Disease Models & Mechanisms 1, 229-240 (2008) doi:10.1242/dmm.000729
RESEARCH ARTICLE
Peptidylarginine deiminase 2 (PAD2) overexpression in transgenic mice leads to myelin loss in the central nervous system Abdiwahab A. Musse1, Zhen Li2, Cameron A. Ackerley3, Dorothee Bienzle4, Helena Lei2, Roberto Poma5, George Harauz1, Mario A. Moscarello2 and Fabrizio G. Mastronardi2,*
Disease Models & Mechanisms DMM
SUMMARY Demyelination in the central nervous system is the hallmark feature in multiple sclerosis (MS). The mechanism resulting in destabilization of myelin is a complex multi-faceted process, part of which involves deimination of myelin basic protein (MBP). Deimination, the conversion of protein-bound arginine to citrulline, is mediated by the peptidylarginine deiminase (PAD) family of enzymes, of which the PAD2 and PAD4 isoforms are present in myelin. To test the hypothesis that PAD contributes to destabilization of myelin in MS, we developed a transgenic mouse line (PD2) containing multiple copies of the cDNA encoding PAD2, under the control of the MBP promoter. Using previously established criteria, clinical signs were more severe in PD2 mice than in their normal littermates. The increase in PAD2 expression and activity in white matter was demonstrated by immunohistochemistry, reverse transcriptase-PCR, enzyme activity assays, and increased deimination of MBP. Light and electron microscopy revealed more severe focal demyelination and thinner myelin in the PD2 homozygous mice compared with heterozygous PD2 mice. Quantitation of the disease-associated molecules GFAP and CD68, as measured by immunoslot blots, were indicative of astrocytosis and macrophage activation. Concurrently, elevated levels of the pro-inflammatory cytokine TNF-α and nuclear histone deimination support initiation of demyelination by increased PAD activity. These data support the hypothesis that elevated PAD levels in white matter represents an early change that precedes demyelination.
INTRODUCTION Multiple sclerosis (MS) is the most common demyelinating disease in young adults, and manifests itself in different ways (Lassmann et al., 2001). Although the cause of MS still remains elusive, the available evidence suggests a complex multi-factorial (genetic, immune and environmental) etiology (Lutton et al., 2004). The triggers that lead to the development of lesions in the ‘normalappearing white matter’ (NAWM) have been hypothesized by us to provide some new pathogenic clues (Mastronardi and Moscarello, 2005; Moscarello et al., 2007). At the molecular level, we have shown that the severity of MS is associated with the degree of myelin basic protein (MBP) citrullination (conversion of arginine to citrulline) (Moscarello et al., 1994; Wood et al., 1996; Kim et al., 2003). In this context, the MBP cationicity found in MS patients was similar to that observed in children 4 years of age or younger, indicative of developmentally immature myelin (Moscarello et al., 1994). Various biochemical and biophysical studies have revealed the presence of increased amounts of citrullinated MBP. Hyperdeimination of MBP results in loss of myelin sheath integrity, which has been recently reviewed (Musse and Harauz, 2007). The conversion of arginine to citrulline in proteins is carried out by the peptidylarginine deiminase (PAD) family of enzymes, of which five isoforms are known, with PAD2 (isoform 2) being the most abundant in the brain (Vossenaar et al., 2003b). The 1
Department of Molecular and Cellular Biology and Biophysics Interdepartmental Group, University of Guelph, Ontario, Canada N1G 2W1 2 Department of Molecular Structure and Function and 3Department of Pathology, Hospital for Sick Children, Ontario, Canada M5G 1X8 4 Department of Pathobiology and 5Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Ontario, Canada N1G 2W1 *Author for correspondence (e-mail:
[email protected]) Disease Models & Mechanisms
involvement of PAD in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis has been established (Suzuki et al., 2003; Vossenaar et al., 2003a; Lundberg et al., 2005; Yamada et al., 2005). Given the increased citrullination of MBP and other proteins in MS (Kim et al., 2003) (reviewed in Harauz and Musse, 2007; Moscarello et al., 2007), we postulated that upregulation of PAD2 also represented an important early molecular change in demyelinating disease, and that increased citrullination of MBP results in reduced myelin stability in MS brain white matter (Moscarello et al., 1994). Evidence for PAD2 involvement has been obtained from the ND4 transgenic mouse model for demyelinating disease, in which increases in both PAD activity and mRNA levels were observed 1 month prior to an increase in citrullinated MBP and the concomitant appearance of any clinical or pathological signs (Moscarello et al., 2002a). We have also recently shown that the PAD2 promoter is hypomethylated in MS brain white matter (Mastronardi et al., 2007a), thereby explaining the increased synthesis of this enzyme. The sequence of events that we propose is as follows: decreased methylation of cytosines in promoter CG sequences by increased activity of a DNA demethylase results in increased transcription of the gene encoding PAD2 (Padi2) (Mastronardi et al., 2007a), an increase in PAD2, increased citrullination of MBP, and a decrease in the net positive charge on MBP and, subsequently, failure of myelin bilayers to compact leading to degradation of myelin and exposure of immunodominant epitopes (Moscarello et al., 2007; Musse and Harauz, 2007). A central player in this sequence is PAD2. Moreover, in a recently described Padi2-knockout mouse line, the amount of citrullination present in the central nervous system (CNS) was significantly diminished (Raijmakers et al., 2006), and demyelination was not observed. These observations were 229
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RESEARCH ARTICLE
suggestive of PAD’s association with disease, but were not indicative of a causal role. In order to further define the involvement of PAD2 in the pathogenesis of demyelinating disease, we demonstrated that upregulation of PAD2 in oligodendrocytes leads to demyelination using a PAD2-overexpressing transgenic mouse line (PD2), in which the heterozygote contained 15 copies of the cDNA encoding PAD2 and the homozygote contained 30 copies. Using a set of previously established clinical criteria (Moscarello et al., 2002b), we compared the progression of the disease signs in transgenic mice with those of normal littermates. The increase in PAD2 expression was demonstrated by enzyme assays and reverse transcriptase (RT)PCR. We used ultrastructural and histochemical analyses to demonstrate demyelination. The PD2 mice thus represent a new model for clinically mild variants of MS (Lassmann et al., 2001). Both clinical signs and morphological studies demonstrated that severity of signs correlated with gene dosage; the homozygote showed more severe signs and greater myelin destruction than the heterozygote. In addition, these data revealed that upregulation of PAD2, either by hypomethylation of the promoter (Mastronardi et al., 2007a) or by increased dosage of the PAD2 gene (in this study), can represent an important contribution to demyelination. RESULTS PAD2 transgenic mice and copy number To determine whether increased expression of PAD2 in oligodendrocytes affected myelin structure, we generated transgenic mice expressing cDNA encoding the PAD2 protein under the regulation of the mouse MBP promoter. The construct used is shown in Fig. 1A. An EcoRI fragment of rat PAD2 cDNA consisting of 2.3 kb of DNA (Watanabe and Senshu, 1989) was inserted into the EcoRI cloning site of the pMG2 plasmid (Gow et al., 1992). The PAD2 minigene was restriction-digested with NotI and injected into eggs from the CD1 mouse strain at the Hospital for Sick Children’s transgenic mouse facility. Founder mice were obtained and assayed for the transgene by Southern analysis of tail clip DNA. The transgene-specific probe was a BglII fragment that straddled a portion of the MBP promoter extending into the 5⬘untranslated region of the PAD2 cDNA (Fig. 1A). The results
PAD2 overexpression causes CNS demyelination
revealed a range in the number of transgene copies incorporated within the founder lines. A Southern blot of tail clip DNA from four mouse lines, numbers 9, 15, 25 and 28, is shown in Fig. 1B. Founder mouse line numbers 9 and 28 contained medium and high copy numbers of the transgene, respectively. To determine the relative abundance of the PAD2 transgene in the founder lines, we used the single copy proteolipid protein (Plp) gene as a loading control. The ratio of expression of the PAD2 transgene over the Plp gene revealed that line numbers 28, 9, 15 and 25 contained 15, 4, 1 and 0 copies of the transgene, respectively. Transgenic line number 28 was maintained by crossing homozygous mice with unrelated normal mice to generate 100% heterozygous mice. When homozygous mice were required, breeding pairs of unrelated heterozygous PD2 mice were used. In these crosses, inheritance of the transgene followed Mendelian ratios, with the progeny being, roughly: 25% homozygous PD2 mice, 50% heterozygous PD2 mice and 25% non-transgenic normal littermates, indicative of autosomal inheritance of the transgene. Clinical phenotype and neurological examination For this study, we maintained the high copy number PAD2 transgenic line, number 28 (containing 15 copies of the transgene), as heterozygous PD2 mice to assess the involvement of PAD2 in demyelinating disease. As a first qualitative approximation of the neurological deficits of the PD2 mice, we documented the clinical disease development, over a 6.0–6.5-month period from birth (longer in the homozygote), as mean clinical scores using previously established phenotypic behavior that included tail droop, tremors, unsteady gait, head shaking, convulsions, reduced physical activity and reduced righting ability (Moscarello et al., 2002b; Mastronardi et al., 2004). For the purpose of assessing the severity of the neurological deficits of the PD2 mice, these phenotypic behavioral displays were also compared with those observed for a previously characterized, spontaneously demyelinating transgenic mouse line, ND4 (DM20 overexpressors) (Mastronardi et al., 1993), which had a more severe clinical course. In our previous studies, we showed that the ND4 transgenic mouse, which contains supernumerary copies of a DM20 transgene, started to develop CNS demyelination 3 months after birth
Fig. 1. PAD2 construct and clinical signs in PAD2 transgenic mice. (A) The rat PAD2 transgene construct. (B) Southern blot analysis of rat PAD2 transgene expression in mouse line numbers 28, 25, 15 and 9, with endogenous PLP used as an internal standard. The ‘+/–’ designation indicates transgene expression in the heterozygotes, whereas ‘–/–’ indicates the absence of the transgene. (C) Progression of clinical disease in PD2 mice (n=10) relative to ND4 mice (n=10). Clinical scores comparing the observed neurological signs of the spontaneously demyelinating ND4 mice with those of PD2 mice are shown. The measured neurological signs included tail droop, tremors, unsteady gait, head shaking, convulsions, physical activity, and righting ability (a measure of strength). The mean clinical signs for normal nontransgenic littermates fell within the range of 0-10, which is represented by the shaded area. The values shown are the aggregated means of the neurological signs. The scores ranged from 0 (no signs) to 40 (severe).
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PAD2 overexpression causes CNS demyelination
(Mastronardi et al., 1993) and displayed clinical signs at 3-4 months of age, which became severe by 5-6 months (Fig. 1C). In our present study, the PD2 mice displayed abnormal clinical signs by 1.5 months (Fig. 1C), at which age ND4 mice displayed no difference compared with their normal littermates. As shown in Fig. 1C, the neurological abnormalities did not progress as quickly in the PD2 mice compared with in the ND4 mice. The severity in mean clinical signs became more apparent as the PD2 mice aged; however, the mean clinical signs were not as severe as for ND4 mice at 6 months of age. The mean clinical scores for the heterozygous PD2 mice were significantly higher than those for normal littermates after 4 months of age. Overall, the mean clinical scores for non-transgenic littermate mice remained within the range of background mean clinical scores (values up to 10, represented by the shaded area of Fig. 1C). The homozygous line showed more severe clinical signs than the heterozygote at about 6 months of age and progressed rapidly thereafter. In addition to the systematic scoring of neurological signs associated with demyelination, an independent, blinded neurological examination was conducted at the Ontario Veterinary College. At 4 months of age, the heterozygous PD2 transgenic mice displayed abnormal gait in all limbs – worse in the hind limbs – including hind-end weakness and proprioceptive positioning deficits. No tremors were observed; however, decreased balance and lack of coordination were evident. Similar signs were observed in the homozygous line but were more exaggerated. These observations were consistent with the more frequent clinical evaluations of the animals at the Hospital for Sick Children.
RESEARCH ARTICLE
On the basis of these observed neurological signs, it was concluded that the lesions in the PD2 mice were localized to the CNS involving, bilaterally, the thalmo-cortex (leading to abnormal mentation, decreased interaction, and dullness). Furthermore, the neurological assessment of the abnormal gait suggested a cortical lesion or a concomitant cervical spinal cord lesion affecting the white matter between the C1-C5 spinal cord segments. Expression of PAD2 protein in PD2 mice To determine whether PAD2 was increased in the white matter tracts of heterozygous PD2 mice, we used a polyclonal antibody generated against whole muscle PAD2 (Takahara et al., 1983; Takahara et al., 1989) to stain mouse brain sections. The antibody labeled white matter tracts in the corpus callosum of both normal and PD2 mice, with a higher degree of labeling in the white matter tracts of the PD2 brain (Fig. 2A, arrows). Cerebellar white matter tracts also displayed higher qualitative labeling with anti-PAD antibody in PD2 mice (supplementary material Fig. S2). Similar increases were observed in spinal cord and cortical white matter (data not shown). For an unambiguous quantitative analysis, the degree of PAD2 enzyme expression was determined from whole brain homogenates from 2-, 4- and 6-month-old heterozygous PD2 transgenic mice, and compared with non-transgenic littermates using anti-PAD2 antibodies by an immunoslot blot method. The loading control for each homogenate was the level of histone H3, which was determined by probing the immunoslot blot with anti-histone H3 antibody. The ratios of PAD2:H3 ± s.d. for each age group were
Fig. 2. PAD2 levels are elevated in PD2 white matter and in brain homogenates from PD2 mice. (A) Immunohistochemical analysis showing the extent of PAD2 expression in normal and heterozygous PD2 mice using anti-PAD2 antibody. Representative corpus callosum regions (arrows) of paraffin-embedded, formalin-fixed, sections of brain from 6-month-old normal control and heterozygous PD2 mice were probed with anti-pan-PAD2 antibody and counterstained with hematoxylin. (B) Immunoslot blot showing quantitative expression levels of PAD2, normalized to histone H3, in normal and heterozygous PD2 mice, at various ages, using anti-PAD2 antibody and anti-histone H3 antibody (loading control). Significantly increased PAD2 levels are found at 2 (P
