UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS

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    Black et al., provide evidence that levels of Paternally Expressed Gene 10 (PEG10) protein are regulated by Ubqln2 and that proteolytic fragments from PEG10 cleavage induce changes in gene expression, in particular genes that encode proteins involved in axon biology. Based on these data and the finding of an increase in PEG10 levels and alterations of proteins regulated by PEG10 in the spinal cord of ALS patients, they propose that abnormal induction of PEG10-regulated genes is involved in ALS. However, the evidence for these claims and PEG10 involvement could be strengthened, and the rigor of the work could be enhanced in places.

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Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS cases are caused by mutation of the proteasome shuttle factor Ubiquilin 2 ( UBQLN2 ), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate that UBQLN2 regulates the domesticated gag-pol retrotransposon ‘paternally expressed gene 10 (PEG10)’ in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated ‘nucleocapsid’ fragment, which uniquely localizes to the nucleus and changes the expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through the regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

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  1. eLife assessment

    Black et al., provide evidence that levels of Paternally Expressed Gene 10 (PEG10) protein are regulated by Ubqln2 and that proteolytic fragments from PEG10 cleavage induce changes in gene expression, in particular genes that encode proteins involved in axon biology. Based on these data and the finding of an increase in PEG10 levels and alterations of proteins regulated by PEG10 in the spinal cord of ALS patients, they propose that abnormal induction of PEG10-regulated genes is involved in ALS. However, the evidence for these claims and PEG10 involvement could be strengthened, and the rigor of the work could be enhanced in places.

  2. Reviewer #1 (Public Review):

    Previous studies from this group reported that PEG10 is increased in the spinal cord from Ubiquilin 2-/- mice as well as PEG10 being elevated in models of Ubiquilin 2-mediated ALS. In this study, the authors provide evidence supporting the concept that the proteasome factor Ubiquilin 2 regulates the activity of the Gag-pol retrotransposon gene (PEG10). Mutations in Ubiquilin 2 underlie a portion of the familial forms of ALS. It is found that in spinal cord tissue from sporadic ALS patients PEG10-pol levels are elevated, leading to the conclusion that altered regulation of PEG10 levels by Ubiquilin 2 and a subsequent alteration in genes regulating axon remodeling may contribute to all forms of ALS pathogenesis. Strengths of this work include the extensive analyses and direct data on the mechanism by which Ubiquilin 2 regulates PEG10 levels in human cells resulting in less PEG10. They further show that peptides generated by the self-cleavage of PEG10 alter the expression of genes involved in axon function. The major weakness of this study is the complete absence of data that directly show that an alteration of PEG10 by Ubiquilin 2 is critical for ALS pathogenesis. As noted by the authors, multiple pathways have been broadly implicated in genetics and sporadic forms of ALS. Thus while the study provides interesting data on the regulation of PEG10 by Ubiquilin 2, the extent to which this pathway underlies ALS pathogenesis and/or progression is speculative.

  3. Reviewer #2 (Public Review):

    This is a follow-up study by the senior author, who previously showed in a 2021 JBC paper that levels of Paternally Expressed Gene 10 (PEG10) protein, among many other protein changes, are increased in the spinal cord of Ubqln2 knockout (KO) animals (JBC 2021). In this report, they provide more direct evidence that PEG10 levels are regulated by ubqln2 and that PEG10 can be proteolytically cleaved generating fragments, which when overexpressed, induce alterations in gene expression. Through proteomic analysis of spinal cord tissue from control and ALS patients, they found that PEG10 levels and the signature of genes regulated by its products are increased in ALS, proposing that elevation in PEG10 is a novel marker and driver of ALS.

    PEG10 resembles a retrotransposon, encoding virus-like gag-pol products. It is only found in eutherian mammals. Although it has lost its ability to transpose, it still retains the retroviral-like translation frameshifting property generating two main products, gag (reading frame 1, RF1) and gag-pol (RF1/2). PEG10 is essential for survival. It plays an important role in RNA-binding and trophoblast stem cell specification, being required for placental development. It is also expressed in several adult tissues, but its function in them is obscure. A recent study showed PEG10 RF1 and RF1/2 bind the deubiquiting enzyme USP9X, and that loss of USP9X destabilizes RF1 but not RF1/2, suggesting USP9X regulates ubiquitination and proteasomal degradation of PEG10 (Abed et al. PLOS One 2021). Additionally, Abed et al. showed PEG10 products support virus-like particle (VLP) assembly and that both RF1 and RF1/2 localize to the cytoplasm, whereas a portion of RF1/2 is found in the nucleus of some cells. They further showed PEG10 binds and regulates RNA expression, most probably through interaction with the 3'-ends of the RNAs but found no common binding motif suggesting interaction could be with the secondary structure.

    As mentioned, the senior author previously reported in a JBC article in 2021 that PEG10 levels are elevated in ubqln2 knock out (KO) mice, but that its levels were slightly decreased in the P497S mouse model of ALS. They validated PEG10 as an interactor of ubqln2 by proximity-dependent biotin labeling. A review of the current manuscript follows.

    1. Evidence that ubqln2 regulates PEG10 accumulation (Fig 1). The authors use human embryonic stem cells to investigate how knockout (KO) of different ubqln isoforms (1, 2, and 4) affects PEG10 accumulation, showing that only KO of ubqln2 increases the RF1/2 product.

    a) There is considerable variation in PEG10 expression in the duplicate sample sets provided, but this is not reflected by the error bars (fig 1 A and B). For example, RF1/2 is quite different in the two ubqln4 KO lysates, yet the error bars do not capture the variation. Better loading and quantification is needed. Also, in the KO cells, gag levels are slightly increased, which is consistent with alterations in proteasomal degradation. Alternatively, the changes in RF1/2 could also result from changes in read-through translation, but this is not investigated. Also, it would be helpful to include blots showing the lower Mol weight PEG10 products, to see how they change relative to Fig 3.

    Fig 1G. The authors examined if removal of the poly proline rich region (PPR) from PEG10 affects RF1/2 regulation by ubqln, confirming its requirement.

    b) The mechanism why deletion of the PPR abolished RF1/2 regulation by ubqlns was not examined. Is it from accelerated degradation? Also, it is not clear why the authors use the triple ubqln KO cells and did not perform that tests in the different ubqln KO cells. The latter comment applies for several of their investigations, leading to uncertainty regarding the specificity of ubqln2 in PEG10 regulation. It is possible that removal of most ubqlns stalls protein degradation affecting PEG10 turnover?

    2. The authors investigated the phylogenetic relationship between PEG10 and ubqln2 demonstrating that PEG10 levels from marsupials that lack a PPR can be increased by appending a PPR from human PEG10. They used triple ubqln KO cells for these investigations.

    a) The change they describe is not obvious in Fig2C and E as they appear quite small. They also conclude that ubqln2 regulates PEG10 by these studies, but really the experiments show it is from loss of all ubqlns, not ubqln2 specifically.

    3. The authors show PEG10 is capable of self-cleavage of the RF1 product, generating 2 detectable N-terminal products, and several other fragments, including a C-terminal nuclear capsid (NC) fragment (Fig3). They show expression of HA-tagged NC fragment localizes to mainly the nucleus, whereas several other PEG10 products and fragments localize to the cytoplasm. They provide strong support that PEG10 is capable of self-cleavage by mutation of an aspartate residue (D) in a DSG motif in the protein to alanine (A to → ASG), which abolished cleavage. They also conducted a nice experiment showing the ASG mutant can be cleaved in trans by introduction of WT PEG10.
    a) The authors never show evidence for liberation and accumulation of the NC fragment, only for an artificially tagged protein by immunofluorescence. Use of a tag to study its localization and affects is problematic as the could influence its properties. They need to show that the fragment is detectable because of their central claim that it is responsible for inducing changes in genes. Biochemical fractionation studies could also reveal the extent of the partitioning of the fragment in the nucleus and cytoplasm. The mechanism by which the NC fragment induces changes in gene expression is not clear.

    4. The authors show differences in gene expression upon transfection of HEK293 cells with PEG10 RF1, RF1/2, and NC expression constructs. They show that two PEG10-regulated genes, DCLK1 and TXNIP, are both increased in the spinal cord in sporadic ALS cases compared to controls.
    a) It is not clear from the studies whether the changes found in ALS are related to changes in PEG10 specifically, or for other reasons. Additionally, more rigorous comparison in many more ALS and controls is needed. PEG10 levels increase upon cell differentiation (Abed et al.) so the changes in ALS may reflect a compensatory and protective response.

    5. To investigate if PEG10 RF1/1 levels are altered by ALS mutations in ubqln2 they transfected ubqln TKO cells with either wt ubqln2, or with mutants carrying either the P497H or P506T ALS mutations. They show PEG10 RF1/2 levels are reduced by overexpression of both the wt and P497H mutant, but not by the P506T mutant. They claim that P497H expression did not affect RF1/2 levels. The authors conducted a proteomic comparison of extracts from the spinal cord of two controls, one P497H ubqln2 case, and six sporadic ALS cases. They found increased levels of RF1/2 in the ALS cases. They also found neurofilament medium and neurogranin were both reduced in the ALS cases. Based on these changes they speculate that PEG10 is a novel marker for ALS.
    a) The conclusion that the P497S mutant did not affect RF1/2 is incorrect. It reduced RF1/2 slightly more than wt ubqln2. In fact, it appears that expression of all three (wt and the 2 ALS mutants) ubqln2 proteins reduce RF1/2 significantly, compared to the TKO cells.
    b) The changes in PEG10 found in the ALS cases are difficult to evaluate because too few controls and ALS cases were used for the comparison. Huge variations in the levels of PEG10 and of the other proteins graphed In Fug 6B-F were seen in the two controls. The comparison needs to be done with many more samples for sound statistical comparison. Were the samples compared from the same region of the spinal cord?

    General comments

    1. In the Discussion the authors write that because ubqln2 is the only ubqln capable of regulating PEG10 RF1/2 levels, the PXX domain that is only present in ubqln2 is likely responsible for the regulation. There is no proof in support of this hypothesis. Only one ALS-causing mutation (P506T) in the PXX domain, but not the P497H mutation in the same PXX domain, affected RF1/2 accumulation, inconsistent with general involvement of the PXX domain in PEG10 regulation.

    2. The authors claim that ubqln2 may have specifically evolved to restrain PEG10 expression, but don't mention USP9X as being another regulator. The common theme that emerges from these studies is that PEG10 levels are regulated by any mechanism that interferes with ubiquitination/proteasomal degradation. Indeed, immunoblots of the gag-pol (RF1/2) in the different ubqln KO cells show a smear at high molecular weight consistent with the accumulation of ubiquitinated PEG10. The authors imply that the transcriptional changes caused by the alteration in PEG10 levels by ubqln2 are responsible for ALS (title of the paper), but this is merely speculation as the effects of the changes are not known. The changes found could be protective. They also claim PEG10 may serve as a novel biomarker for ALS, but such a claim is not justified from the limited analysis conducted so far, which will require more extensive proof.

  4. Reviewer #3 (Public Review):

    Ubiquilin 2 (UBQLN2) encoded by a familial predisposition gene for Amyotrophic Lateral Sclerosis (ALS) is a proteosome shuttle protein shown to associate with Cxx2 and mammalian Ty3/gypsy-like retrotransposon PEG10 in previous work by this author. Other work has shown that PEG10 is expressed from an imprinted gene required for placental development and in adrenal and brain tissues. Increases in PEG10 expression are also implicated in some cancers. Building on previous work by the senior authors (Whitely et al., 2021) which showed that PEG10 interacts with components of the UBQLN machiner and is elevated in UBQUILN TKO human and mouse cells, this work focuses on the interaction of UBQLN proteins and PEG10 target. Using a HEK human kidney cell line, authors show first that targeting of PEG10 depends upon a proline-rich repeat at the carboxy terminus of Gag-Pol unique to eutherian animals; second, that the aspartyl protease previously implicated in gag-pol processing can release the gag carboxyl-terminal CHCC zinc knuckle nucleocapsid to concentrate in the nucleus and correlates with changes in expression of genes related to neuronal development. Finally, they show that PEG10 is elevated in human spinal cord neuron cell lines.


    The primary strengths of this manuscript lie in the multiple experiments linking UBQLN2 activity to the target PEG10 PPR motif and in potentially linking Gag-Pol and NC production to changes in cellular gene expression. The authors knock out multiple UBQLN genes in various species and demonstrate the phylogenetic correspondence between UBQLN2 PEG10 levels.


    Although this manuscript links elevated PEG10 protein levels to fALS mutated UBQLN2 and changes in neuronal gene expression, it does not as the title suggests demonstrate that UBQLN2 control of PEG10 is required for "neuronal health in ALS". This is an awkward title and the link between neuronal health and the ability to turnover PEG10 is not clearly established since most of the experiments were conducted in non-neuronal human cell lines.

    Authors could more completely set the context for their work including their own work (Whiteley et al., 2021) and findings in Angelman (UBE3A, Pandya et al., 2021) and Parkinsons (Sakharkar et al., 2019) which, rather than detracting from their work, would confer greater interest. In addition, they mention in passing that in the absence of familial predisposition mutations, in ALS UBQLN2 can be inactivated by trapping in aggregates. This undermines their comparison of fALS and sALS cells.

    The multiple western blots while consistent with authors conclusions, do not show greater than two-fold differences in PEG10 protein levels in the absence of UBQLN2 proteins so that there are likely other factors besides UBQLN2 influencing PEG10 levels. For example, the authors do not comment on PEG10 extracellular VLP production which occurs in some cells or that other proteins previously implicated as targets of PEG10 could be influencing the neuronal phenotypes of fALS. In addition, clarification of the different phenotypes of fALS mutations in the UBQLN2 hotspot would have addressed concerns that more than UBQLN2 is involved in the phenotype.