Deletion of a splicing enhancer disrupts PLP1/DM20 ratio and myelin stability Erming Wang a, 1 , Neviana Dimova a, 1 , Karen Sperle b, 1 , Zhong Huang c , Leslie Lock d , Mailis C. McCulloch e , Julia M. Edgar e , Grace M. Hobson b , Franca Cambi a, ⁎ a Department of Neurology, University of Kentucky, Lexington, KY, USA b Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE, USA c Department of Surgery, Jefferson Medical College, Philadelphia, PA, USA d Sue and Bill Gross Stem Cell Research Center, Department of Biological Chemistry, and Developmental and Cell Biology, Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine, CA 92697 e Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, Scotland, UK abstract article info Article history: Received 11 April 2008 Revised 21 August 2008 Accepted 2 September 2008 Available online 18 September 2008 Keywords: Pelizaeus-Merzbacher disease Myelin Proteolipid protein Alternative splicing Intronic splicing enhancers PLP1 and DM20, major myelin proteins, are generated by developmentally regulated alternative splicing. In the post-natal brain, PLP1 is the predominant product. Deletion of a splicing enhancer in PLP1 intron 3 causes a mild form of Pelizaeus-Merzbacher disease and reduces PLP1 specific splicing in vitro (Hobson, G. M., Huang, Z., Sperle, K., Stabley, D. L., Marks, H. G., and Cambi, F., 2002. A PLP splicing abnormality is associated with an unusual presentation of PMD. Ann. Neurol. 52, 477–488). We sought to investigate the pathogenic role of the mutation and to determine the consequences on the developmental regulation of PLP1 alternative splicing and myelin stability and function in vivo. We have generated a knockin mouse that carries deletion of the intronic splicing enhancer and have characterized the PLP1/DM20 ratio by Real Time RT-PCR and Western blot analysis in the developing and mature brain and examined the clinical and pathological phenotype by motor testing and electron microscopy. The deletion impairs the increase in the PLP1/DM20 transcript and protein ratio at the time of myelination and in adulthood and results in a PLP1 hypomorph. Electron microscopy shows abnormal myelin wraps with fragmented myelin whorls, which are progressive with age, suggesting a defect in myelin stability. Phenotypic characterization of the knockin mouse shows a defect in motor coordination. The data indicate that the intronic splicing enhancer is necessary for the developmental increase in PLP1/DM20 ratio and that full PLP1 dosage is necessary for myelin stability and brain function. This knockin mouse represents a useful model to investigate the mechanisms of disease in human disorders in which PLP1 expression is reduced. © 2008 Elsevier Inc. All rights reserved. Introduction Proteolipid protein 1 (PLP1), the major central nervous system (CNS) myelin protein, is necessary to maintain both normal myelin structure and axonal integrity (Griffiths et al., 1998a, 1998b; Yool et al., 2001). The PLP1 gene gives rise to two protein isoforms, PLP1 and DM20, from a single gene by alternative utilization of competing 5′ splice sites within exon 3 (Nave et al., 1987). PLP1 and DM20 are identical except for the presence of a 35 amino acid sequence within the intracellular loop of PLP1 which is absent in DM20 (Nave et al., 1987). Alternative splicing of the PLP1 gene is regulated during myelination in oligodendrocytes, the myelinating glia of the CNS, so that PLP1 expression exceeds that of DM20 by approximately three- fold (Campagnoni and Macklin, 1988; Timsit et al., 1992). Mature mRNA formation depends on the precise recognition of exon–intron boundaries through consensus sequences at 5′ splice donor site, 3′ acceptor site and branch points that allow efficient and accurate removal of introns from pre-mRNAs. Alternative splicing is regulated by elements located in introns and exons that act as either enhancers or silencers (Matlin et al., 2005). PLP alternative splicing is regulated by a complex array of regulatory elements in exon 3B and intron 3, in addition to the strength of the PLP and DM20 5′ splice sites (Hobson et al., 2002, 2006; Wang et al., 2006, 2007). Mutations in the PLP1/DM20 gene are associated with a broad spectrum of X-linked neurological disorders ranging from severe connatal forms of Pelizaeus-Merzbacher disease (PMD, MIM 312080) to mild forms of Spastic Paraplegia 2 (SPG2, MIM 312920) (Garbern, 2002). The PLP1/DM20 gene mutations range from duplications of the entire gene (60–70%), to nucleotide changes/deletions (15–20%) and deletions of the entire gene (1–2%) (Cailloux et al., 2000; Garbern, 2002; Mimault et al., 1999; Shy et al., 2003). A growing Experimental Neurology 214 (2008) 322–330 ⁎ Corresponding author. University of Kentucky, Department of Neurology, KY Clinic L445, Lexington, KY 40536, USA. Fax: +1 859 323 5943. E-mail address: franca.cambi@uky.edu (F. Cambi). 1 These authors contributed equally to this work. 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.09.001 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr