The author has requested to add support from the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM152335. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The following was added to the funding section of the published article.
\"Institutional support is provided to T.M.G. through a NIH Center grant from National Institute of General Medical Sciences (NIGMS; P20GM152335).\"
","date":"Mon Dec 22 2025","correctionType":"corrigendum"}],"versions":[{"id":"db95a9aa-cf91-4252-90b7-1eafe411f0c4","decisionLetter":"Dear Teresa M. Gunn,
Your article \"Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy\" has been accepted, with additional data, which may entail further experiments. If gathering more data or doing more experiments is not possible, please include a caveat that the relevant conclusion is preliminary.
Recommendation: accept, with additional data/experiment or a caveat concerning the conclusion given the missing information, as well as any additional minor or major modifications
Review
This short communication describes the effects of conditional deletion of the Tsg101 gene, encoding a component of the ESCRT-1 complex, within Schwann cells in the mouse. The authors report that inactivation of Tsg101 within Schwann cells leads to a postnatal lethality (usually within 3 weeks postnatal) associated with tremor and arthrogryposis. At the histological level, the sciatic nerves of the conditional knockouts contain Schwann cells, but show reduced MBP+ myelin sheaths and the presence of onion bulb structures. These findings provide a novel demonstration of the necessity for Tsg101 (and, by extension, endosomal sorting) for maintenance of PNS myelin.
The article is well-written, clearly outlining the relevant background literature of other human variants or mouse mutants linking endosomal trafficking to dysmyelination/demyelination. The phenotype of the conditional knockouts seems to be quite robust, with the authors reporting on the postnatal lethality of 89 conditional knockouts (out of 427 pups born). My only concern is that in spite of this large number of available animals, the histology shown is of one control and one conditional knockout nerve at postnatal day 15 and 21, with no quantification of the myelin phenotypes. Given the strength of the phenotype I do not anticipate that replication and quantification of the histology would alter the conclusions of the piece, so will leave it to editorial discretion as to whether the figure meets microPublication guidelines in the absence of replication/quantification.
Editorial Comment:
For this article we consider the key result to be the ~25% of affected animals with tremor, which we find appropriately supported. Nonetheless, we acknowledge the reviewer's concern with the lack of replication of the histology results. However, we consider the reported histology to be backup evidence. To continue, the authors should say either \"we looked at one mouse\" or \"we report on the mouse, but in more casual observations of others, we saw consistent results, so that the reviewer's concerns are addressed.
We kindly ask you to address each point and summarize your changes in line with the reviewer's response in the 'Comments to Editor' section on the platform. In order to expedite the processing of your revised manuscript, please be as specific as possible in your responses.
The microPublication Editorial Team
","decision":"revise","submitted":true,"abstract":"Myelinating Schwann cells are particularly susceptible to defects in endosomal trafficking. TSG101 is a component of the endosomal trafficking machinery that mediates the sorting of ubiquitinated receptors into multivesicular bodies. We previously demonstrated that deleting Tsg101 from mouse oligodendrocytes in the central nervous system causes rapid onset de/dys-myelination and vacuolation of white matter, suggesting an important role for TSG101-dependent trafficking in myelination. Here, we show that TSG101 is also required for normal myelination in the peripheral nervous system.
","acknowledgements":"We thank Anita Pecukonis and the McLaughlin Institute Animal Resource Center for excellent animal care and Dr. John R. Bermingham Jr for instruction on sciatic nerve dissection.
","authors":[{"affiliations":["McLaughlin Research Institute"],"credit":["investigation","writing_reviewEditing"],"email":"derek@mclaughlinresearch.org","firstName":"Derek","lastName":"Silvius","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","writing_reviewEditing"],"email":"edwardhu@buffalo.edu","firstName":"Edward","lastName":"Hurley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Albany Medical College"],"credit":["writing_reviewEditing"],"email":"poitely@amc.edu","firstName":"Yannick","lastName":"Poitelon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Wayne State University, Detroit, Michigan, United States"],"credit":["resources","writing_reviewEditing"],"email":"kuwagner@wayne.edu","firstName":"Kay-Uwe","lastName":"Wagner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","supervision","validation"],"email":"mamillig@buffalo.edu","firstName":"M. Laura","lastName":"Feltri","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","methodology","fundingAcquisition"],"email":"tmg@mclaughlinresearch.org","firstName":"Teresa M.","lastName":"Gunn","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2688-6420"}],"comments":"Thank you for considering our microPublication.
","dataTable":null,"disclaimer":true,"funding":"This work was funded by the McLaughlin Research Institute and its generous supporters. Kay-Uwe Wagner is the Lloyd and Marilyn Smith Endowed Professor of Breast Cancer Research at the Karmanos Comprehensive Cancer Center.
","image":{"name":"Fig 1 Tsg101 SC null.png","url":"https://portal.micropublication.org/uploads/c7ad46ba5ce67475b19a471661ada5c2.png"},"imageCaption":"(A-C) Semi-thin cross sections of sciatic nerves from control (Tsg101fl/+; P0-Cre+) and Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) IHC for myelin basic protein (MBP) on 7m paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. (G-H) Semi-thin cross sections of sciatic nerves of control and Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
","imageTitle":"Histopathology of sciatic nerves lacking TSG101 in Schwann cells.
","laboratory":{"name":"","WBId":""},"methods":"Animals
All studies were approved by the McLaughlin Research Institute’s Institutional Animal Care and Use Committee and adhered to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Mice homozygous for a Tsg101 conditional knockout allele (129X1/SvJ-Tsg101tm1Kuw, referred to herein as Tsg101fl; RRID: MMRRC_037407-MU), in which exon 1 is flanked by a floxed neo cassette inserted approximately 3 kb upstream and a single loxP site inserted 230 bp into intron 1 (Wagner et al., 2003), were mated to mice hemizygous for Tg(Mpz-cre)26Mes/J (obtained from the Jackson Laboratory and referred to herein as P0-cre; RRID: IMSR_JAX:017928). P0-cre transgenic mice express cre recombinase in Schwann cells, under the control of the myelin protein zero (P0, Mpz) promoter, by embryonic day 14 (Feltri et al., 1999). Tsg101fl/+; P0-cre+ F1 offspring were backcrossed to Tsg101fl/fl mice and Tsg101fl/fl; P0-cre+ and control (Tsg101fl/+; P0-cre+ ad Tsg101fl/+; P0-cre-neg) pups were obtained from intercrossing N1 or N1F1 Tsg101fl/+; P0-cre+ x Tsg101fl/fl; P0-cre-neg sibs.
Histology
Sciatic nerve segments were sampled from approximately the same position for each animal. Contralateral nerves were either fixed in 4% buffered paraformaldehyde, for immunohistochemistry (IHC), or 2% buffered glutaraldehyde with postfixation in 1% osmium tetroxide (for semi- and ultra-thin sections).
For IHC, specimens were embedded in paraffin following standard protocols and sectioned at 5 microns. Following deparaffinization and rehydration, sections were permeabilized in a % Triton-X100 solution in PBS, then subjected to antigen retrieval in 10mM sodium citrate (pH 6.0, 100 C, 10 minutes). Slides were blocked in 10% serum, then incubated with an antibody against myelin basic protein (MBP; Covance Cat# SMI-99, RRID:AB_2314772) at 1:1000, followed by horseradish peroxidase conjugated anti-mouse secondary antibody (BD Pharmingen horseradish peroxidase-conjugated anti-mouse Ig, cat# 554002, RRID: AB_395198) at 1:100 and chromogenic visualization using NovaRed substrate (Vector Labs, CA). Slides were counterstained with hematoxylin prior to coverslipping, and then examined on a Zeiss AxioImagerM1 light microscope.
Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996). Briefly, sciatic nerves were fixed in 2% buffered glutaraldehyde, then postfixed in 1% osmium tetroxide. After alcohol dehydration, nerves were submerged in propylene oxide, and then in a 1:1 mixture of Epon-propylene oxide. Nerves were embedded in 100% Epon, and resin was allowed to polymerize. Semithin transverse sections were sliced 0.5-μm-thick using Leica UC7, stained with 2% toluidine blue, and then examined by light microscopy with Leica DM6000B. EM transverse sections were sliced 700–900 Å-thick using Leica UC7, stained with uranyl acetate and lead citrate, and then examined with an electron microscope (model FEI BioTwin). Analyzed sections were sliced from the distal end of embedded sciatic nerve. Images acquired from semithins and EMs were nonoverlapping and comprehensive.
","reagents":"","patternDescription":"The endosomal pathway traffics receptor proteins and lipids into early endosomes and directs them either to recycling endosomes for trafficking back to the cell membrane or sorting into intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which fuse with lysosomes to degrade their contents or with the plasma membrane to secrete ILVs as exosomes (reviewed in Scott, Vacca, & Gruenberg, 2014). The timing of these events is important to cellular processes since receptors can continue to activate downstream signaling pathways while they remain on early endosomes. Several forms of demyelinating CMT are caused by mutations in genes encoding proteins involved in membrane dynamics and endosomal trafficking, including N-myc downstream regulated gene 1 (NDRG1), myotubularin related protein 2 (MTMR2), SET binding factor 2 (SBF2/MTMR13),SH3 domain and tetratricopeptide repeats 2 (SH3TC2), dynamin 2 (DNM2), FIG4 phosphoinositide 5-phosphatase (FIG4), and LPS-induced TN factor (LITAF/SIMPLE) (reviewed by Markworth, Bähr, & Burk, 2021). For example, mutations in SH3TC2 that cause CMT type 4C cause impaired recycling of membrane components necessary for Schwann cell function (Roberts et al., 2010; Stendel et al., 2010). Furthermore, mouse models with Schwann cell-specific deletion of Fig4, HGF-regulated tyrosine kinase substrate (Hgs/Hrs), or phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) show mild peripheral hypo/de/dys-myelination associated with altered ERBB2/3 signaling (Logan et al., 2017; McLean et al., 2022; Vaccari et al., 2015). In Schwann cells, neuregulin 1 (NRG1) signaling through erb-b2 receptor tyrosine kinases 2 and 3 (ERBB2/B3) mediates myelination, and endosomal trafficking of NRG1-bound ERBB2/B3 regulates receptor down-regulation and recycling (Newbern & Birchmeier, 2010; Salzer, 2015). Disrupted endosomal sorting can result in sustained activation of downstream pathways, including the ERK1/2 signaling cascade, to negatively impact Schwann cell myelin integrity (Newbern & Birchmeier, 2010; Salzer, 2015). Thus, there is support for the idea that endosomal trafficking defects can cause Schwann cell dysfunction and demyelination, reinforcing the relevance of this pathway to myelination.
Tumor susceptibility gene 101 (TSG101) encodes a component of the Endosomal Sorting Complex Required for Transport-1 (ESCRT-I), which helps mediate the sorting of ubiquitinated receptors onto ILVs of MVBs. The ESCRT-0 protein, HGF-regulated tyrosine kinase substrate, HGS (formerly referred to as HRS) mediates the initial recruitment of ESCRT-I to endosomes (via interaction with TSG101), but in multiple mammalian cell lines, siRNA depletion of TSG101 or HGS had significantly different effects on endosomal and MVB morphology (Bache, Brech, Mehlum, & Stenmark, 2003; Lu, Hope, Brasch, Reinhard, & Cohen, 2003; Raiborg, Malerod, Pedersen, & Stenmark, 2008; Razi & Futter, 2006). Specifically, knockdown of TSG101 inhibited epidermal growth factor degradation and MVB formation and caused tubulation of the vacuolar domains of early endosomes, while depletion of HGS had only a modest effect on EGF degradation, did not induce tubulation of early endosomes, and resulted in the production of enlarged MVBs containing few ILVs but that could still fuse with the lysosome (Bishop, Horman, & Woodman, 2002; Doyotte, Russell, Hopkins, & Woodman, 2005; Razi & Futter, 2006). These data suggest that HGS and TSG101 have distinct roles in the endosomal trafficking pathway, with TSG101 being required for the formation of stable vacuolar domains within the early endosome that subsequently develop into MVBs and HGS being more important in the formation and/or accumulation of ILVs within MVBs. Mice lacking HGS in Schwann cells developed mild motor and sensory defects, a reduced number of myelinated axons and thinner myelin sheaths in the sciatic nerve, as well as aberrantly folded myelin sheaths (McLean et al., 2022).
TSG101 and HGS also both interact and partially colocalize with LITAF (Lee, Olzmann, Chin, & Li, 2011), which is expressed in Schwann cells and mutations in it cause dominant demyelinating peripheral neuropathy, CMT1C (Bennett et al., 2004). Although one study showed that CMT1C-associated LITAF mutations did not effect on its subcellular localization or association with TSG101 (Shirk, Anderson, Hashemi, Chance, & Bennett, 2005), another study showed a dominant negative effect on EGFR degradation and lysosomal trafficking of EGF, associated with reduced membrane association of HGS and TSG101 (Lee et al., 2011). In the latter study, expression of CMT1C-associated LITAF mutants in Schwann cells caused prolonged activation of ERK1/2 signaling, presumably downstream of NRG1-ERBB2/3 signaling.
Deleting Tsg101 from oligodendroglia in the central nervous system resulted in severe, rapid-onset myelination defects and vacuolation (Walker, Oehler, Edinger, Wagner, & Gunn, 2016), suggesting an important role for TSG101-dependent trafficking in signaling pathways that regulate myelination. To test whether TSG101 is also required for normal myelination in the peripheral nervous system, we investigated the consequences of deleting Tsg101 in Schwann cells. We predicted this would cause a more severe peripheral neuropathy than deleting Hgs, given the stronger effect of Tsg101 depletion on endosomal/MVB phenotypes in cultured cells.
Tsg101 conditional knockout mice (Tsg101tm1KuW, referred to here as Tsg101fl) were mated to P0-Cre transgenic mice, which express cre recombinase specifically in Schwann cells starting on embryonic day 13.5. Cre-positive Tsg101fl/+ offspring were backcrossed to Tsg101fl/fl animals. Pups that were homozygous for the Tsg101 conditional allele and carried the P0-Cre transgene (referred to herein as Tsg101-Schwann cell null, or Tsg101SC-null animals) were smaller than their littermates, developed a tremor by 12 days of age, had abnormal posture of their fore- and hind-limbs (arthrogryposis), and failed to thrive. They died throughout the postnatal period, with very few surviving to 3 weeks of age. We recorded 89 affected animals out of 427 pups born from 15 different breeder pairs, for a frequency of ~21% affected. This is significantly different from the 25% expected (c2= 4.04, p = 0.044) and is likely due to the loss of some affected animals prior to them being observed and recorded.
Histological analysis of the sciatic nerves of Tsg101SC-null animals at postnatal days 15 and 21 revealed striking de- and dys-myelination (Fig. 1). Toluidine blue-stained semi-thin cross sections showed reduced myelin, enlargement of the interstitial space, and presence of onion bulb structures in the sciatic nerves of Tsg101SC-null mice by postnatal day 15 (P15, Fig. 1A-C). The sciatic nerves of some Tsg101SC-null animals had fewer, thinner myelin sheaths, while others showed an almost complete absence of myelin, as detected by toluidine blue staining on semithin sections (Fig. 1A-C) and immunohistochemistry for myelin basic protein (MBP) on paraffin sections (Fig. 1D-F). The myelination defects associated with loss of TSG101 were progressive, with the sciatic nerves of P21 Tsg101SC-null animals consistently showing severe hypomyelination and presence of onion bulb formations, (Fig. G-H). Ultrathin electron micrograph (EM) analysis of P21 sciatic nerve cross-sections revealed axons with thin myelin sheaths and multiple cell layers adjacent to nucleus of Schwann cells. These “onion bulbs” comprise concentric layers of Schwann cell processes and connective tissue (collagen) arranged around axons and are consistent with multiple rounds of de- and remyelination (Iwata, Kunimoto, & Inoue, 1998; Tracy et al., 2019; Webster, Schröder, Asbury, & Adams, 1967). This suggests that TSG101 is not essential for myelin production, but it is required for the maintenance of stable myelin sheaths. Schwann cell nuclei were still present by P21 and did not appear pyknotic (Fig. 1F), suggesting that loss of TSG101 did not disrupt myelination by triggering Schwann cell apoptosis.
As predicted, based on the differences observed in cellular phenotypes and EGF/EGFR degradation when TSG101 or HGS was depleted from mammalian cells by siRNA, the phenotype of Tsg101SC-null mice was more severe than that observed in HgsSC-null mice. Onion bulb formations are characteristic features in CMT1A and other demyelinating CMTs. Since TSG101 and ESCRT proteins are critical for endosomal sorting, their dysfunction could impair myelin production and turnover, exacerbating the cycles of demyelination and remyelination that contribute to onion bulb pathology. In the future, proteomic studies may shed light on the specific signaling pathways disrupted in Tsg101SC-null that contribute to the severe peripheral neuropathy phenotype. The variable expressivity of myelination defects and survival of Tsg101SC-null mice likely reflects their mixed genetic background (129S1/Sv x C57BL/6) and an effect of modifier genes, although inter-animal differences in P0-cre expression and Tsg101 deletion cannot be ruled out. Identifying the genes and pathways that influence disease severity in these mice could reveal druggable targets to treat some forms of CMT or other peripheral neuropathies.
","references":[{"reference":"Bache KG, Brech A, Mehlum A, Stenmark H. 2003. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. The Journal of Cell Biology 162: 435-442.
","pubmedId":"","doi":"doi: 10.1083/jcb.200302131"},{"reference":"Bennett CL, Shirk AJ, Huynh HM, Street VA, Nelis E, Van Maldergem L, et al., Chance. 2004. SIMPLE mutation in demyelinating neuropathy and distribution in sciatic nerve. Annals of Neurology 55: 713-720.
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","pubmedId":"","doi":"doi: 10.1083/jcb.200112080"},{"reference":"Doyotte A, Russell MRG, Hopkins CR, Woodman PG. 2005. Depletion of TSG101 forms a mammalian `Class E' compartment: a multicisternal early endosome with multiple sorting defects. Journal of Cell Science 118: 3003-3017.
","pubmedId":"","doi":"doi.org/10.1242/jcs.02421"},{"reference":"Feltri ML, D'Antonio M, Previtali S, Fasolini M, Messing A, Wrabetz L. 1999. P0-Cre transgenic mice for inactivation of adhesion molecules in Schwann cells. Ann N Y Acad Sci 883: 116-23.
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","pubmedId":"21896645","doi":""},{"reference":"Logan AM, Mammel AE, Robinson DC, Chin AL, Condon AF, Robinson FL. 2017. Schwann cell‐specific deletion of the endosomal PI 3‐kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2‐ErbB3 tyrosine kinase signaling. Glia 65: 1452-1470.
","pubmedId":"","doi":"10.1002/glia.23173"},{"reference":"Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN. 2003. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proceedings of the National Academy of Sciences 100: 7626-7631.
","pubmedId":"","doi":"10.1073/pnas.0932599100"},{"reference":"Markworth R, Bähr M, Burk K. 2021. Held Up in Traffic—Defects in the Trafficking Machinery in Charcot-Marie-Tooth Disease. Frontiers in Molecular Neuroscience 14: 10.3389/fnmol.2021.695294.
","pubmedId":"","doi":"10.3389/fnmol.2021.695294"},{"reference":"McLean JW, Wilson JA, Tian T, Watson JA, VanHart M, Bean AJ, et al., Wilson. 2022. Disruption of Endosomal Sorting in Schwann Cells Leads to Defective Myelination and Endosomal Abnormalities Observed in Charcot-Marie-Tooth Disease. The Journal of Neuroscience 42: 5085-5101.
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","pubmedId":"","doi":"10.1002/mus.26452"},{"reference":"Vaccari I, Carbone A, Previtali SC, Mironova YA, Alberizzi V, Noseda R, et al., Bolino. 2014. Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy. Human Molecular Genetics 24: 383-396.
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","pubmedId":"","doi":"10.1128/mcb.23.1.150-162.2003"},{"reference":"Walker WP, Oehler A, Edinger AL, Wagner KU, Gunn TM. 2016. Oligodendroglial deletion of ESCRT‐I component TSG101 causes spongiform encephalopathy. Biology of the Cell 108: 324-337.
","pubmedId":"","doi":"10.1111/boc.201600014"},{"reference":"deF. Webster H, Schröder JM, Asbury AK, Adams RD. 1967. The Role of Schwann Cells in the Formation of “Onion Bulbs” Found in Chronic Neuropathies***†. Journal of Neuropathology & Experimental Neurology 26: 276-299.
","pubmedId":"","doi":"10.1097/00005072-196704000-00008"}],"suggestedReviewer":{"name":"Dr. Scott Wilson, Department of Neurobiology, Evelyn F. McKnight Brain Institute, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294. Email: livvy01@uab.edu
","WBId":""},"title":"Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy
","reviews":[{"reviewer":{"displayName":"Ben Emery"},"openAcknowledgement":false,"status":{"submitted":true}}]},{"id":"d3f13d1d-503f-419a-8b4b-c75111f85c68","decisionLetter":null,"decision":"edit","submitted":true,"abstract":"Myelinating Schwann cells are particularly susceptible to defects in endosomal trafficking. TSG101 is a component of the endosomal trafficking machinery that mediates the sorting of ubiquitinated receptors into multivesicular bodies. We previously demonstrated that deleting Tsg101 from mouse oligodendrocytes in the central nervous system causes rapid onset de/dys-myelination and vacuolation of white matter, suggesting an important role for TSG101-dependent trafficking in myelination. Here, we show that TSG101 is also required for normal myelination in the peripheral nervous system.
","acknowledgements":"We thank Anita Pecukonis and the McLaughlin Institute Animal Resource Center for excellent animal care and Dr. John R. Bermingham Jr for instruction on sciatic nerve dissection.
","authors":[{"affiliations":["McLaughlin Research Institute"],"credit":["investigation","writing_reviewEditing"],"email":"derek@mclaughlinresearch.org","firstName":"Derek","lastName":"Silvius","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","writing_reviewEditing"],"email":"edwardhu@buffalo.edu","firstName":"Edward","lastName":"Hurley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Albany Medical College"],"credit":["writing_reviewEditing"],"email":"poitely@amc.edu","firstName":"Yannick","lastName":"Poitelon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Wayne State University, Detroit, Michigan, United States"],"credit":["resources","writing_reviewEditing"],"email":"kuwagner@wayne.edu","firstName":"Kay-Uwe","lastName":"Wagner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","supervision","validation"],"email":"mamillig@buffalo.edu","firstName":"M. Laura","lastName":"Feltri","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","methodology","fundingAcquisition"],"email":"tmg@mclaughlinresearch.org","firstName":"Teresa M.","lastName":"Gunn","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2688-6420"}],"comments":"Dear microPublication Editorial Team,
Thank you for the opportunity to revise our manuscript. We appreciate the reviewer’s comments, especially those noting that this work is “a novel demonstration of the necessity for Tsg101 (and, by extension, endosomal sorting) for maintenance of PNS myelin” and that “The article is well-written, clearly outlining the relevant background literature of other human variants or mouse mutants linking endosomal trafficking to dysmyelination/demyelination.”
We apologize that we did not include sample sizes to make it clear that we show seminthin data from 2 different P15 and 1 P21 conditional knockout nerves, and performed IHC for MBP on a larger number of samples (5 mutants) and show representative images of that data in the figure. As Dr. Laura Feltri has passed away and the analysis her lab performed took place quite a few years ago, we are unable to find all the necessary records to determine whether they looked at more than 2 x P15 and 1 x P21 nerves; that is all the data I was sent at the time. However, I hope the fact that this is a total of 3 different mice at two ages, and we looked at nerves from 5 mutant mice with MBP staining on paraffin sections, will satisfy the reviewer and the editorial team. The myelination phenotype was so robust and qualitatively obvious that we do not think it necessary to quantify it.
We have addressed the issues raised by editing the Methods and Figure Legend to clearly indicate sample sizes for the seminthin, EM and IHC analyses, as follows (additions are underlined):
Methods –Histology section:
2nd paragraph: “For IHC, specimens from five Tsg101SC-null and four control animals were embedded in…”
3rd paragraph: “Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age).”
Revised Figure legend:
Fig. 1. Histopathology of sciatic nerves lacking TSG101 in Schwann cells. (A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7 paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed. (G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
We hope the Editorial Team will agree that these revisions address the reviewer’s concerns and look forward to your response.
Thank you again for considering our manuscript for microPublication.
Sincerely,
Teresa Gunn
","dataTable":null,"disclaimer":true,"funding":"This work was funded by the McLaughlin Research Institute and its generous supporters. Kay-Uwe Wagner is the Lloyd and Marilyn Smith Endowed Professor of Breast Cancer Research at the Karmanos Comprehensive Cancer Center.
","image":{"name":"Fig 1 Tsg101 SC null.png","url":"https://portal.micropublication.org/uploads/c7ad46ba5ce67475b19a471661ada5c2.png"},"imageCaption":"(A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7m paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed.(G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
","imageTitle":"Histopathology of sciatic nerves lacking TSG101 in Schwann cells.
","laboratory":{"name":"","WBId":""},"methods":"Animals
All studies were approved by the McLaughlin Research Institute’s Institutional Animal Care and Use Committee and adhered to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Mice homozygous for a Tsg101 conditional knockout allele (129X1/SvJ-Tsg101tm1Kuw, referred to herein as Tsg101fl; RRID: MMRRC_037407-MU), in which exon 1 is flanked by a floxed neo cassette inserted approximately 3 kb upstream and a single loxP site inserted 230 bp into intron 1 (Wagner et al., 2003), were mated to mice hemizygous for Tg(Mpz-cre)26Mes/J (obtained from the Jackson Laboratory and referred to herein as P0-cre; RRID: IMSR_JAX:017928). P0-cre transgenic mice express cre recombinase in Schwann cells, under the control of the myelin protein zero (P0, Mpz) promoter, by embryonic day 14 (Feltri et al., 1999). Tsg101fl/+; P0-cre+ F1 offspring were backcrossed to Tsg101fl/fl mice and Tsg101fl/fl; P0-cre+ and control (Tsg101fl/+; P0-cre+ ad Tsg101fl/+; P0-cre-neg) pups were obtained from intercrossing N1 or N1F1 Tsg101fl/+; P0-cre+ x Tsg101fl/fl; P0-cre-neg sibs.
Histology
Sciatic nerve segments were sampled from approximately the same position for each animal. Contralateral nerves were either fixed in 4% buffered paraformaldehyde, for immunohistochemistry (IHC), or 2% buffered glutaraldehyde with postfixation in 1% osmium tetroxide (for semi- and ultra-thin sections).
For IHC, specimens from five Tsg101SC-null and four control animals were embedded in paraffin following standard protocols and sectioned at 5 microns. Following deparaffinization and rehydration, sections were permeabilized in a % Triton-X100 solution in PBS, then subjected to antigen retrieval in 10mM sodium citrate (pH 6.0, 100 C, 10 minutes). Slides were blocked in 10% serum, then incubated with an antibody against myelin basic protein (MBP; Covance Cat# SMI-99, RRID:AB_2314772) at 1:1000, followed by horseradish peroxidase conjugated anti-mouse secondary antibody (BD Pharmingen horseradish peroxidase-conjugated anti-mouse Ig, cat# 554002, RRID: AB_395198) at 1:100 and chromogenic visualization using NovaRed substrate (Vector Labs, CA). Slides were counterstained with hematoxylin prior to coverslipping, and then examined on a Zeiss AxioImagerM1 light microscope.
Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age). Briefly, sciatic nerves were fixed in 2% buffered glutaraldehyde, then postfixed in 1% osmium tetroxide. After alcohol dehydration, nerves were submerged in propylene oxide, and then in a 1:1 mixture of Epon-propylene oxide. Nerves were embedded in 100% Epon, and resin was allowed to polymerize. Semithin transverse sections were sliced 0.5-μm-thick using Leica UC7, stained with 2% toluidine blue, and then examined by light microscopy with Leica DM6000B. EM transverse sections were sliced 700–900 Å-thick using Leica UC7, stained with uranyl acetate and lead citrate, and then examined with an electron microscope (model FEI BioTwin). Analyzed sections were sliced from the distal end of embedded sciatic nerve. Images acquired from semithins and EMs were nonoverlapping and comprehensive.
","reagents":"","patternDescription":"The endosomal pathway traffics receptor proteins and lipids into early endosomes and directs them either to recycling endosomes for trafficking back to the cell membrane or sorting into intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which fuse with lysosomes to degrade their contents or with the plasma membrane to secrete ILVs as exosomes (reviewed in Scott, Vacca, & Gruenberg, 2014). The timing of these events is important to cellular processes since receptors can continue to activate downstream signaling pathways while they remain on early endosomes. Several forms of demyelinating CMT are caused by mutations in genes encoding proteins involved in membrane dynamics and endosomal trafficking, including N-myc downstream regulated gene 1 (NDRG1), myotubularin related protein 2 (MTMR2), SET binding factor 2 (SBF2/MTMR13),SH3 domain and tetratricopeptide repeats 2 (SH3TC2), dynamin 2 (DNM2), FIG4 phosphoinositide 5-phosphatase (FIG4), and LPS-induced TN factor (LITAF/SIMPLE) (reviewed by Markworth, Bähr, & Burk, 2021). For example, mutations in SH3TC2 that cause CMT type 4C cause impaired recycling of membrane components necessary for Schwann cell function (Roberts et al., 2010; Stendel et al., 2010). Furthermore, mouse models with Schwann cell-specific deletion of Fig4, HGF-regulated tyrosine kinase substrate (Hgs/Hrs), or phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) show mild peripheral hypo/de/dys-myelination associated with altered ERBB2/3 signaling (Logan et al., 2017; McLean et al., 2022; Vaccari et al., 2015). In Schwann cells, neuregulin 1 (NRG1) signaling through erb-b2 receptor tyrosine kinases 2 and 3 (ERBB2/B3) mediates myelination, and endosomal trafficking of NRG1-bound ERBB2/B3 regulates receptor down-regulation and recycling (Newbern & Birchmeier, 2010; Salzer, 2015). Disrupted endosomal sorting can result in sustained activation of downstream pathways, including the ERK1/2 signaling cascade, to negatively impact Schwann cell myelin integrity (Newbern & Birchmeier, 2010; Salzer, 2015). Thus, there is support for the idea that endosomal trafficking defects can cause Schwann cell dysfunction and demyelination, reinforcing the relevance of this pathway to myelination.
Tumor susceptibility gene 101 (TSG101) encodes a component of the Endosomal Sorting Complex Required for Transport-1 (ESCRT-I), which helps mediate the sorting of ubiquitinated receptors onto ILVs of MVBs. The ESCRT-0 protein, HGF-regulated tyrosine kinase substrate, HGS (formerly referred to as HRS) mediates the initial recruitment of ESCRT-I to endosomes (via interaction with TSG101), but in multiple mammalian cell lines, siRNA depletion of TSG101 or HGS had significantly different effects on endosomal and MVB morphology (Bache, Brech, Mehlum, & Stenmark, 2003; Lu, Hope, Brasch, Reinhard, & Cohen, 2003; Raiborg, Malerod, Pedersen, & Stenmark, 2008; Razi & Futter, 2006). Specifically, knockdown of TSG101 inhibited epidermal growth factor degradation and MVB formation and caused tubulation of the vacuolar domains of early endosomes, while depletion of HGS had only a modest effect on EGF degradation, did not induce tubulation of early endosomes, and resulted in the production of enlarged MVBs containing few ILVs but that could still fuse with the lysosome (Bishop, Horman, & Woodman, 2002; Doyotte, Russell, Hopkins, & Woodman, 2005; Razi & Futter, 2006). These data suggest that HGS and TSG101 have distinct roles in the endosomal trafficking pathway, with TSG101 being required for the formation of stable vacuolar domains within the early endosome that subsequently develop into MVBs and HGS being more important in the formation and/or accumulation of ILVs within MVBs. Mice lacking HGS in Schwann cells developed mild motor and sensory defects, a reduced number of myelinated axons and thinner myelin sheaths in the sciatic nerve, as well as aberrantly folded myelin sheaths (McLean et al., 2022).
TSG101 and HGS also both interact and partially colocalize with LITAF (Lee, Olzmann, Chin, & Li, 2011), which is expressed in Schwann cells and mutations in it cause dominant demyelinating peripheral neuropathy, CMT1C (Bennett et al., 2004). Although one study showed that CMT1C-associated LITAF mutations did not effect on its subcellular localization or association with TSG101 (Shirk, Anderson, Hashemi, Chance, & Bennett, 2005), another study showed a dominant negative effect on EGFR degradation and lysosomal trafficking of EGF, associated with reduced membrane association of HGS and TSG101 (Lee et al., 2011). In the latter study, expression of CMT1C-associated LITAF mutants in Schwann cells caused prolonged activation of ERK1/2 signaling, presumably downstream of NRG1-ERBB2/3 signaling.
Deleting Tsg101 from oligodendroglia in the central nervous system resulted in severe, rapid-onset myelination defects and vacuolation (Walker, Oehler, Edinger, Wagner, & Gunn, 2016), suggesting an important role for TSG101-dependent trafficking in signaling pathways that regulate myelination. To test whether TSG101 is also required for normal myelination in the peripheral nervous system, we investigated the consequences of deleting Tsg101 in Schwann cells. We predicted this would cause a more severe peripheral neuropathy than deleting Hgs, given the stronger effect of Tsg101 depletion on endosomal/MVB phenotypes in cultured cells.
Tsg101 conditional knockout mice (Tsg101tm1KuW, referred to here as Tsg101fl) were mated to P0-Cre transgenic mice, which express cre recombinase specifically in Schwann cells starting on embryonic day 13.5. Cre-positive Tsg101fl/+ offspring were backcrossed to Tsg101fl/fl animals. All pups that were homozygous for the Tsg101 conditional allele and carried the P0-Cre transgene (referred to herein as Tsg101-Schwann cell null, or Tsg101SC-null animals) were smaller than their littermates, developed a tremor by 12 days of age, had abnormal posture of their fore- and hind-limbs (arthrogryposis), and failed to thrive. They died throughout the postnatal period, with very few surviving to 3 weeks of age. We recorded 89 affected animals out of 427 pups born from 15 different breeder pairs, for a frequency of ~21% affected. This is significantly different from the 25% expected (c2= 4.04, p = 0.044) and is likely due to the loss of some affected animals prior to them being observed and recorded.
Histological analysis of the sciatic nerves of Tsg101SC-null animals at postnatal days 15 and 21 revealed striking de- and dys-myelination (Fig. 1). Toluidine blue-stained semi-thin cross sections showed reduced myelin, enlargement of the interstitial space, and presence of onion bulb structures in the sciatic nerves of Tsg101SC-null mice by postnatal day 15 (P15, Fig. 1A-C). The sciatic nerves of some Tsg101SC-null animals had fewer, thinner myelin sheaths, while others showed an almost complete absence of myelin, as detected by toluidine blue staining on semithin sections (Fig. 1A-C) and immunohistochemistry for myelin basic protein (MBP) on paraffin sections (Fig. 1D-F). The myelination defects associated with loss of TSG101 were progressive, with the sciatic nerve of a P21 Tsg101SC-null animal showing severe hypomyelination and presence of onion bulb formations, (Fig. G-H). Ultrathin electron micrograph (EM) analysis of P21 sciatic nerve cross-sections revealed axons with thin myelin sheaths and multiple cell layers adjacent to nucleus of Schwann cells. These “onion bulbs” comprise concentric layers of Schwann cell processes and connective tissue (collagen) arranged around axons and are consistent with multiple rounds of de- and remyelination (Iwata, Kunimoto, & Inoue, 1998; Tracy et al., 2019; Webster, Schröder, Asbury, & Adams, 1967). This suggests that TSG101 is not essential for myelin production, but it is required for the maintenance of stable myelin sheaths. Schwann cell nuclei were still present by P21 and did not appear pyknotic (Fig. 1F), suggesting that loss of TSG101 did not disrupt myelination by triggering Schwann cell apoptosis.
As predicted, based on the differences observed in cellular phenotypes and EGF/EGFR degradation when TSG101 or HGS was depleted from mammalian cells by siRNA, the phenotype of Tsg101SC-null mice was more severe than that observed in HgsSC-null mice. Onion bulb formations are characteristic features in CMT1A and other demyelinating CMTs. Since TSG101 and ESCRT proteins are critical for endosomal sorting, their dysfunction could impair myelin production and turnover, exacerbating the cycles of demyelination and remyelination that contribute to onion bulb pathology. In the future, proteomic studies may shed light on the specific signaling pathways disrupted in Tsg101SC-null that contribute to the severe peripheral neuropathy phenotype. The variable expressivity of myelination defects and survival of Tsg101SC-null mice likely reflects their mixed genetic background (129S1/Sv x C57BL/6) and an effect of modifier genes, although inter-animal differences in P0-cre expression and Tsg101 deletion cannot be ruled out. Identifying the genes and pathways that influence disease severity in these mice could reveal druggable targets to treat some forms of CMT or other peripheral neuropathies.
","references":[{"reference":"Bache KG, Brech A, Mehlum A, Stenmark H. 2003. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. The Journal of Cell Biology 162: 435-442.
","pubmedId":"","doi":"doi: 10.1083/jcb.200302131"},{"reference":"Bennett CL, Shirk AJ, Huynh HM, Street VA, Nelis E, Van Maldergem L, et al., Chance. 2004. SIMPLE mutation in demyelinating neuropathy and distribution in sciatic nerve. Annals of Neurology 55: 713-720.
","pubmedId":"","doi":"doi: 10.1002/ana.20094"},{"reference":"Bishop N, Horman A, Woodman P. 2002. Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein–ubiquitin conjugates. The Journal of Cell Biology 157: 91-102.
","pubmedId":"","doi":"doi: 10.1083/jcb.200112080"},{"reference":"Doyotte A, Russell MRG, Hopkins CR, Woodman PG. 2005. Depletion of TSG101 forms a mammalian `Class E' compartment: a multicisternal early endosome with multiple sorting defects. Journal of Cell Science 118: 3003-3017.
","pubmedId":"","doi":"doi.org/10.1242/jcs.02421"},{"reference":"Feltri ML, D'Antonio M, Previtali S, Fasolini M, Messing A, Wrabetz L. 1999. P0-Cre transgenic mice for inactivation of adhesion molecules in Schwann cells. Ann N Y Acad Sci 883: 116-23.
","pubmedId":"10586237","doi":""},{"reference":"Iwata A, Kunimoto M, Inoue K. 1998. Schwann cell proliferation as the cause of peripheral neuropathy in neurofibromatosis-2. Journal of the Neurological Sciences 156: 201-204.
","pubmedId":"","doi":"doi: 10.1016/s0022-510x(98)00032-x"},{"reference":"Lee SM, Olzmann JA, Chin LS, Li L. 2011. Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome-autophagy pathways. J Cell Sci 124(Pt 19): 3319-31.
","pubmedId":"21896645","doi":""},{"reference":"Logan AM, Mammel AE, Robinson DC, Chin AL, Condon AF, Robinson FL. 2017. Schwann cell‐specific deletion of the endosomal PI 3‐kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2‐ErbB3 tyrosine kinase signaling. Glia 65: 1452-1470.
","pubmedId":"","doi":"10.1002/glia.23173"},{"reference":"Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN. 2003. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proceedings of the National Academy of Sciences 100: 7626-7631.
","pubmedId":"","doi":"10.1073/pnas.0932599100"},{"reference":"Markworth R, Bähr M, Burk K. 2021. Held Up in Traffic—Defects in the Trafficking Machinery in Charcot-Marie-Tooth Disease. Frontiers in Molecular Neuroscience 14: 10.3389/fnmol.2021.695294.
","pubmedId":"","doi":"10.3389/fnmol.2021.695294"},{"reference":"McLean JW, Wilson JA, Tian T, Watson JA, VanHart M, Bean AJ, et al., Wilson. 2022. Disruption of Endosomal Sorting in Schwann Cells Leads to Defective Myelination and Endosomal Abnormalities Observed in Charcot-Marie-Tooth Disease. The Journal of Neuroscience 42: 5085-5101.
","pubmedId":"","doi":"10.1523/jneurosci.2481-21.2022"},{"reference":"Newbern J, Birchmeier C. 2010. Nrg1/ErbB signaling networks in Schwann cell development and myelination. Seminars in Cell & Developmental Biology 21: 922-928.
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","pubmedId":"","doi":"10.1016/j.yexcr.2007.10.014"},{"reference":"Razi M, Futter CE. 2006. Distinct Roles for Tsg101 and Hrs in Multivesicular Body Formation and Inward Vesiculation. Molecular Biology of the Cell 17: 3469-3483.
","pubmedId":"","doi":"10.1091/mbc.e05-11-1054"},{"reference":"Roberts RC, Peden AA, Buss F, Bright NA, Latouche M, Reilly MM, Kendrick-Jones J, Luzio JP. 2009. Mistargeting of SH3TC2 away from the recycling endosome causes Charcot–Marie–Tooth disease type 4C. Human Molecular Genetics 19: 1009-1018.
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","pubmedId":"","doi":"10.1093/brain/awq168"},{"reference":"Tracy JA, Dyck PJ, Klein CJ, Engelstad JK, Meyer JE, Dyck PJB. 2019. Onion‐bulb patterns predict acquired or inherited demyelinating polyneuropathy. Muscle & Nerve 59: 665-670.
","pubmedId":"","doi":"10.1002/mus.26452"},{"reference":"Vaccari I, Carbone A, Previtali SC, Mironova YA, Alberizzi V, Noseda R, et al., Bolino. 2014. Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy. Human Molecular Genetics 24: 383-396.
","pubmedId":"","doi":"10.1093/hmg/ddu451"},{"reference":"Wagner KU, Krempler A, Qi Y, Park K, Henry MD, Triplett AA, et al., Hennighausen. 2003. Tsg101 Is Essential for Cell Growth, Proliferation, and Cell Survival of Embryonic and Adult Tissues. Molecular and Cellular Biology 23: 150-162.
","pubmedId":"","doi":"10.1128/mcb.23.1.150-162.2003"},{"reference":"Walker WP, Oehler A, Edinger AL, Wagner KU, Gunn TM. 2016. Oligodendroglial deletion of ESCRT‐I component TSG101 causes spongiform encephalopathy. Biology of the Cell 108: 324-337.
","pubmedId":"","doi":"10.1111/boc.201600014"},{"reference":"deF. Webster H, Schröder JM, Asbury AK, Adams RD. 1967. The Role of Schwann Cells in the Formation of “Onion Bulbs” Found in Chronic Neuropathies***†. Journal of Neuropathology & Experimental Neurology 26: 276-299.
","pubmedId":"","doi":"10.1097/00005072-196704000-00008"}],"suggestedReviewer":{"name":"Dr. Scott Wilson, Department of Neurobiology, Evelyn F. McKnight Brain Institute, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294. Email: livvy01@uab.edu
","WBId":""},"title":"Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy
","reviews":[]},{"id":"05f4bba8-f73e-4822-904a-6ce8490122e2","decisionLetter":"\n\n Dear Teresa M. Gunn,\n
\n\n We are happy to let you know that your article has been accepted for publication. Congratulations!\n
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\n ","decision":"accept","submitted":true,"abstract":"Myelinating Schwann cells are particularly susceptible to defects in endosomal trafficking. TSG101 is a component of the endosomal trafficking machinery that mediates the sorting of ubiquitinated receptors into multivesicular bodies. We previously demonstrated that deleting Tsg101 from mouse oligodendrocytes in the central nervous system causes rapid onset de/dys-myelination and vacuolation of white matter, suggesting an important role for TSG101-dependent trafficking in myelination. Here, we show that TSG101 is also required for normal myelination in the peripheral nervous system.
","acknowledgements":"We thank Anita Pecukonis and the McLaughlin Institute Animal Resource Center for excellent animal care and Dr. John R. Bermingham Jr for instruction on sciatic nerve dissection.
","authors":[{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States"],"credit":["investigation","writing_reviewEditing"],"email":"derek@mclaughlinresearch.org","firstName":"Derek","lastName":"Silvius","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","writing_reviewEditing"],"email":"edwardhu@buffalo.edu","firstName":"Edward","lastName":"Hurley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Albany Medical College, Albany, New York, United States"],"credit":["writing_reviewEditing"],"email":"poitely@amc.edu","firstName":"Yannick","lastName":"Poitelon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Wayne State University, Detroit, Michigan, United States"],"credit":["resources","writing_reviewEditing"],"email":"kuwagner@wayne.edu","firstName":"Kay-Uwe","lastName":"Wagner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","supervision","validation"],"email":"mamillig@buffalo.edu","firstName":"M. Laura","lastName":"Feltri","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","methodology","fundingAcquisition"],"email":"tmg@mclaughlinresearch.org","firstName":"Teresa M.","lastName":"Gunn","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2688-6420"}],"comments":"Dear microPublication Editorial Team,
Thank you for the opportunity to revise our manuscript. We appreciate the reviewer’s comments, especially those noting that this work is “a novel demonstration of the necessity for Tsg101 (and, by extension, endosomal sorting) for maintenance of PNS myelin” and that “The article is well-written, clearly outlining the relevant background literature of other human variants or mouse mutants linking endosomal trafficking to dysmyelination/demyelination.”
We apologize that we did not include sample sizes to make it clear that we show seminthin data from 2 different P15 and 1 P21 conditional knockout nerves, and performed IHC for MBP on a larger number of samples (5 mutants) and show representative images of that data in the figure. As Dr. Laura Feltri has passed away and the analysis her lab performed took place quite a few years ago, we are unable to find all the necessary records to determine whether they looked at more than 2 x P15 and 1 x P21 nerves; that is all the data I was sent at the time. However, I hope the fact that this is a total of 3 different mice at two ages, and we looked at nerves from 5 mutant mice with MBP staining on paraffin sections, will satisfy the reviewer and the editorial team. The myelination phenotype was so robust and qualitatively obvious that we do not think it necessary to quantify it.
We have addressed the issues raised by editing the Methods and Figure Legend to clearly indicate sample sizes for the seminthin, EM and IHC analyses, as follows (additions are underlined):
Methods –Histology section:
2nd paragraph: “For IHC, specimens from five Tsg101SC-null and four control animals were embedded in…”
3rd paragraph: “Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age).”
Revised Figure legend:
Fig. 1. Histopathology of sciatic nerves lacking TSG101 in Schwann cells. (A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7 paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed. (G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
We hope the Editorial Team will agree that these revisions address the reviewer’s concerns and look forward to your response.
Thank you again for considering our manuscript for microPublication.
Sincerely,
Teresa Gunn
","dataTable":null,"disclaimer":true,"funding":"This work was funded by the McLaughlin Research Institute and its generous supporters. Kay-Uwe Wagner is the Lloyd and Marilyn Smith Endowed Professor of Breast Cancer Research at the Karmanos Comprehensive Cancer Center.
","image":{"name":"Fig 1 Tsg101 SC null.png","url":"https://portal.micropublication.org/uploads/c7ad46ba5ce67475b19a471661ada5c2.png"},"imageCaption":"(A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7m paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed.(G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
","imageTitle":"Histopathology of sciatic nerves lacking TSG101 in Schwann cells.
","laboratory":{"name":"","WBId":""},"methods":"Animals
All studies were approved by the McLaughlin Research Institute’s Institutional Animal Care and Use Committee and adhered to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Mice homozygous for a Tsg101 conditional knockout allele (129X1/SvJ-Tsg101tm1Kuw, referred to herein as Tsg101fl; RRID: MMRRC_037407-MU), in which exon 1 is flanked by a floxed neo cassette inserted approximately 3 kb upstream and a single loxP site inserted 230 bp into intron 1 (Wagner et al., 2003), were mated to mice hemizygous for Tg(Mpz-cre)26Mes/J (obtained from the Jackson Laboratory and referred to herein as P0-cre; RRID: IMSR_JAX:017928). P0-cre transgenic mice express cre recombinase in Schwann cells, under the control of the myelin protein zero (P0, Mpz) promoter, by embryonic day 14 (Feltri et al., 1999). Tsg101fl/+; P0-cre+ F1 offspring were backcrossed to Tsg101fl/fl mice and Tsg101fl/fl; P0-cre+ and control (Tsg101fl/+; P0-cre+ ad Tsg101fl/+; P0-cre-neg) pups were obtained from intercrossing N1 or N1F1 Tsg101fl/+; P0-cre+ x Tsg101fl/fl; P0-cre-neg sibs.
Histology
Sciatic nerve segments were sampled from approximately the same position for each animal. Contralateral nerves were either fixed in 4% buffered paraformaldehyde, for immunohistochemistry (IHC), or 2% buffered glutaraldehyde with postfixation in 1% osmium tetroxide (for semi- and ultra-thin sections).
For IHC, specimens from five Tsg101SC-null and four control animals were embedded in paraffin following standard protocols and sectioned at 5 microns. Following deparaffinization and rehydration, sections were permeabilized in a % Triton-X100 solution in PBS, then subjected to antigen retrieval in 10mM sodium citrate (pH 6.0, 100 C, 10 minutes). Slides were blocked in 10% serum, then incubated with an antibody against myelin basic protein (MBP; Covance Cat# SMI-99, RRID:AB_2314772) at 1:1000, followed by horseradish peroxidase conjugated anti-mouse secondary antibody (BD Pharmingen horseradish peroxidase-conjugated anti-mouse Ig, cat# 554002, RRID: AB_395198) at 1:100 and chromogenic visualization using NovaRed substrate (Vector Labs, CA). Slides were counterstained with hematoxylin prior to coverslipping, and then examined on a Zeiss AxioImagerM1 light microscope.
Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age). Briefly, sciatic nerves were fixed in 2% buffered glutaraldehyde, then postfixed in 1% osmium tetroxide. After alcohol dehydration, nerves were submerged in propylene oxide, and then in a 1:1 mixture of Epon-propylene oxide. Nerves were embedded in 100% Epon, and resin was allowed to polymerize. Semithin transverse sections were sliced 0.5-μm-thick using Leica UC7, stained with 2% toluidine blue, and then examined by light microscopy with Leica DM6000B. EM transverse sections were sliced 700–900 Å-thick using Leica UC7, stained with uranyl acetate and lead citrate, and then examined with an electron microscope (model FEI BioTwin). Analyzed sections were sliced from the distal end of embedded sciatic nerve. Images acquired from semithins and EMs were nonoverlapping and comprehensive.
","reagents":"","patternDescription":"The endosomal pathway traffics receptor proteins and lipids into early endosomes and directs them either to recycling endosomes for trafficking back to the cell membrane or sorting into intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which fuse with lysosomes to degrade their contents or with the plasma membrane to secrete ILVs as exosomes (reviewed in (Scott et al., 2014)). The timing of these events is important to cellular processes since receptors can continue to activate downstream signaling pathways while they remain on early endosomes. Several forms of demyelinating CMT are caused by mutations in genes encoding proteins involved in membrane dynamics and endosomal trafficking, including N-myc downstream regulated gene 1 (NDRG1), myotubularin related protein 2 (MTMR2), SET binding factor 2 (SBF2/MTMR13),SH3 domain and tetratricopeptide repeats 2 (SH3TC2), dynamin 2 (DNM2), FIG4 phosphoinositide 5-phosphatase (FIG4), and LPS-induced TN factor (LITAF/SIMPLE) (reviewed in (Markworth et al., 2021)). For example, mutations in SH3TC2 that cause CMT type 4C cause impaired recycling of membrane components necessary for Schwann cell function (Roberts et al., 2010; Stendel et al., 2010). Furthermore, mouse models with Schwann cell-specific deletion of Fig4, HGF-regulated tyrosine kinase substrate (Hgs/Hrs), or phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) show mild peripheral hypo/de/dys-myelination associated with altered ERBB2/3 signaling (Logan et al., 2017; McLean et al., 2022; Vaccari et al., 2015). In Schwann cells, neuregulin 1 (NRG1) signaling through erb-b2 receptor tyrosine kinases 2 and 3 (ERBB2/B3) mediates myelination, and endosomal trafficking of NRG1-bound ERBB2/B3 regulates receptor down-regulation and recycling (Newbern and Birchmeier, 2010; Salzer, 2015). Disrupted endosomal sorting can result in sustained activation of downstream pathways, including the ERK1/2 signaling cascade, to negatively impact Schwann cell myelin integrity (Newbern and Birchmeier, 2010; Salzer, 2015). Thus, there is support for the idea that endosomal trafficking defects can cause Schwann cell dysfunction and demyelination, reinforcing the relevance of this pathway to myelination.
Tumor susceptibility gene 101 (TSG101) encodes a component of the Endosomal Sorting Complex Required for Transport-1 (ESCRT-I), which helps mediate the sorting of ubiquitinated receptors onto ILVs of MVBs. The ESCRT-0 protein, HGF-regulated tyrosine kinase substrate, HGS (formerly referred to as HRS) mediates the initial recruitment of ESCRT-I to endosomes (via interaction with TSG101), but in multiple mammalian cell lines, siRNA depletion of TSG101 or HGS had significantly different effects on endosomal and MVB morphology (Bache et al., 2003; Lu et al., 2003; Raiborg et al., 2008; Razi and Futter, 2006). Specifically, knockdown of TSG101 inhibited epidermal growth factor degradation and MVB formation and caused tubulation of the vacuolar domains of early endosomes, while depletion of HGS had only a modest effect on EGF degradation, did not induce tubulation of early endosomes, and resulted in the production of enlarged MVBs containing few ILVs but that could still fuse with the lysosome (Bishop et al., 2002; Doyotte et al., 2005; Razi and Futter, 2006). These data suggest that HGS and TSG101 have distinct roles in the endosomal trafficking pathway, with TSG101 being required for the formation of stable vacuolar domains within the early endosome that subsequently develop into MVBs and HGS being more important in the formation and/or accumulation of ILVs within MVBs. Mice lacking HGS in Schwann cells developed mild motor and sensory defects, a reduced number of myelinated axons and thinner myelin sheaths in the sciatic nerve, as well as aberrantly folded myelin sheaths (McLean et al., 2022).
TSG101 and HGS also both interact and partially colocalize with LITAF (Lee et al., 2011), which is expressed in Schwann cells and mutations in it cause dominant demyelinating peripheral neuropathy, CMT1C (Bennett et al., 2004). Although one study showed that CMT1C-associated LITAF mutations did not effect on its subcellular localization or association with TSG101 (Shirk et al., 2005), another study showed a dominant negative effect on EGFR degradation and lysosomal trafficking of EGF, associated with reduced membrane association of HGS and TSG101 (Lee et al., 2011). In the latter study, expression of CMT1C-associated LITAF mutants in Schwann cells caused prolonged activation of ERK1/2 signaling, presumably downstream of NRG1-ERBB2/3 signaling.
Deleting Tsg101 from oligodendroglia in the central nervous system resulted in severe, rapid-onset myelination defects and vacuolation (Walker et al., 2016), suggesting an important role for TSG101-dependent trafficking in signaling pathways that regulate myelination. To test whether TSG101 is also required for normal myelination in the peripheral nervous system, we investigated the consequences of deleting Tsg101 in Schwann cells. We predicted this would cause a more severe peripheral neuropathy than deleting Hgs, given the stronger effect of Tsg101 depletion on endosomal/MVB phenotypes in cultured cells.
Tsg101 conditional knockout mice (Tsg101tm1KuW, referred to here as Tsg101fl) were mated to P0-Cre transgenic mice, which express cre recombinase specifically in Schwann cells starting on embryonic day 13.5. Cre-positive Tsg101fl/+ offspring were backcrossed to Tsg101fl/fl animals. All pups that were homozygous for the Tsg101 conditional allele and carried the P0-Cre transgene (referred to herein as Tsg101-Schwann cell null, or Tsg101SC-null animals) were smaller than their littermates, developed a tremor by 12 days of age, had abnormal posture of their fore- and hind-limbs (arthrogryposis), and failed to thrive. They died throughout the postnatal period, with very few surviving to 3 weeks of age. We recorded 89 affected animals out of 427 pups born from 15 different breeder pairs, for a frequency of ~21% affected. This is significantly different from the 25% expected (c2= 4.04, p = 0.044) and is likely due to the loss of some affected animals prior to them being observed and recorded.
Histological analysis of the sciatic nerves of Tsg101SC-null animals at postnatal days 15 and 21 revealed striking de- and dys-myelination (Fig. 1). Toluidine blue-stained semi-thin cross sections showed reduced myelin, enlargement of the interstitial space, and presence of onion bulb structures in the sciatic nerves of Tsg101SC-null mice by postnatal day 15 (P15, Fig. 1A-C). The sciatic nerves of some Tsg101SC-null animals had fewer, thinner myelin sheaths, while others showed an almost complete absence of myelin, as detected by toluidine blue staining on semithin sections (Fig. 1A-C) and immunohistochemistry for myelin basic protein (MBP) on paraffin sections (Fig. 1D-F). The myelination defects associated with loss of TSG101 were progressive, with the sciatic nerve of a P21 Tsg101SC-null animal showing severe hypomyelination and presence of onion bulb formations, (Fig. G-H). Ultrathin electron micrograph (EM) analysis of P21 sciatic nerve cross-sections revealed axons with thin myelin sheaths and multiple cell layers adjacent to nucleus of Schwann cells. These “onion bulbs” comprise concentric layers of Schwann cell processes and connective tissue (collagen) arranged around axons and are consistent with multiple rounds of de- and remyelination (Iwata et al., 1998; Tracy et al., 2019; Webster et al., 1967). This suggests that TSG101 is not essential for myelin production, but it is required for the maintenance of stable myelin sheaths. Schwann cell nuclei were still present by P21 and did not appear pyknotic (Fig. 1F), suggesting that loss of TSG101 did not disrupt myelination by triggering Schwann cell apoptosis.
As predicted, based on the differences observed in cellular phenotypes and EGF/EGFR degradation when TSG101 or HGS was depleted from mammalian cells by siRNA, the phenotype of Tsg101SC-null mice was more severe than that observed in HgsSC-null mice. Onion bulb formations are characteristic features in CMT1A and other demyelinating CMTs. Since TSG101 and ESCRT proteins are critical for endosomal sorting, their dysfunction could impair myelin production and turnover, exacerbating the cycles of demyelination and remyelination that contribute to onion bulb pathology. In the future, proteomic studies may shed light on the specific signaling pathways disrupted in Tsg101SC-null that contribute to the severe peripheral neuropathy phenotype. The variable expressivity of myelination defects and survival of Tsg101SC-null mice likely reflects their mixed genetic background (129S1/Sv x C57BL/6) and an effect of modifier genes, although inter-animal differences in P0-cre expression and Tsg101 deletion cannot be ruled out. Identifying the genes and pathways that influence disease severity in these mice could reveal druggable targets to treat some forms of CMT or other peripheral neuropathies.
","references":[{"reference":"Bache KG, Brech A, Mehlum A, Stenmark H. 2003. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. The Journal of Cell Biology 162: 435-442.
","pubmedId":"","doi":"10.1083/jcb.200302131"},{"reference":"Bennett CL, Shirk AJ, Huynh HM, Street VA, Nelis E, Van Maldergem L, et al., Chance. 2004. SIMPLE mutation in demyelinating neuropathy and distribution in sciatic nerve. Annals of Neurology 55: 713-720.
","pubmedId":"","doi":"10.1002/ana.20094"},{"reference":"Bishop N, Horman A, Woodman P. 2002. Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein–ubiquitin conjugates. The Journal of Cell Biology 157: 91-102.
","pubmedId":"","doi":"10.1083/jcb.200112080"},{"reference":"Doyotte A, Russell MRG, Hopkins CR, Woodman PG. 2005. Depletion of TSG101 forms a mammalian `Class E' compartment: a multicisternal early endosome with multiple sorting defects. Journal of Cell Science 118: 3003-3017.
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","pubmedId":"10586237","doi":""},{"reference":"Iwata A, Kunimoto M, Inoue K. 1998. Schwann cell proliferation as the cause of peripheral neuropathy in neurofibromatosis-2. Journal of the Neurological Sciences 156: 201-204.
","pubmedId":"","doi":"10.1016/s0022-510x(98)00032-x"},{"reference":"Lee SM, Olzmann JA, Chin LS, Li L. 2011. Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome-autophagy pathways. J Cell Sci 124(Pt 19): 3319-31.
","pubmedId":"21896645","doi":""},{"reference":"Logan AM, Mammel AE, Robinson DC, Chin AL, Condon AF, Robinson FL. 2017. Schwann cell‐specific deletion of the endosomal PI 3‐kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2‐ErbB3 tyrosine kinase signaling. Glia 65: 1452-1470.
","pubmedId":"","doi":"10.1002/glia.23173"},{"reference":"Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN. 2003. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proceedings of the National Academy of Sciences 100: 7626-7631.
","pubmedId":"","doi":"10.1073/pnas.0932599100"},{"reference":"Markworth R, Bähr M, Burk K. 2021. Held Up in Traffic—Defects in the Trafficking Machinery in Charcot-Marie-Tooth Disease. Frontiers in Molecular Neuroscience 14: 10.3389/fnmol.2021.695294.
","pubmedId":"","doi":"10.3389/fnmol.2021.695294"},{"reference":"McLean JW, Wilson JA, Tian T, Watson JA, VanHart M, Bean AJ, et al., Wilson. 2022. Disruption of Endosomal Sorting in Schwann Cells Leads to Defective Myelination and Endosomal Abnormalities Observed in Charcot-Marie-Tooth Disease. The Journal of Neuroscience 42: 5085-5101.
","pubmedId":"","doi":"10.1523/jneurosci.2481-21.2022"},{"reference":"Newbern J, Birchmeier C. 2010. Nrg1/ErbB signaling networks in Schwann cell development and myelination. Seminars in Cell & Developmental Biology 21: 922-928.
","pubmedId":"","doi":"10.1016/j.semcdb.2010.08.008"},{"reference":"Raiborg C, Malerød L, Pedersen NM, Stenmark H. 2008. Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking. Experimental Cell Research 314: 801-813.
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","pubmedId":"","doi":"10.1091/mbc.e05-11-1054"},{"reference":"Roberts RC, Peden AA, Buss F, Bright NA, Latouche M, Reilly MM, Kendrick-Jones J, Luzio JP. 2009. Mistargeting of SH3TC2 away from the recycling endosome causes Charcot–Marie–Tooth disease type 4C. Human Molecular Genetics 19: 1009-1018.
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","pubmedId":"","doi":"10.1093/brain/awq168"},{"reference":"Tracy JA, Dyck PJ, Klein CJ, Engelstad JK, Meyer JE, Dyck PJB. 2019. Onion‐bulb patterns predict acquired or inherited demyelinating polyneuropathy. Muscle & Nerve 59: 665-670.
","pubmedId":"","doi":"10.1002/mus.26452"},{"reference":"Vaccari I, Carbone A, Previtali SC, Mironova YA, Alberizzi V, Noseda R, et al., Bolino. 2014. Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy. Human Molecular Genetics 24: 383-396.
","pubmedId":"","doi":"10.1093/hmg/ddu451"},{"reference":"Wagner KU, Krempler A, Qi Y, Park K, Henry MD, Triplett AA, et al., Hennighausen. 2003. Tsg101 Is Essential for Cell Growth, Proliferation, and Cell Survival of Embryonic and Adult Tissues. Molecular and Cellular Biology 23: 150-162.
","pubmedId":"","doi":"10.1128/mcb.23.1.150-162.2003"},{"reference":"Walker WP, Oehler A, Edinger AL, Wagner KU, Gunn TM. 2016. Oligodendroglial deletion of ESCRT‐I component TSG101 causes spongiform encephalopathy. Biology of the Cell 108: 324-337.
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","pubmedId":"5336778","doi":"10.1097/00005072-196704000-00008"}],"suggestedReviewer":{"name":"Dr. Scott Wilson, Department of Neurobiology, Evelyn F. McKnight Brain Institute, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294. Email: livvy01@uab.edu
","WBId":""},"title":"Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy
","reviews":[]},{"id":"3134eee1-54f0-48a3-b983-7df5a20ef16f","decisionLetter":"\n\n Dear Authors,\n
\n\n Congratulations on your new publication! We are pleased to let you know that your microPublication is \n now available online. You can access it here: https://micropublication.org/journals/biology/micropub-biology-001406\n
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\n\n For your records, this is your article's citation:
\n \"Silvius, D; Hurley, E; Poitelon, Y; Wagner, KU; Feltri, ML; Gunn, TM (2025). Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy. microPublication Biology. 10.17912/micropub.biology.001406.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"Myelinating Schwann cells are particularly susceptible to defects in endosomal trafficking. TSG101 is a component of the endosomal trafficking machinery that mediates the sorting of ubiquitinated receptors into multivesicular bodies. We previously demonstrated that deleting Tsg101 from mouse oligodendrocytes in the central nervous system causes rapid onset de/dys-myelination and vacuolation of white matter, suggesting an important role for TSG101-dependent trafficking in myelination. Here, we show that TSG101 is also required for normal myelination in the peripheral nervous system.
","acknowledgements":"We thank Anita Pecukonis and the McLaughlin Institute Animal Resource Center for excellent animal care and Dr. John R. Bermingham Jr for instruction on sciatic nerve dissection.
","authors":[{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States"],"credit":["investigation","writing_reviewEditing"],"email":"derek@mclaughlinresearch.org","firstName":"Derek","lastName":"Silvius","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","writing_reviewEditing"],"email":"edwardhu@buffalo.edu","firstName":"Edward","lastName":"Hurley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Albany Medical College, Albany, New York, United States"],"credit":["writing_reviewEditing"],"email":"poitely@amc.edu","firstName":"Yannick","lastName":"Poitelon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Wayne State University, Detroit, Michigan, United States"],"credit":["resources","writing_reviewEditing"],"email":"kuwagner@wayne.edu","firstName":"Kay-Uwe","lastName":"Wagner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","supervision","validation"],"email":"mamillig@buffalo.edu","firstName":"M. Laura","lastName":"Feltri","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States","Touro University College of Osteopathic Medicine - Montana, Great Falls, Montana, United States"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","methodology","fundingAcquisition"],"email":"tmg@mclaughlinresearch.org","firstName":"Teresa M.","lastName":"Gunn","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2688-6420"}],"comments":"Dear microPublication Editorial Team,
Thank you for the opportunity to revise our manuscript. We appreciate the reviewer’s comments, especially those noting that this work is “a novel demonstration of the necessity for Tsg101 (and, by extension, endosomal sorting) for maintenance of PNS myelin” and that “The article is well-written, clearly outlining the relevant background literature of other human variants or mouse mutants linking endosomal trafficking to dysmyelination/demyelination.”
We apologize that we did not include sample sizes to make it clear that we show seminthin data from 2 different P15 and 1 P21 conditional knockout nerves, and performed IHC for MBP on a larger number of samples (5 mutants) and show representative images of that data in the figure. As Dr. Laura Feltri has passed away and the analysis her lab performed took place quite a few years ago, we are unable to find all the necessary records to determine whether they looked at more than 2 x P15 and 1 x P21 nerves; that is all the data I was sent at the time. However, I hope the fact that this is a total of 3 different mice at two ages, and we looked at nerves from 5 mutant mice with MBP staining on paraffin sections, will satisfy the reviewer and the editorial team. The myelination phenotype was so robust and qualitatively obvious that we do not think it necessary to quantify it.
We have addressed the issues raised by editing the Methods and Figure Legend to clearly indicate sample sizes for the seminthin, EM and IHC analyses, as follows (additions are underlined):
Methods –Histology section:
2nd paragraph: “For IHC, specimens from five Tsg101SC-null and four control animals were embedded in…”
3rd paragraph: “Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age).”
Revised Figure legend:
Fig. 1. Histopathology of sciatic nerves lacking TSG101 in Schwann cells. (A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7 paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed. (G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
We hope the Editorial Team will agree that these revisions address the reviewer’s concerns and look forward to your response.
Thank you again for considering our manuscript for microPublication.
Sincerely,
Teresa Gunn
","dataTable":null,"disclaimer":true,"funding":"This work was funded by the McLaughlin Research Institute and its generous supporters. Kay-Uwe Wagner is the Lloyd and Marilyn Smith Endowed Professor of Breast Cancer Research at the Karmanos Comprehensive Cancer Center.
","image":{"name":"Fig 1 Tsg101 SC null.png","url":"https://portal.micropublication.org/uploads/c7ad46ba5ce67475b19a471661ada5c2.png"},"imageCaption":"(A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7m paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed. (G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mouse at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
","imageTitle":"Histopathology of sciatic nerves lacking TSG101 in Schwann cells.
","laboratory":{"name":"","WBId":""},"methods":"Animals
All studies were approved by the McLaughlin Research Institute’s Institutional Animal Care and Use Committee and adhered to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Mice homozygous for a Tsg101 conditional knockout allele (129X1/SvJ-Tsg101tm1Kuw, referred to herein as Tsg101fl; RRID: MMRRC_037407-MU), in which exon 1 is flanked by a floxed neo cassette inserted approximately 3 kb upstream and a single loxP site inserted 230 bp into intron 1 (Wagner et al., 2003), were mated to mice hemizygous for Tg(Mpz-cre)26Mes/J (obtained from the Jackson Laboratory and referred to herein as P0-cre; RRID: IMSR_JAX:017928). P0-cre transgenic mice express cre recombinase in Schwann cells, under the control of the myelin protein zero (P0, Mpz) promoter, by embryonic day 14 (Feltri et al., 1999). Tsg101fl/+; P0-cre+ F1 offspring were backcrossed to Tsg101fl/fl mice and Tsg101fl/fl; P0-cre+ and control (Tsg101fl/+; P0-cre+ and Tsg101fl/fl; P0-cre-neg) pups were obtained from intercrossing N1 or N1F1 Tsg101fl/+; P0-cre+ x Tsg101fl/fl; P0-cre-neg sibs.
Histology
Sciatic nerve segments were sampled from approximately the same position for each animal. Contralateral nerves were either fixed in 4% buffered paraformaldehyde, for immunohistochemistry (IHC), or 2% buffered glutaraldehyde with postfixation in 1% osmium tetroxide (for semi- and ultra-thin sections).
For IHC, specimens from five Tsg101SC-null and four control animals were embedded in paraffin following standard protocols and sectioned at 5 microns. Following deparaffinization and rehydration, sections were permeabilized in a 0.2% Triton-X100 solution in PBS, then subjected to antigen retrieval in 10mM sodium citrate (pH 6.0, 100 C, 10 minutes). Slides were blocked in 10% serum, then incubated with an antibody against myelin basic protein (MBP; Covance Cat# SMI-99, RRID:AB_2314772) at 1:1000, followed by horseradish peroxidase conjugated anti-mouse secondary antibody (BD Pharmingen horseradish peroxidase-conjugated anti-mouse Ig, cat# 554002, RRID: AB_395198) at 1:100 and chromogenic visualization using NovaRed substrate (Vector Labs, CA). Slides were counterstained with hematoxylin prior to coverslipping, and then examined on a Zeiss AxioImagerM1 light microscope.
Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age). Briefly, sciatic nerves were fixed in 2% buffered glutaraldehyde, then postfixed in 1% osmium tetroxide. After alcohol dehydration, nerves were submerged in propylene oxide, and then in a 1:1 mixture of Epon-propylene oxide. Nerves were embedded in 100% Epon, and resin was allowed to polymerize. Semithin transverse sections were sliced 0.5-μm-thick using Leica UC7, stained with 2% toluidine blue, and then examined by light microscopy with Leica DM6000B. EM transverse sections were sliced 700–900 Å-thick using Leica UC7, stained with uranyl acetate and lead citrate, and then examined with an electron microscope (model FEI BioTwin). Analyzed sections were sliced from the distal end of embedded sciatic nerve. Images acquired from semithins and EMs were nonoverlapping and comprehensive.
","reagents":"","patternDescription":"The endosomal pathway traffics receptor proteins and lipids into early endosomes and directs them either to recycling endosomes for trafficking back to the cell membrane or sorts them into intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which fuse with lysosomes to degrade their contents or with the plasma membrane to secrete ILVs as exosomes (reviewed in (Scott et al., 2014)). The timing of these events is important to cellular processes since receptors can continue to activate downstream signaling pathways while they remain on early endosomes. Several forms of demyelinating CMT are caused by mutations in genes encoding proteins involved in membrane dynamics and endosomal trafficking, including N-myc downstream regulated gene 1 (NDRG1), myotubularin related protein 2 (MTMR2), SET binding factor 2 (SBF2/MTMR13),SH3 domain and tetratricopeptide repeats 2 (SH3TC2), dynamin 2 (DNM2), FIG4 phosphoinositide 5-phosphatase (FIG4), and LPS-induced TN factor (LITAF/SIMPLE) (reviewed in (Markworth et al., 2021)). For example, mutations in SH3TC2 that cause CMT type 4C cause impaired recycling of membrane components necessary for Schwann cell function (Roberts et al., 2010; Stendel et al., 2010). Furthermore, mouse models with Schwann cell-specific deletion of Fig4, HGF-regulated tyrosine kinase substrate (Hgs/Hrs), or phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) show mild peripheral hypo/de/dys-myelination associated with altered ERBB2/3 signaling (Logan et al., 2017; McLean et al., 2022; Vaccari et al., 2015). In Schwann cells, neuregulin 1 (NRG1) signaling through erb-b2 receptor tyrosine kinases 2 and 3 (ERBB2/B3) mediates myelination, and endosomal trafficking of NRG1-bound ERBB2/B3 regulates receptor down-regulation and recycling (Newbern and Birchmeier, 2010; Salzer, 2015). Disrupted endosomal sorting can result in sustained activation of downstream pathways, including the ERK1/2 signaling cascade, to negatively impact Schwann cell myelin integrity (Newbern and Birchmeier, 2010; Salzer, 2015). Thus, there is support for the idea that endosomal trafficking defects can cause Schwann cell dysfunction and demyelination, reinforcing the relevance of this pathway to myelination.
Tumor susceptibility gene 101 (TSG101) encodes a component of the Endosomal Sorting Complex Required for Transport-1 (ESCRT-I), which helps mediate the sorting of ubiquitinated receptors onto ILVs of MVBs. The ESCRT-0 protein, HGF-regulated tyrosine kinase substrate, HGS (formerly referred to as HRS) mediates the initial recruitment of ESCRT-I to endosomes (via interaction with TSG101), but in multiple mammalian cell lines, siRNA depletion of TSG101 or HGS had significantly different effects on endosomal and MVB morphology (Bache et al., 2003; Lu et al., 2003; Raiborg et al., 2008; Razi and Futter, 2006). Specifically, knockdown of TSG101 inhibited epidermal growth factor degradation and MVB formation and caused tubulation of the vacuolar domains of early endosomes, while depletion of HGS had only a modest effect on EGF degradation, did not induce tubulation of early endosomes, and resulted in the production of enlarged MVBs containing few ILVs but that could still fuse with the lysosome (Bishop et al., 2002; Doyotte et al., 2005; Razi and Futter, 2006). These data suggest that HGS and TSG101 have distinct roles in the endosomal trafficking pathway, with TSG101 being required for the formation of stable vacuolar domains within the early endosome that subsequently develop into MVBs and HGS being more important in the formation and/or accumulation of ILVs within MVBs. Mice lacking HGS in Schwann cells developed mild motor and sensory defects, a reduced number of myelinated axons and thinner myelin sheaths in the sciatic nerve, as well as aberrantly folded myelin sheaths (McLean et al., 2022).
TSG101 and HGS also both interact and partially colocalize with LITAF (Lee et al., 2011), which is expressed in Schwann cells and mutations in it cause dominant demyelinating peripheral neuropathy, CMT1C (Bennett et al., 2004). Although one study showed that CMT1C-associated LITAF mutations did not effect on its subcellular localization or association with TSG101 (Shirk et al., 2005), another study showed a dominant negative effect on EGFR degradation and lysosomal trafficking of EGF, associated with reduced membrane association of HGS and TSG101 (Lee et al., 2011). In the latter study, expression of CMT1C-associated LITAF mutants in Schwann cells caused prolonged activation of ERK1/2 signaling, presumably downstream of NRG1-ERBB2/3 signaling.
Deleting Tsg101 from oligodendroglia in the central nervous system resulted in severe, rapid-onset myelination defects and vacuolation (Walker et al., 2016), suggesting an important role for TSG101-dependent trafficking in signaling pathways that regulate myelination. To test whether TSG101 is also required for normal myelination in the peripheral nervous system, we investigated the consequences of deleting Tsg101 in Schwann cells. We predicted this would cause a more severe peripheral neuropathy than deleting Hgs, given the stronger effect of Tsg101 depletion on endosomal/MVB phenotypes in cultured cells.
Tsg101 conditional knockout mice (Tsg101tm1KuW, referred to here as Tsg101fl) were mated to P0-Cre transgenic mice, which express cre recombinase specifically in Schwann cells starting on embryonic day 13.5. Cre-positive Tsg101fl/+ offspring were backcrossed to Tsg101fl/fl animals. All pups that were homozygous for the Tsg101 conditional allele and carried the P0-Cre transgene (referred to herein as Tsg101-Schwann cell null, or Tsg101SC-null animals) were smaller than their littermates, developed a tremor by 12 days of age, had abnormal posture of their fore- and hind-limbs (arthrogryposis), and failed to thrive. They died throughout the postnatal period, with very few surviving to 3 weeks of age. We recorded 89 affected animals out of 427 pups born from 15 different breeder pairs, for a frequency of ~21% affected. This is significantly different from the 25% expected (χ2= 4.04, p = 0.044) and is likely due to the loss of some affected animals prior to them being observed and recorded.
Histological analysis of the sciatic nerves of Tsg101SC-null animals at postnatal days 15 and 21 revealed striking de- and dys-myelination (Fig. 1). Toluidine blue-stained semi-thin cross sections showed reduced myelin, enlargement of the interstitial space, and presence of onion bulb structures in the sciatic nerves of Tsg101SC-null mice by postnatal day 15 (P15, Fig. 1A-C). The sciatic nerves of some Tsg101SC-null animals had fewer, thinner myelin sheaths, while others showed an almost complete absence of myelin, as detected by toluidine blue staining on semithin sections (Fig. 1A-C) and immunohistochemistry for myelin basic protein (MBP) on paraffin sections (Fig. 1D-F). The myelination defects associated with loss of TSG101 were progressive, with the sciatic nerve of a P21 Tsg101SC-null animal showing severe hypomyelination and presence of onion bulb formations, (Fig. G-H). Ultrathin electron micrograph (EM) analysis of P21 sciatic nerve cross-sections revealed axons with thin myelin sheaths and multiple cell layers adjacent to nucleus of Schwann cells. These “onion bulbs” comprise concentric layers of Schwann cell processes and connective tissue (collagen) arranged around axons and are consistent with multiple rounds of de- and remyelination (Iwata et al., 1998; Tracy et al., 2019; Webster et al., 1967). This suggests that TSG101 is not essential for myelin production, but it is required for the maintenance of stable myelin sheaths. Schwann cell nuclei were still present by P21 and did not appear pyknotic (Fig. 1F), suggesting that loss of TSG101 did not disrupt myelination by triggering Schwann cell apoptosis.
As predicted, based on the differences observed in cellular phenotypes and EGF/EGFR degradation when TSG101 or HGS was depleted from mammalian cells by siRNA, the phenotype of Tsg101SC-null mice was more severe than that observed in HgsSC-null mice. Onion bulb formations are characteristic features in CMT1A and other demyelinating CMTs. Since TSG101 and ESCRT proteins are critical for endosomal sorting, their dysfunction could impair myelin production and turnover, exacerbating the cycles of demyelination and remyelination that contribute to onion bulb pathology. In the future, proteomic studies may shed light on the specific signaling pathways disrupted in Tsg101SC-null that contribute to the severe peripheral neuropathy phenotype. The variable expressivity of myelination defects and survival of Tsg101SC-null mice likely reflects their mixed genetic background (129S1/Sv x C57BL/6) and an effect of modifier genes, although inter-animal differences in P0-cre expression and Tsg101 deletion cannot be ruled out. Identifying the genes and pathways that influence disease severity in these mice could reveal druggable targets to treat some forms of CMT or other peripheral neuropathies.
","references":[{"reference":"Bache KG, Brech A, Mehlum A, Stenmark H. 2003. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. The Journal of Cell Biology 162: 435-442.
","pubmedId":"","doi":"10.1083/jcb.200302131"},{"reference":"Bennett CL, Shirk AJ, Huynh HM, Street VA, Nelis E, Van Maldergem L, et al., Chance. 2004. SIMPLE mutation in demyelinating neuropathy and distribution in sciatic nerve. Annals of Neurology 55: 713-720.
","pubmedId":"","doi":"10.1002/ana.20094"},{"reference":"Bishop N, Horman A, Woodman P. 2002. Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein–ubiquitin conjugates. The Journal of Cell Biology 157: 91-102.
","pubmedId":"","doi":"10.1083/jcb.200112080"},{"reference":"Doyotte A, Russell MRG, Hopkins CR, Woodman PG. 2005. Depletion of TSG101 forms a mammalian `Class E' compartment: a multicisternal early endosome with multiple sorting defects. Journal of Cell Science 118: 3003-3017.
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","pubmedId":"21896645","doi":""},{"reference":"Logan AM, Mammel AE, Robinson DC, Chin AL, Condon AF, Robinson FL. 2017. Schwann cell‐specific deletion of the endosomal PI 3‐kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2‐ErbB3 tyrosine kinase signaling. Glia 65: 1452-1470.
","pubmedId":"","doi":"10.1002/glia.23173"},{"reference":"Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN. 2003. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proceedings of the National Academy of Sciences 100: 7626-7631.
","pubmedId":"","doi":"10.1073/pnas.0932599100"},{"reference":"Markworth R, Bähr M, Burk K. 2021. Held Up in Traffic—Defects in the Trafficking Machinery in Charcot-Marie-Tooth Disease. Frontiers in Molecular Neuroscience 14: 10.3389/fnmol.2021.695294.
","pubmedId":"","doi":"10.3389/fnmol.2021.695294"},{"reference":"McLean JW, Wilson JA, Tian T, Watson JA, VanHart M, Bean AJ, et al., Wilson. 2022. Disruption of Endosomal Sorting in Schwann Cells Leads to Defective Myelination and Endosomal Abnormalities Observed in Charcot-Marie-Tooth Disease. The Journal of Neuroscience 42: 5085-5101.
","pubmedId":"","doi":"10.1523/jneurosci.2481-21.2022"},{"reference":"Newbern J, Birchmeier C. 2010. Nrg1/ErbB signaling networks in Schwann cell development and myelination. Seminars in Cell & Developmental Biology 21: 922-928.
","pubmedId":"","doi":"10.1016/j.semcdb.2010.08.008"},{"reference":"Raiborg C, Malerød L, Pedersen NM, Stenmark H. 2008. Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking. Experimental Cell Research 314: 801-813.
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","pubmedId":"","doi":"10.1091/mbc.e05-11-1054"},{"reference":"Roberts RC, Peden AA, Buss F, Bright NA, Latouche M, Reilly MM, Kendrick-Jones J, Luzio JP. 2009. Mistargeting of SH3TC2 away from the recycling endosome causes Charcot–Marie–Tooth disease type 4C. Human Molecular Genetics 19: 1009-1018.
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","pubmedId":"","doi":"10.1093/brain/awq168"},{"reference":"Tracy JA, Dyck PJ, Klein CJ, Engelstad JK, Meyer JE, Dyck PJB. 2019. Onion‐bulb patterns predict acquired or inherited demyelinating polyneuropathy. Muscle & Nerve 59: 665-670.
","pubmedId":"","doi":"10.1002/mus.26452"},{"reference":"Vaccari I, Carbone A, Previtali SC, Mironova YA, Alberizzi V, Noseda R, et al., Bolino. 2014. Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy. Human Molecular Genetics 24: 383-396.
","pubmedId":"","doi":"10.1093/hmg/ddu451"},{"reference":"Wagner KU, Krempler A, Qi Y, Park K, Henry MD, Triplett AA, et al., Hennighausen. 2003. Tsg101 Is Essential for Cell Growth, Proliferation, and Cell Survival of Embryonic and Adult Tissues. Molecular and Cellular Biology 23: 150-162.
","pubmedId":"","doi":"10.1128/mcb.23.1.150-162.2003"},{"reference":"Walker WP, Oehler A, Edinger AL, Wagner KU, Gunn TM. 2016. Oligodendroglial deletion of ESCRT‐I component TSG101 causes spongiform encephalopathy. Biology of the Cell 108: 324-337.
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","pubmedId":"5336778","doi":"10.1097/00005072-196704000-00008"}],"suggestedReviewer":{"name":"Dr. Scott Wilson, Department of Neurobiology, Evelyn F. McKnight Brain Institute, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294. Email: livvy01@uab.edu
","WBId":""},"title":"Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy
","reviews":[]},{"id":"df7ceb17-3179-42c0-9ae3-abca09b740c0","decisionLetter":"\n\n Dear Authors,\n
\n\n Congratulations on your new publication! We are pleased to let you know that your microPublication is \n now available online. You can access it here: https://micropublication.org/journals/biology/micropub-biology-001406\n
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\n \"Silvius, D; Hurley, E; Poitelon, Y; Wagner, KU; Feltri, ML; Gunn, TM (2025). Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy. microPublication Biology. 10.17912/micropub.biology.001406.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"Myelinating Schwann cells are particularly susceptible to defects in endosomal trafficking. TSG101 is a component of the endosomal trafficking machinery that mediates the sorting of ubiquitinated receptors into multivesicular bodies. We previously demonstrated that deleting Tsg101 from mouse oligodendrocytes in the central nervous system causes rapid onset de/dys-myelination and vacuolation of white matter, suggesting an important role for TSG101-dependent trafficking in myelination. Here, we show that TSG101 is also required for normal myelination in the peripheral nervous system.
","acknowledgements":"We thank Anita Pecukonis and the McLaughlin Institute Animal Resource Center for excellent animal care and Dr. John R. Bermingham Jr for instruction on sciatic nerve dissection.
","authors":[{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States"],"credit":["investigation","writing_reviewEditing"],"email":"derek@mclaughlinresearch.org","firstName":"Derek","lastName":"Silvius","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","writing_reviewEditing"],"email":"edwardhu@buffalo.edu","firstName":"Edward","lastName":"Hurley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Albany Medical College, Albany, New York, United States"],"credit":["writing_reviewEditing"],"email":"poitely@amc.edu","firstName":"Yannick","lastName":"Poitelon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Wayne State University, Detroit, Michigan, United States"],"credit":["resources","writing_reviewEditing"],"email":"kuwagner@wayne.edu","firstName":"Kay-Uwe","lastName":"Wagner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University at Buffalo, State University of New York, Buffalo, New York, United States"],"credit":["investigation","supervision","validation"],"email":"mamillig@buffalo.edu","firstName":"M. Laura","lastName":"Feltri","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["McLaughlin Research Institute, Great Falls, Montana, United States","Touro University College of Osteopathic Medicine - Montana, Great Falls, Montana, United States"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","methodology","fundingAcquisition"],"email":"tmg@mclaughlinresearch.org","firstName":"Teresa M.","lastName":"Gunn","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2688-6420"}],"comments":"Dear microPublication Editorial Team,
Thank you for the opportunity to revise our manuscript. We appreciate the reviewer’s comments, especially those noting that this work is “a novel demonstration of the necessity for Tsg101 (and, by extension, endosomal sorting) for maintenance of PNS myelin” and that “The article is well-written, clearly outlining the relevant background literature of other human variants or mouse mutants linking endosomal trafficking to dysmyelination/demyelination.”
We apologize that we did not include sample sizes to make it clear that we show seminthin data from 2 different P15 and 1 P21 conditional knockout nerves, and performed IHC for MBP on a larger number of samples (5 mutants) and show representative images of that data in the figure. As Dr. Laura Feltri has passed away and the analysis her lab performed took place quite a few years ago, we are unable to find all the necessary records to determine whether they looked at more than 2 x P15 and 1 x P21 nerves; that is all the data I was sent at the time. However, I hope the fact that this is a total of 3 different mice at two ages, and we looked at nerves from 5 mutant mice with MBP staining on paraffin sections, will satisfy the reviewer and the editorial team. The myelination phenotype was so robust and qualitatively obvious that we do not think it necessary to quantify it.
We have addressed the issues raised by editing the Methods and Figure Legend to clearly indicate sample sizes for the seminthin, EM and IHC analyses, as follows (additions are underlined):
Methods –Histology section:
2nd paragraph: “For IHC, specimens from five Tsg101SC-null and four control animals were embedded in…”
3rd paragraph: “Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age).”
Revised Figure legend:
Fig. 1. Histopathology of sciatic nerves lacking TSG101 in Schwann cells. (A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7 paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed. (G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mice at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
We hope the Editorial Team will agree that these revisions address the reviewer’s concerns and look forward to your response.
Thank you again for considering our manuscript for microPublication.
Sincerely,
Teresa Gunn
","dataTable":null,"disclaimer":true,"funding":"This work was funded by the McLaughlin Research Institute and its generous supporters. Kay-Uwe Wagner is the Lloyd and Marilyn Smith Endowed Professor of Breast Cancer Research at the Karmanos Comprehensive Cancer Center. Institutional support is provided to T.M.G. through a NIH Center grant from National Institute of General Medical Sciences (NIGMS; P20GM152335).
","image":{"name":"Fig 1 Tsg101 SC null.png","url":"https://portal.micropublication.org/uploads/c7ad46ba5ce67475b19a471661ada5c2.png"},"imageCaption":"(A-C) Representative semi-thin cross sections of sciatic nerves from one control (Tsg101fl/+; P0-Cre+) and two different Tsg101SC-null (Tsg101fl/fl; P0-Cre+) postnatal day 15 (P15) pups showing reduction of myelin and presence of onion bulb structures in nerves from Tsg101SC-null mice. Scale bars: 5µm. (D-F) Representative IHC for myelin basic protein (MBP) on 7m paraffin-embedded cross sections on contralateral nerves from animals shown in A-C (B and E from same animal, C and F from same animal). Sections are counterstained with hematoxylin. Scale bars: 10µm. Note the difference in severity of myelination defects between the two Tsg101SC-null animals. Sciatic nerves from 5 Tsg101SC-null mice were examined, the images shown represent the spectrum of the phenotypes observed. (G-H) Representative semi-thin cross sections of sciatic nerves of 1 control and 1 Tsg101SC-null mouse at P21 showing similar but more severe myelination defects compared to P15 nerves. Scale bars: 5µm. (I) Electron micrograph (4800X) of sciatic nerve cross-section from a P21 Tsg101SC-null mouse. Scale bar: 500nm. Note Schwann cell layers adjacent to nucleus, indicating multiple rounds of de- and remyelination.
","imageTitle":"Histopathology of sciatic nerves lacking TSG101 in Schwann cells.
","laboratory":{"name":"","WBId":""},"methods":"Animals
All studies were approved by the McLaughlin Research Institute’s Institutional Animal Care and Use Committee and adhered to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Mice homozygous for a Tsg101 conditional knockout allele (129X1/SvJ-Tsg101tm1Kuw, referred to herein as Tsg101fl; RRID: MMRRC_037407-MU), in which exon 1 is flanked by a floxed neo cassette inserted approximately 3 kb upstream and a single loxP site inserted 230 bp into intron 1 (Wagner et al., 2003), were mated to mice hemizygous for Tg(Mpz-cre)26Mes/J (obtained from the Jackson Laboratory and referred to herein as P0-cre; RRID: IMSR_JAX:017928). P0-cre transgenic mice express cre recombinase in Schwann cells, under the control of the myelin protein zero (P0, Mpz) promoter, by embryonic day 14 (Feltri et al., 1999). Tsg101fl/+; P0-cre+ F1 offspring were backcrossed to Tsg101fl/fl mice and Tsg101fl/fl; P0-cre+ and control (Tsg101fl/+; P0-cre+ and Tsg101fl/fl; P0-cre-neg) pups were obtained from intercrossing N1 or N1F1 Tsg101fl/+; P0-cre+ x Tsg101fl/fl; P0-cre-neg sibs.
Histology
Sciatic nerve segments were sampled from approximately the same position for each animal. Contralateral nerves were either fixed in 4% buffered paraformaldehyde, for immunohistochemistry (IHC), or 2% buffered glutaraldehyde with postfixation in 1% osmium tetroxide (for semi- and ultra-thin sections).
For IHC, specimens from five Tsg101SC-null and four control animals were embedded in paraffin following standard protocols and sectioned at 5 microns. Following deparaffinization and rehydration, sections were permeabilized in a 0.2% Triton-X100 solution in PBS, then subjected to antigen retrieval in 10mM sodium citrate (pH 6.0, 100 C, 10 minutes). Slides were blocked in 10% serum, then incubated with an antibody against myelin basic protein (MBP; Covance Cat# SMI-99, RRID:AB_2314772) at 1:1000, followed by horseradish peroxidase conjugated anti-mouse secondary antibody (BD Pharmingen horseradish peroxidase-conjugated anti-mouse Ig, cat# 554002, RRID: AB_395198) at 1:100 and chromogenic visualization using NovaRed substrate (Vector Labs, CA). Slides were counterstained with hematoxylin prior to coverslipping, and then examined on a Zeiss AxioImagerM1 light microscope.
Routine semithin and ultrathin (electron micrograph [EM]) analyses were performed as described (Quattrini et al., 1996) on sciatic nerves from Tsg101SC-null animals (n=2 at P15, n=1 at P21) and controls (n=1-2 at each age). Briefly, sciatic nerves were fixed in 2% buffered glutaraldehyde, then postfixed in 1% osmium tetroxide. After alcohol dehydration, nerves were submerged in propylene oxide, and then in a 1:1 mixture of Epon-propylene oxide. Nerves were embedded in 100% Epon, and resin was allowed to polymerize. Semithin transverse sections were sliced 0.5-μm-thick using Leica UC7, stained with 2% toluidine blue, and then examined by light microscopy with Leica DM6000B. EM transverse sections were sliced 700–900 Å-thick using Leica UC7, stained with uranyl acetate and lead citrate, and then examined with an electron microscope (model FEI BioTwin). Analyzed sections were sliced from the distal end of embedded sciatic nerve. Images acquired from semithins and EMs were nonoverlapping and comprehensive.
","reagents":"","patternDescription":"The endosomal pathway traffics receptor proteins and lipids into early endosomes and directs them either to recycling endosomes for trafficking back to the cell membrane or sorts them into intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which fuse with lysosomes to degrade their contents or with the plasma membrane to secrete ILVs as exosomes (reviewed in (Scott et al., 2014)). The timing of these events is important to cellular processes since receptors can continue to activate downstream signaling pathways while they remain on early endosomes. Several forms of demyelinating CMT are caused by mutations in genes encoding proteins involved in membrane dynamics and endosomal trafficking, including N-myc downstream regulated gene 1 (NDRG1), myotubularin related protein 2 (MTMR2), SET binding factor 2 (SBF2/MTMR13),SH3 domain and tetratricopeptide repeats 2 (SH3TC2), dynamin 2 (DNM2), FIG4 phosphoinositide 5-phosphatase (FIG4), and LPS-induced TN factor (LITAF/SIMPLE) (reviewed in (Markworth et al., 2021)). For example, mutations in SH3TC2 that cause CMT type 4C cause impaired recycling of membrane components necessary for Schwann cell function (Roberts et al., 2010; Stendel et al., 2010). Furthermore, mouse models with Schwann cell-specific deletion of Fig4, HGF-regulated tyrosine kinase substrate (Hgs/Hrs), or phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) show mild peripheral hypo/de/dys-myelination associated with altered ERBB2/3 signaling (Logan et al., 2017; McLean et al., 2022; Vaccari et al., 2015). In Schwann cells, neuregulin 1 (NRG1) signaling through erb-b2 receptor tyrosine kinases 2 and 3 (ERBB2/B3) mediates myelination, and endosomal trafficking of NRG1-bound ERBB2/B3 regulates receptor down-regulation and recycling (Newbern and Birchmeier, 2010; Salzer, 2015). Disrupted endosomal sorting can result in sustained activation of downstream pathways, including the ERK1/2 signaling cascade, to negatively impact Schwann cell myelin integrity (Newbern and Birchmeier, 2010; Salzer, 2015). Thus, there is support for the idea that endosomal trafficking defects can cause Schwann cell dysfunction and demyelination, reinforcing the relevance of this pathway to myelination.
Tumor susceptibility gene 101 (TSG101) encodes a component of the Endosomal Sorting Complex Required for Transport-1 (ESCRT-I), which helps mediate the sorting of ubiquitinated receptors onto ILVs of MVBs. The ESCRT-0 protein, HGF-regulated tyrosine kinase substrate, HGS (formerly referred to as HRS) mediates the initial recruitment of ESCRT-I to endosomes (via interaction with TSG101), but in multiple mammalian cell lines, siRNA depletion of TSG101 or HGS had significantly different effects on endosomal and MVB morphology (Bache et al., 2003; Lu et al., 2003; Raiborg et al., 2008; Razi and Futter, 2006). Specifically, knockdown of TSG101 inhibited epidermal growth factor degradation and MVB formation and caused tubulation of the vacuolar domains of early endosomes, while depletion of HGS had only a modest effect on EGF degradation, did not induce tubulation of early endosomes, and resulted in the production of enlarged MVBs containing few ILVs but that could still fuse with the lysosome (Bishop et al., 2002; Doyotte et al., 2005; Razi and Futter, 2006). These data suggest that HGS and TSG101 have distinct roles in the endosomal trafficking pathway, with TSG101 being required for the formation of stable vacuolar domains within the early endosome that subsequently develop into MVBs and HGS being more important in the formation and/or accumulation of ILVs within MVBs. Mice lacking HGS in Schwann cells developed mild motor and sensory defects, a reduced number of myelinated axons and thinner myelin sheaths in the sciatic nerve, as well as aberrantly folded myelin sheaths (McLean et al., 2022).
TSG101 and HGS also both interact and partially colocalize with LITAF (Lee et al., 2011), which is expressed in Schwann cells and mutations in it cause dominant demyelinating peripheral neuropathy, CMT1C (Bennett et al., 2004). Although one study showed that CMT1C-associated LITAF mutations did not effect on its subcellular localization or association with TSG101 (Shirk et al., 2005), another study showed a dominant negative effect on EGFR degradation and lysosomal trafficking of EGF, associated with reduced membrane association of HGS and TSG101 (Lee et al., 2011). In the latter study, expression of CMT1C-associated LITAF mutants in Schwann cells caused prolonged activation of ERK1/2 signaling, presumably downstream of NRG1-ERBB2/3 signaling.
Deleting Tsg101 from oligodendroglia in the central nervous system resulted in severe, rapid-onset myelination defects and vacuolation (Walker et al., 2016), suggesting an important role for TSG101-dependent trafficking in signaling pathways that regulate myelination. To test whether TSG101 is also required for normal myelination in the peripheral nervous system, we investigated the consequences of deleting Tsg101 in Schwann cells. We predicted this would cause a more severe peripheral neuropathy than deleting Hgs, given the stronger effect of Tsg101 depletion on endosomal/MVB phenotypes in cultured cells.
Tsg101 conditional knockout mice (Tsg101tm1KuW, referred to here as Tsg101fl) were mated to P0-Cre transgenic mice, which express cre recombinase specifically in Schwann cells starting on embryonic day 13.5. Cre-positive Tsg101fl/+ offspring were backcrossed to Tsg101fl/fl animals. All pups that were homozygous for the Tsg101 conditional allele and carried the P0-Cre transgene (referred to herein as Tsg101-Schwann cell null, or Tsg101SC-null animals) were smaller than their littermates, developed a tremor by 12 days of age, had abnormal posture of their fore- and hind-limbs (arthrogryposis), and failed to thrive. They died throughout the postnatal period, with very few surviving to 3 weeks of age. We recorded 89 affected animals out of 427 pups born from 15 different breeder pairs, for a frequency of ~21% affected. This is significantly different from the 25% expected (χ2= 4.04, p = 0.044) and is likely due to the loss of some affected animals prior to them being observed and recorded.
Histological analysis of the sciatic nerves of Tsg101SC-null animals at postnatal days 15 and 21 revealed striking de- and dys-myelination (Fig. 1). Toluidine blue-stained semi-thin cross sections showed reduced myelin, enlargement of the interstitial space, and presence of onion bulb structures in the sciatic nerves of Tsg101SC-null mice by postnatal day 15 (P15, Fig. 1A-C). The sciatic nerves of some Tsg101SC-null animals had fewer, thinner myelin sheaths, while others showed an almost complete absence of myelin, as detected by toluidine blue staining on semithin sections (Fig. 1A-C) and immunohistochemistry for myelin basic protein (MBP) on paraffin sections (Fig. 1D-F). The myelination defects associated with loss of TSG101 were progressive, with the sciatic nerve of a P21 Tsg101SC-null animal showing severe hypomyelination and presence of onion bulb formations, (Fig. G-H). Ultrathin electron micrograph (EM) analysis of P21 sciatic nerve cross-sections revealed axons with thin myelin sheaths and multiple cell layers adjacent to nucleus of Schwann cells. These “onion bulbs” comprise concentric layers of Schwann cell processes and connective tissue (collagen) arranged around axons and are consistent with multiple rounds of de- and remyelination (Iwata et al., 1998; Tracy et al., 2019; Webster et al., 1967). This suggests that TSG101 is not essential for myelin production, but it is required for the maintenance of stable myelin sheaths. Schwann cell nuclei were still present by P21 and did not appear pyknotic (Fig. 1F), suggesting that loss of TSG101 did not disrupt myelination by triggering Schwann cell apoptosis.
As predicted, based on the differences observed in cellular phenotypes and EGF/EGFR degradation when TSG101 or HGS was depleted from mammalian cells by siRNA, the phenotype of Tsg101SC-null mice was more severe than that observed in HgsSC-null mice. Onion bulb formations are characteristic features in CMT1A and other demyelinating CMTs. Since TSG101 and ESCRT proteins are critical for endosomal sorting, their dysfunction could impair myelin production and turnover, exacerbating the cycles of demyelination and remyelination that contribute to onion bulb pathology. In the future, proteomic studies may shed light on the specific signaling pathways disrupted in Tsg101SC-null that contribute to the severe peripheral neuropathy phenotype. The variable expressivity of myelination defects and survival of Tsg101SC-null mice likely reflects their mixed genetic background (129S1/Sv x C57BL/6) and an effect of modifier genes, although inter-animal differences in P0-cre expression and Tsg101 deletion cannot be ruled out. Identifying the genes and pathways that influence disease severity in these mice could reveal druggable targets to treat some forms of CMT or other peripheral neuropathies.
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","pubmedId":"5336778","doi":"10.1097/00005072-196704000-00008"}],"suggestedReviewer":{"name":"Dr. Scott Wilson, Department of Neurobiology, Evelyn F. McKnight Brain Institute, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294. Email: livvy01@uab.edu
","WBId":""},"title":"Schwann cell deletion of Tumor Susceptibility Gene 101 (Tsg101) in mice results in severe peripheral neuropathy
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