The wmN1 Enhancer Region of the Mouse Myelin Proteolipid Protein Gene (mPlp1) is Indispensable for Expression of an mPlp1-lacZ Transgene in Both the CNS and PNS

  • Pankaj Patyal
  • Neriman T. Kockara
  • Patricia A. WightEmail author
Original Paper


The myelin proteolipid protein gene (PLP1) encodes the most abundant protein in CNS myelin. Expression of the gene must be strictly regulated, as evidenced by human X-linked leukodystrophies resulting from variations in PLP1 copy number, including elevated dosages as well as deletions. Recently, we showed that the wmN1 region in human PLP1 (hPLP1) intron 1 is required to promote high levels of an hPLP1-lacZ transgene in mice, using a Cre-lox approach. The current study tests whether loss of the wmN1 region from a related transgene containing mouse Plp1 (mPlp1) DNA produces similar results. In addition, we investigated the effects of loss of another region (ASE) in mPlp1 intron 1. Previous studies have shown that the ASE is required to promote high levels of mPlp1-lacZ expression by transfection analysis, but had no effect when removed from the native gene in mouse. Whether this is due to compensation by another regulatory element in mPlp1 that was not included in the mPlp1-lacZ constructs, or to differences in methodology, is unclear. Two transgenic mouse lines were generated that harbor mPLP(+)Z/FL. The parental transgene utilizes mPlp1 sequences (proximal 2.3 kb of 5ʹ-flanking DNA to the first 37 bp of exon 2) to drive expression of a lacZ reporter cassette. Here we demonstrate that mPLP(+)Z/FL is expressed in oligodendrocytes, oligodendrocyte precursor cells, olfactory ensheathing cells and neurons in brain, and Schwann cells in sciatic nerve. Loss of the wmN1 region from the parental transgene abolished expression, whereas removal of the ASE had no effect.


Myelin proteolipid protein gene (Plp1Gene regulation Enhancer Cre/loxP and Flp/Frt recombination lacZ Transgenic mouse 



The authors are grateful to Joseph J. Goellner and Dr. Charles A. O’Brien at the UAMS Transgenic Mouse Core Facility for generation of the mPLP(+)Z/FL founder mice. The core is supported by a COBRE grant from the National Institutes of Health (P20GM125503) and Tobacco Settlement Funds.


This work was supported by National Institutes of Health Grants R01NS106179 (PAW) and UL1TR000039, and a gift from the Rampy MS Research Foundation.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

11064_2019_2919_MOESM1_ESM.pptx (1.1 mb)
Supplementary material 1 (PPTX 1101 kb)
11064_2019_2919_MOESM2_ESM.pptx (527 kb)
Supplementary material 2 (PPTX 527 kb)


  1. 1.
    Diehl HJ, Schaich M, Budzinski RM, Stoffel W (1986) Individual exons encode the integral membrane domains of human myelin proteolipid protein. Proc Natl Acad Sci USA 83:9807–9811PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Macklin WB, Campagnoni CW, Deininger PL, Gardinier MV (1987) Structure and expression of the mouse myelin proteolipid protein gene. J Neurosci Res 18:383–394PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Ikenaka K, Furuichi T, Iwasaki Y, Moriguchi A, Okano H, Mikoshiba K (1988) Myelin proteolipid protein gene structure and its regulation of expression in normal and jimpy mutant mice. J Mol Biol 199:587–596PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Wight PA, Dobretsova A (1997) The first intron of the myelin proteolipid protein gene confers cell type-specific expression by a transcriptional repression mechanism in non-expressing cell types. Gene 201:111–117PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Hamdan H, Kockara NT, Jolly LA, Haun S, Wight PA (2015) Control of human PLP1 expression through transcriptional regulatory elements and alternatively spliced exons in intron 1. ASN Neuro 7:1–12CrossRefGoogle Scholar
  6. 6.
    Jahn O, Tenzer S, Werner HB (2009) Myelin proteomics: molecular anatomy of an insulating sheath. Mol Neurobiol 40:55–72PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Dickinson PJ, Griffiths IR, Barrie JM, Kyriakides E, Pollock GF, Barnett SC (1997) Expression of the dm-20 isoform of the plp gene in olfactory nerve ensheathing cells: evidence from developmental studies. J Neurocytol 26:181–189PubMedCrossRefGoogle Scholar
  8. 8.
    Miller MJ, Kangas CD, Macklin WB (2009) Neuronal expression of the proteolipid protein gene in the medulla of the mouse. J Neurosci Res 87:2842–2853PubMedCrossRefGoogle Scholar
  9. 9.
    Griffiths IR, Dickinson P, Montague P (1995) Expression of the proteolipid protein gene in glial cells of the post-natal peripheral nervous system of rodents. Neuropathol Appl Neurobiol 21:97–110PubMedCrossRefGoogle Scholar
  10. 10.
    Rao M, Nelms BD, Dong L, Salinas-Rios V, Rutlin M, Gershon MD, Corfas G (2015) Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system. Glia 63:2040–2057PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Grundmann D, Loris E, Maas-Omlor S, Huang W, Scheller A, Kirchhoff F, Schäfer KH (2019) Enteric Glia: S100, GFAP, and Beyond. Anat Rec 302:1333–1344CrossRefGoogle Scholar
  12. 12.
    Inoue K (2005) PLP1-related inherited dysmyelinating disorders: pelizaeus-Merzbacher disease and spastic paraplegia type 2. Neurogenetics 6:1–16PubMedCrossRefGoogle Scholar
  13. 13.
    Garbern JY (2007) Pelizaeus-Merzbacher disease: genetic and cellular pathogenesis. Cell Mol Life Sci 64:50–65PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Hobson GM, Garbern JY (2012) Pelizaeus-Merzbacher disease, Pelizaeus-Merzbacher-like disease 1, and related hypomyelinating disorders. Semin Neurol 32:62–67PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Hobson GM, Kamholz J (2013) PLP1-related disorders. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K (eds) GeneReviews®. University of Washington, Seattle, pp 1993–2019Google Scholar
  16. 16.
    Regis S, Grossi S, Corsolini F, Biancheri R, Filocamo M (2009) PLP1 gene duplication causes overexpression and alteration of the PLP/DM20 splicing balance in fibroblasts from Pelizaeus-Merzbacher disease patients. Biochim Biophys Acta 1792:548–554PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Clark K, Sakowski L, Sperle K, Banser L, Landel CP, Bessert DA, Skoff RP, Hobson GM (2013) Gait abnormalities and progressive myelin degeneration in a new murine model of Pelizaeus-Merzbacher disease with tandem genomic duplication. J Neurosci 33:11788–11799PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Laukka JJ, Kamholz J, Bessert D, Skoff RP (2016) Novel pathologic findings in patients with Pelizaeus-Merzbacher disease. Neurosci Lett 627:222–232PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Readhead C, Schneider A, Griffiths I, Nave KA (1994) Premature arrest of myelin formation in transgenic mice with increased proteolipid protein gene dosage. Neuron 12:583–595PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Kagawa T, Ikenaka K, Inoue Y, Kuriyama S, Tsujii T, Nakao J, Nakajima K, Aruga J, Okano H, Mikoshiba K (1994) Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene. Neuron 13:427–442PubMedCrossRefGoogle Scholar
  21. 21.
    Simons M, Kramer EM, Macchi P, Rathke-Hartlieb S, Trotter J, Nave KA, Schulz JB (2002) Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease. J Cell Biol 157:327–336PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Karim SA, Barrie JA, McCulloch MC, Montague P, Edgar JM, Kirkham D, Anderson TJ, Nave KA, Griffiths IR, McLaughlin M (2007) PLP overexpression perturbs myelin protein composition and myelination in a mouse model of Pelizaeus-Merzbacher disease. Glia 55:341–351PubMedCrossRefGoogle Scholar
  23. 23.
    Karim SA, Barrie JA, McCulloch MC, Montague P, Edgar JM, Iden DL, Anderson TJ, Nave KA, Griffiths IR, McLaughlin M (2010) PLP/DM20 expression and turnover in a transgenic mouse model of Pelizaeus-Merzbacher disease. Glia 58:1727–1738PubMedCrossRefGoogle Scholar
  24. 24.
    Hüttemann M, Zhang Z, Mullins C, Bessert D, Lee I, Nave KA, Appikatla S, Skoff RP (2009) Different proteolipid protein mutants exhibit unique metabolic defects. ASN Neuro 1(3):e00014. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Somayajulu M, Bessert DA, Hüttemann M, Sohi J, Kamholz J, Skoff RP (2018) Insertion of proteolipid protein into mitochondria but not DM20 regulates metabolism of cells. Neurosci Lett 678:90–98PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Tatar CL, Appikatla S, Bessert DA, Paintlia AS, Singh I, Skoff RP (2010) Increased Plp1 gene expression leads to massive microglial cell activation and inflammation throughout the brain. ASN Neuro 2(4):e00043. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Marteyn A, Baron-Van Evercooren A (2016) Is involvement of inflammation underestimated in Pelizaeus-Merzbacher disease? J Neurosci Res 94:1572–1578PubMedCrossRefGoogle Scholar
  28. 28.
    Rosenbluth J, Nave KA, Mierzwa A, Schiff R (2006) Subtle myelin defects in PLP-null mice. Glia 54:172–182PubMedCrossRefGoogle Scholar
  29. 29.
    Gruenenfelder FI, Thomson G, Penderis J, Edgar JM (2011) Axon-glial interaction in the CNS: what we have learned from mouse models of Pelizaeus-Merzbacher disease. J Anat 219:33–43PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Werner HB, Kuhlmann K, Shen S, Uecker M, Schardt A, Dimova K, Orfaniotou F, Dhaunchak A, Brinkmann BG, Möbius W, Guarente L, Casaccia-Bonnefil P, Jahn O, Nave KA (2007) Proteolipid protein is required for transport of sirtuin 2 into CNS myelin. J Neurosci 27:7717–7730PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Lüders KA, Nessler S, Kusch K, Patzig J, Jung RB, Möbius W, Nave KA, Werner HB (2019) Maintenance of high proteolipid protein level in adult central nervous system myelin is required to preserve the integrity of myelin and axons. Glia 67:634–649PubMedCrossRefGoogle Scholar
  32. 32.
    Li S, Moore CL, Dobretsova A, Wight PA (2002) Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain. J Neurochem 83:193–201PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Hamdan H, Patyal P, Kockara NT, Wight PA (2018) The wmN1 enhancer region in intron 1 is required for expression of human Plp1. Glia 66:1763–1774PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Tuason MC, Rastikerdar A, Kuhlmann T, Goujet-Zalc C, Zalc B, Dib S, Friedman H, Peterson A (2008) Separate proteolipid protein/DM20 enhancers serve different lineages and stages of development. J Neurosci 28:6895–6903PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Sarret C, Combes P, Micheau P, Gelot A, Boespflug-Tanguy O, Vaurs-Barriere C (2010) Novel neuronal proteolipid protein isoforms encoded by the human myelin proteolipid protein 1 gene. Neuroscience 166:522–538PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Dobretsova A, Kokorina NA, Wight PA (2000) Functional characterization of a cis-acting DNA antisilencer region that modulates myelin proteolipid protein gene expression. J Neurochem 75:1368–1376PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Dobretsova A, Kokorina NA, Wight PA (2004) Potentiation of myelin proteolipid protein (Plp) gene expression is mediated through AP-1-like binding sites. J Neurochem 90:1500–1510PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Pereira GB, Meng F, Kockara NT, Yang B, Wight PA (2013) Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination. J Neurochem 124:454–465PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Wight PA, Duchala CS, Readhead C, Macklin WB (1993) A myelin proteolipid protein-lacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice. J Cell Biol 123:443–454PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8:e1000412PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Truett G, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29:52–54PubMedCrossRefGoogle Scholar
  42. 42.
    Stratman JL, Barnes WM, Simon TC (2003) Universal PCR genotyping assay that achieves single copy sensitivity with any primer pair. Transgenic Res 12:521–522PubMedCrossRefGoogle Scholar
  43. 43.
    Young DC, Kingsley SD, Ryan KA, Dutko FJ (1993) Selective inactivation of eukaryotic β-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial β-galactosidase as a reporter enzyme. Anal Biochem 215:24–30PubMedCrossRefGoogle Scholar
  44. 44.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Bongarzone ER, Campagnoni CW, Kampf K, Jacobs EC, Handley VW, Schonmann V, Campagnoni AT (1999) Identification of a new exon in the myelin proteolipid protein gene encoding novel protein isoforms that are restricted to the somata of oligodendrocytes and neurons. J Neurosci 19:8349–8357PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Jacobs EC, Bongarzone ER, Campagnoni CW, Kampf K, Campagnoni AT (2003) Soma-restricted products of the myelin proteolipid gene are expressed primarily in neurons in the developing mouse nervous system. Dev Neurosci 25:96–104PubMedCrossRefGoogle Scholar
  47. 47.
    Jacobs EC, Bongarzone ER, Campagnoni CW, Campagnoni AT (2004) Embryonic expression of the soma-restricted products of the myelin proteolipid gene in motor neurons and muscle. Neurochem Res 29:997–1002PubMedCrossRefGoogle Scholar
  48. 48.
    Kitsis RN, Leinwand LA (1992) Discordance between gene regulation in vitro and in vivo. Gene Expr 2:313–318PubMedGoogle Scholar
  49. 49.
    Recillas-Targa F (2006) Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals. Mol Biotechnol 34:337–354PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Physiology and BiophysicsUniversity of Arkansas for Medical SciencesLittle RockUSA

Personalised recommendations