons (e.g. co-label with motor neuron-specific markers). Were there any other cellular defects in these motor neurons alongside changes in neurite number (e.g. evidence for activation of cell death pathways and/or changes in the size of the neuronal soma)? Also, these data should be quantified in the same way the zebrafish axonal branching defects were quantified.
2) For both the in vitro and in vivo experiments in Figure 2, the authors need to provide data showing the level of overexpression achieved in their UBQLN4-WT and UBQLN4D90A cells/animals. Were the UBQLN4D90A animals otherwise healthy?
3) Example raw GFP images should be provided to accompany Figure 3A.
4) The dose(s) of quercetin need to be clearly stated in the results and legend to Figure 4. Was there any evidence for a dose-dependent effect of quercetin? This is a particularly interesting finding of the manuscript, so elaboration with either experiments or discussion will be of strong interest to the readers.
5) Is the in vivo developmental branching phenotype observed in zebrafish in the current study similar to any other published zebrafish models of ALS, or does this appear to be a UBQLN4D90A specific phenotype? The similarity to zebrafish models of spinal muscular atrophy is quite striking, but did the authors find any evidence for truncated motor axons, as well as aberrantly-branched axons (c.f. McWhorter et al. 2003 J Cell Biol; Wishart et al. 2014 JCI)? If this can be addressed experimentally with further analysis of the data on hand, this should be added.https://doi.org/10.7554/eLife.25453.011
- Yongchao C Ma
- Yongchao C Ma
- Yongchao C Ma
- Yongchao C Ma
- Teepu Siddique
- Teepu Siddique
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
This work was supported by NIH grants NS094564, AG043970 and grants from The Hartwell Foundation and Whitehall Foundation (YCM); NS078504, NS078504, Les Turner ALS Foundation, the Foglia Family Fund for ALS Research, Les Turner ALS Foundation/Herbert C Wenske Foundation Professorship, Vena E Schaff ALS Research Fund, and SP Foundation (TS). YCM is Ann Marie and Francis Klocke MD Research Scholar supported by the Joseph and Bessie Feinberg Foundation.
Human subjects: The use of human subjects in this study has been approved by the Northwestern University Institutional Review Board (IRB). Informed consent was obtained from all subjects.
Animal experimentation: All animal use in this study has been approved by the Institutional Animal Care and Use Committee (IACUC) of the Lurie Children's Hospital of Chicago (protocols 14-012 and 15-006). All studies were conducted in accordance with the US Public Health Service's Policy on Humane Care and Use of Laboratory Animals.
- Anne E West, Reviewing Editor, Duke University School of Medicine, United States
© 2017, Edens et al
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