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Research Report

White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion Yanina Tsenkinaa,b,n, Alexey Ruzova,c, Catherine Gliddona,d, Karen Horsburghe, Paul A. De Sousaa,f,nn a

Centre for Clinical Brain Sciences, School of Clinical Sciences, Chancellor’s Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK b The Miami Project to Cure Paralysis, Lois Pope Life Center, University of Miami, 1095 NW 14th Terrace, Miami, FL 33136, USA c Wolfson Centre for Stem Cells, Tissue Engineering and Modeling (STEM), Division of Cancer and Stem Cells, School of Medicine, Center for Biomolecular Sciences, University Park, University of Nottingham, University Park NG7 2RD, UK d Pharmacology and Toxicology Department, Otago School of Medical Sciences, University of Otago, Dunedin, NZ e Centre for Neuroregeneration, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK f Roslin Cells, Scottish Centre for Regenerative Medicine, 5 Little France Drive, Edinburgh EH16 4UX, UK

ar t ic l e in f o

abs tra ct

Article history:

White matter abnormalities due to age-related cerebrovascular alterations is a common

Accepted 25 September 2014

pathological hallmark associated with functional impairment in the elderly which has

Available online 11 October 2014

been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been

Keywords: Chronic cerebral hypoperfusion Epigenetics 5-Hydroxymethylcytosine 5-Methylcytosine TET proteins White matter

recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (po0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract

Abbreviations: CC1,

adenomatous polyposis coli; CNS, central nervous system; DIV, days in vitro; DNMT1, DNA methyltransferase 1;

GFAP, glial fibrillary acidic protein; HCl, hydrochloric acid; HDAC1, histone deacetylase 1; H&E, haemotoxylin and eosin; 5hmC, 5-hydroxymethylcytosine; Iba1, ionized calcium binding antigen 1; IFNγ, interferron γ; LPS, lipopolysaccaride; 5mC, 5-methylcytosine; NG2, chondroitin sulfate proteoglycan; OPCs, oligodendroglial progenitor cells; PBS, phosphate buffer saline; PLP, proteolipid protein; ssDNA, single stranded DNA; TET1, ten–eleven translocation protein 1; TET2, ten–eleven translocation protein 2; TET3, ten–eleven translocation protein 3 n Corresponding author at: University of Miami, The Miami Project to Cure Paralysis, Lois Pope Life Center, 1095 NW 14th Terrace, Miami, FL 33136, USA. nn Corresponding author at: Centre for Clinical Brain Sciences, School of Clinical Sciences, Chancellor’s Building, University of Edinburgh, 49, Little France Crescent, Edinburgh EH16 4SB, UK. E-mail addresses: [email protected] (Y. Tsenkina), [email protected] (P.A. De Sousa). http://dx.doi.org/10.1016/j.brainres.2014.09.060 0006-8993/Published by Elsevier B.V.

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there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p ¼0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice. Published by Elsevier B.V.

1.

Introduction

White matter abnormalities are a common pathological feature of the ageing brain and are closely linked with cognitive impairment particularly a reduction in the speed of information processing (O’Sullivan et al., 2001; Charlton et al., 2006; Grieve et al., 2007; Kennedy and Raz, 2009; Bolandzadeh et al., 2012). Chronic cerebral hypoperfusion is closely associated with white matter alterations in ageing and in disease (Kalaria, 1996; Tang et al., 1997; de Leeuw et al., 2000; Farkas and Luiten, 2001; Fernando et al., 2006; Holland et al., 2008; DeCarli, 2013). To experimentally study the association between reductions in the cerebral blood supply and white matter pathology, animal models of chronic cerebral hypoperfusion have been developed initially in the rat and recently in the mouse. In the rat, chronic cerebral hypoperfusion is induced by the permanent bilateral ligation of the common carotid leading to 30–60% of baseline level initial reductions of the cerebral blood supply with a gradual flow recovery by 4 weeks post-surgery and the development of both white and grey matter pathology (Pappas et al., 1996; Abraham and Lazar, 2000; Wakita et al., 2002; Farkas et al., 2004, 2007; Otori et al., 2003; Tomimoto et al., 2003). Because of the occurrence of mixed white and grey matter abnormalities, it is difficult to examine the selective effects of chronic cerebral hypoperfusion on white matter integrity in chronically hypoperfused rats. Recently, ours and other groups developed a new mouse model of chronic cerebral hypoperfusion where wire microcoils with internal diameter of 0.18 mm are implanted around the common carotid arteries leading to mild (  20–30% of baseline levels) reductions of the cerebral blood supply and the development of a selective white matter pathology one month post-surgery (Shibata et al., 2004, 2007; Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014). The observed pathological differences between chronically hypoperfused rats and mice could be explained by (1) the above mentioned methodological differences in the induction of chronic cerebral hypoperfusion as well as by (2) species differences in the cerebrovasculature (e.g. the Willy’s polygon). In the rat, the Willy’s Polygon is morphologically similar to this cerebrovascular structure in humans, whereas in the mouse the Willy’s Polygon is significantly underdeveloped. The reported progressive recovery of the cerebral blood flow in chronically hypoperfused rats is explained by compensatory mechanisms such

as enlargement of the posterior vessels constituting the Willy’s Polygon (the posterior cerebral artery, the posterior communicating artery, and the basilar artery) between 3 and 6 months after the induction of chronic cerebral hypoperfusion (Olendorf, 1989; Choy et al., 2006). Since selective white matter pathology in the absence of grey matter abnormalities develops in chronically hypoperfused mice (Shibata et al., 2004, 2007; Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014), we used this animal model to examine molecular alterations occurring in the hypoperfused white matter. Specifically, molecular analysis of the underlying gene alterations evoked by hypoperfusion in the white matter demonstrated significant changes in the expression of 129 genes and highlighted aberrations in pathways linked to oxidative stress and inflammation (Reimer et al., 2011). However, the exact molecular mechanisms leading to transcriptional alterations in the hypoperfused white matter remain unclear. One possible explanation may rely on hypoperfusion-induced epigenetic changes as it is known that during normal development as well as under different pathological conditions, epigenetic marks can switch genes on and off (Murrell et al., 2013; MacDonald and Roskams, 2009). 5-Methylcytosine (5mC) is an epigenetic modification generated by addition of a methyl group to the 50 carbon of cytosine that occurs predominantly in a CpG dinucleotide context (Bird, 2002). DNA methylation is mainly associated with transcriptional repression modulating cellular function during lifespan and disease. With ageing, dynamic global and gene-specific changes in 5mC distribution are observed in grey and white matter (West et al., 1995; Mehler, 2008; Zawia et al., 2009; Chouliaras et al., 2010; Penner et al., 2010; Hernandez et al., 2011; Chouliaras et al., 2012b; Coppieters et al., 2014). Several mechanisms may impact on DNA methylation with increasing age including cerebrovascular conditions (e.g. stroke, chronic cerebral hypoperfusion) and the resulting hypoxic-ischemic environment accompanied by increased excitotoxicity, oxidative stress, and inflammation (Endres et al., 2000; Westberry et al., 2008). Severe reductions of cerebral blood flow occurring as a result of experimental stroke and focal ischemia are known to reduce the levels of DNA methylation in the brain (Endres et al., 2000; Westberry et al., 2008). The exact functional role of DNA methylation in the injured central nervous system (CNS) is still controversial. For instance, although pharmacological and genetic inhibition

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of DNA methyltransferase 1 (Dnmt1), the enzyme maintaining methylation levels, reduces lesion size and increases neuronal survival in an animal model of stroke according to some

studies (Endres et al., 2000; Endres et al., 2001), other reports imply that decreased 5mC levels could also lead to neuronal cell death (Fan et al., 2001; Rhee et al., 2012).

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In addition to 5mC, another form of modified cytosine, 5hydroxymethylcytosine (5hmC) was recently rediscovered in the mammalian genome (Kriaucionis and Heintz, 2009). The Ten-Eleven Translocation proteins (TET1, TET2, TET3) can convert 5mC to 5hmC in the presence of oxygen and iron (Tahiliani et al., 2009; Ito et al., 2010). Genome-wide studies using human brain tissue (Jin et al., 2011) and mouse embryonic stem cells (Ficz et al., 2011) demonstrated that, in contrast to 5mC, which is predominantly located at heterochromatic regions, 5hmC is abundant in euchromatin suggesting that this epigenetic mark is likely to be involved in facilitating gene expression. Although 5hmC can be detected in a range of mammalian tissues at different developmental stages, it is particularly enriched in the early embryo as well as in the developing and adult brain (Li and Liu, 2011; Ruzov et al., 2011). Specifically, 5hmC levels are high in brain grey matter areas where 5hmC content increases with chronological age (Munzel et al., 2010; Orr et al., 2012; Song et al., 2010; Szulwach et al., 2011; Chen et al., 2012; Chouliaras et al., 2012a). Epigenetic changes have also been reported to occur in the ageing white matter where increases in promoter methylation of the Klotho gene have been evidenced in the rhesus monkey (King et al., 2012). Additionally, oligodendrocytes constituting the myelin sheet also exhibit dynamic age-related changes in their epigenome. TET proteins catalyzing the conversion of 5mC to 5hmC show dynamic alterations during oligodendroglial maturation in the developing CNS (Zhao et al., 2014). Furthermore, at the difference from their progenitor cells, mature oligodendrocytes usually present compact chromatin structure and increased histone deacetylase 1 (HDAC1) expression (Liu and Casaccia, 2010). However, with ageing the oligodendroglial cells show decreases in histone deacetylation and methylation (Shen et al., 2008a) which affects their ability to efficiently produce myelin and remyelinate under pathological conditions (Shen et al., 2008b). The present study aimed at investigating methylation and hydroxymethylation in white matter and selected control grey matter areas under normal physiological conditions vs. one month after chronic cerebral hypoperfusion in mice. We report a specific elevation of 5hmC positive cells associated with increases in activated microglia but not oligodendroglial progenitor cells (OPCs) or mature oligodendrocytes in the corpus collosum of chronically hypoperfused mice. Our study supports future investigations of a mechanistic role for genomic hydroxymethylation in relation to white matter pathology in the hypoperfused brain and the associated cognitive impairment.

2.

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Results

2.1. 5hmC levels are significantly increased in white matter (the corpus callosum) upon chronic cerebral hypoperfusion To determine whether methylation and hydroxymethylation were altered in white and control grey matter areas after chronic cerebral hypoperfusion, we immunohistochemically evaluated the distribution of 5mC, 5hmC and one of the TET family members, TET2 in the adult mouse brain. Immunostaining for both 5mC and 5hmC coincided with that for antissDNA immunostained genomic DNA in examined white (the corpus callosum, external capsule, internal capsule) and control grey matter (the cortex, CA1) regions (Fig. 1A and B; Fig. 2A and B; Fig. 3A and B). Under normal physiological conditions, 5mC and TET2 were visually equally distributed among white (the corpus callosum, external capsule, internal capsule) and grey (the cortex, CA1) matter areas (Fig. 1A and C; Fig. 2A and C; Fig. 3A and C), whereas 5hmC signal intensity was considerably higher in cortical and subcortical (CA1) grey matter regions than in white matter tracts (the corpus callosum, external capsule, internal capsule) (Fig. 1B; Fig. 2B; Fig. 3B). Interestingly, under normal physiological conditions, not all the cells situated in white matter areas were 5hmC positive (arrows in Fig. 1B). TET2 immunoreactivity was evidenced in both the nuclei and cytoplasm of glial cells, but it was restricted to the cytoplasm of neurons (Fig. 1C; Fig. 2C; Fig. 3C). Chronic cerebral hypoperfusion led to a significant increase in the proportion of 5hmC positive cells in the corpus callosum (t¼ 2.214, p¼ 0.0427; sham (0.5070.1) vs. hypoperfused (0.8470.0)) (Fig. 1B1 and B2) in the absence of significant alterations in the proportions of 5mC (t¼0.6800, p¼ 0.5068; sham (0.7870.1) vs. hypoperfused (0.8370.0)) (Fig. 1A1 and A2) and TET2 (t¼0.07560, p¼ 0.9407; sham (0.3170.1) vs. hypoperfused (0.3170.1) (Fig. 1C1 and C2)) positive cells in the same white matter tract (Table 1). Additionally, a significant up-regulation in the proportion of TET2 positive cells was evidenced in the hypoperfused cortex (t¼2.190, p¼ 0.0447; sham (0.3070.0) vs. hypoperfused (0.4070.0)) (Fig. 3C1 and C2), whereas the proportion of 5mC (t¼0.1461, p¼ 0.8858; sham (0.9170.0) vs. hypoperfused (0.9070.0 )) (Fig. 3A1 and A2) and 5hmC (t¼1.479, p ¼0.1598; sham (0.8970.0) vs. hypoperfused ( 0.9270.0)) (Fig. 3B1 and B2) positive cells remained unaltered in cortical grey matter one

Fig. 1 – 5mC, 5hmC and TET2 immunoreactivity in the corpus callosum of sham (A–C) and chronically hypoperfused (A1–C1) mice. 5mC, 5hmC, and TET2 were detected in the cellular nuclei as demonstrated by immunocolocalization with ssDNA (A– A1; B–B1) and DAPI (C–C1) in sham (A–C) and hypoperfused (A1–C1) mice. 5mC (A–A1) and TET2 (C–C1) were equally distributed among cells in the corpus callosum, whereas 5hmC was present in some (white arrow in (B) and absent in other cells (blue arrow in B). One month after chronic cerebral hypoperfusion there was a significant increase in the proportion of 5hmC (po0.05) (B2), but not 5mC (A2) and TET2 (C2) (p40.05) immunopositive cells in the corpus callosum. A schematic representation of a coronal brain section at bregma—2.12 with the corpus callosum delineated in pink (D).Scale bar equals 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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month following hypoperfusion (Table 1). The rest of the examined white and grey matter areas did not exhibit significant group differences in the proportion of 5mC, 5hmC or TET2 positive cells (Fig. 2A–A2, B–B2, C–C2; Table 1). The numbers of 5mC, 5hmC and TET2 positive cells as well as the total numbers of assessed cells in white matter, cortical and subcortical regions of interest did not significantly differ between hypoperfused mice compared with controls (sham animals) (p40.05) (Supplementary materials Table S1). To determine the existence of potential association (s) between 5hmC, 5mC, and TET2 in white matter of the adult mouse brain, we assessed for correlations in the proportions of cells immunopositive for these epigenetic markers in the corpus callosum. The Pearson’s statistical analysis revealed no significant correlation between hydroxymethylation and methylation (r¼ 0.42, p ¼0.08) or between hydroxymethylation and TET2 (r¼ 0.08, p¼ 0.75) in this white matter tract.

2.2. Inflammatory microglia are increased in the hypoperfused corpus callosum exhibiting significant associations with 5hmC To determine the cellular basis accounting for the observed significant 5hmC up-regulation in the hypoperfused corpus callosum, we investigated the number and proportion of cells positive for CC1 (mature oligodendrocytes), NG2 (OPCs) and Iba1 (microglia) glial-specific markers. Although the statistical analysis demonstrated that the total regional numbers of glial cells in all white matter areas were not affected by hypoperfusion (Supplementary materials Table S2), we observed significant increases in the number and proportion of NG2 (corpus callosum t¼ 3.217, p ¼0.0058; sham (0.1670.1) vs. hypoperfused (0.4270.1); internal capsule (t¼ 2.199, p¼ 0.0439; sham (0.1970.1) vs. hypoperfused (0.3770.1)) and Iba1 (corpus callosum t¼2.538, p¼ 0.0228; sham (0.0870.0) vs. hypoperfused (0.1470.0); internal capsule (t¼ 2.834, p¼ 0.0126; sham (0.0670.0) vs hypoperfused (0.1270.0)), but not CC1 (corpus callosum t ¼1.354, p¼ 0.1957; sham (0.6870.1) vs hypoperfused (0.5170.1); internal capsule (t¼ 1.546, p¼ 0.1430; sham (0.7470.0) vs hypoperfused (0.6170.1)) positive glial cells in white matter tracts of hypoperfused mice (Fig. 4A–A2, B–B2, C–C2; Table 1; Supplementary materials Table S2). This was due to significant increases in the number of OPCs and microglia in the absence of significant alterations in mature oligodendrocytes in the hypoperfused white matter (po0.05) (Supplementary materials Table S2). To find out if the increased 5hmC levels observed only in the corpus callosum are associated with a specific glial cell, we correlated the proportions of 5hmC positive cells with the proportions of mature oligodendrocytes (CC1), OPCs (NG2) and

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microglia (Iba1) in this white matter tract across individual mice from the two treatment groups. The Pearson’s analysis demonstrated that hydroxymethylation correlated significantly with inflammatory microglia (r¼ 0.60, p¼ 0.01), but not with mature oligodendrocytes (r¼ 0.17, p¼ 0.52) or OPCs (r¼0.37, p¼ 0.14).

2.3. 5hmC

Both OPCs and microglial cells are highly enriched in

Since co-localization of 5hmC with CC1 and NG2 on paraffinembedded sections was not technically possible due to incompatibility of the corresponding antigen retrieval methods, we used in vitro systems of oligodendroglial maturation and microglial activation to assess whether 5hmC could vary during these processes in an effort to understand the cellular component of the observed elevation of proportions of 5hmC positive cells in white matter tracts of chronically hypoperfused mice in vivo. 5hmC immunoreactivity decreased with oligodendroglial maturation over 6 days in vitro (DIV) (Fig. 5A1–A2). By contrast IFNγ/LPS activated and nonactivated microglial cells were both strongly 5hmC immunopositive (Fig. 5B–B1).

2.4. Dynamic changes in TET proteins with oligodendroglial maturation and oxidative stress in vitro Because chronic cerebral hypoperfusion is accompanied by increased oxidative stress in vivo (Kašparová et al., 2005) that could negatively affect the oligodendroglial lineage, we further examined the effects of oxidation (staurosporine treatment, Pong et al., 2001) on TET1, TET2, TET3 protein levels in OPCs and mature oligodendrocytes in vitro. The analysis demonstrated dynamic changes in TET proteins with both oligodendroglial maturation and oxidative insult (po0.001). We observed significant decreases in TET1 protein levels with oligodendroglial maturation under normal culture conditions from 2 to 6 DIV (F(3, 11) ¼ 11.85; po0.05). However, when OPCs and mature oligodendrocytes were subjected to staurosporineinduced oxidative stress, TET1 protein levels increased with significant group differences observed only between OPCs treated with staurosporine and mature oligodendrocytes cultured under normal conditions (po0.01) (Fig. 6A and B). TET2 protein levels did not change significantly with oligodendroglial maturation in vitro (F(3, 11) ¼ 206.1; p40.05). However, TET2 was significantly decreased in both OPCs and mature oligodendrocytes subjected to an oxidative insult when compared with cells cultured under normal conditions (po0.001). Interestingly, following staurosporine treatment, TET2 protein levels were significantly lower in OPCs than in mature oligodendrocytes (po0.001) (Fig. 6A and C).

Fig. 2 – 5mC, 5hmC and TET2 immunoreactivity in the internal capsule of sham (A–C) and chronically hypoperfused (A1–C1) mice. 5mC, 5hmC, and TET2 were detected in the cellular nuclei as demonstrated by immunocolocalization with ssDNA (A–A1; B–B1) and DAPI (C–C1) in sham (A–C) and hypoperfused (A1–C1) mice. One month after chronic cerebral hypoperfusion, the proportion of 5mC (A2) and TET2 (C2) positive cells remained unaltered in the internal capsule, whereas there was a nonsignificant decrease in the proportion of 5hmC (B2) in the same white matter tract (p40.05). A schematic representation of a coronal brain section at bregma—2.12 with the internal capsule delineated in blue (D). Scale bar equals 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Inversely to TET2, TET3 protein levels increased significantly with oligodendroglial maturation in vitro (F(3, 11) ¼ 980.1; po0.001) and following staurosporine treatment the

levels of this protein were significantly lower in mature oligodendrocytes than in OPCs cultured under the same condition (po0.001) (Fig. 6A and D).

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Table 1 – Regional group proportions of 5mC, 5hmC, TET2, CC1, NG2, Iba1 positive cells in sham and hypoperfused mice with the corresponding t-test statistical analysis. Biomarker

5mC

5hmC

TET2

CC1

NG2

Iba1

Region of interest

Corpus callosum (CC) External capsule (EC) Internal capsule (IC) CA1 Cortex (Cx) Corpus callosum (CC) External capsule (EC) Internal capsule (IC) CA1 Cortex (Cx) Corpus callosum (CC) External capsule (EC) Internal capsule (IC) CA1 Cortex (Cx) Corpus callosum (CC) External capsule (EC) Internal capsule (IC) Corpus callosum (CC) External capsule (EC) Internal capsule (IC) Corpus callosum (CC) External capsule (EC) Internal capsule (IC) CA1 Cortex (Cx)

Proportion of biomarker positive cells (mean7SE) Sham (n ¼8)

Hypoperfused (n¼ 9)

0.7870.1 0.7770.1 0.8870.1 0.9670.0 0.9170.0 0.5070.1 0.5170.1 0.8670.1 0.7970.1 0.8970.0 0.3170.1 0.2970.1 0.4970.1 0.5870.0 0.3070.0 0.6870.1 0.5570.1 0.7470.0 0.1670.1 0.4270.1 0.1970.1 0.0870.0 0.0870.0 0.0670.0 0.0170.0 0.0570.0

0.8370.0 0.7670.1 0.8470.0 0.9970.0 0.9070.0 0.8470.0n 0.6070.1 0.7470.1 0.8970.1 0.9270.0 0.3170.1 0.2870.1 0.4670.1 0.5770.0 0.4070.0n 0.5170.1 0.4970.1 0.6170.1 0.4270.1nn 0.5770.1 0.3770.1n 0.1470.0n 0.0970.0 0.1270.0n 0.1170.1 0.1170.0

Group statistics

t ¼0.6800, p¼ 0.5068 t ¼0.1208, p¼ 0.9054 t ¼0.01288, p¼ 0.9904 t ¼0.3089, p¼ 0.7616 t ¼0.1461, p¼ 0.8858 t ¼2.214, p¼ 0.0427 t ¼0.2283, p¼ 0.8225 t ¼1.315, p¼ 0.2081 t ¼1.146, p¼ 0.2699 t ¼1.479, p¼ 0.1598 t ¼0.07560, p¼ 0.9407 t ¼0.08406, p¼ 0.9341 t ¼0.4457, p¼ 0.6621 t ¼0.2312, p¼ 0.8203 t ¼2.190, p¼ 0.0447 t ¼1.354, p¼ 0.1957 t ¼0.5096, p¼ 0.6178 t ¼1.546, p¼ 0.1430 t ¼3.217, p¼ 0.0058 t ¼1.661, p¼ 0.1174 t ¼2.199, p¼ 0.0439 t ¼2.538, p¼ 0.0228 t ¼0.5695, p¼ 0.5774 t ¼2.834, p¼ 0.0126 t ¼1.526, p¼ 0.1478 t ¼1.583, p¼ 0.1343

Significant group differences as given by the t-tests statistical analysis. n (po0.05). nn (po0.01).

Additionally, we examined the expression of OPCs-specific (NG2) and mature oligodendrocyte-specific (PLP) markers on the same samples to confirm the maturation state of our cell preparations. NG2 protein levels were significantly higher in OPCs than in mature oligodendrocytes cultured under both normal and oxidative conditions (F(3, 11) ¼497.1; po0.001). Interestingly, staurosporine-induced oxidative stress in vitro was associated with significantly increased levels of NG2 protein in mature oligodendrocytes (po0.01), but not in OPCs (p40.05) (Fig. 6A and E). PLP protein levels were significantly higher in mature oligodendrocytes cultured in the presence (F(3, 11) ¼ 28.47; po0.001) and absence (po0.01) of staurosporine when compared with OPCs cultured under the same conditions. Staurosporine treatment did not seem to significantly affect PLP protein levels in OPCs and mature oligodendrocytes when

compared with their respective control cultured under normal conditions (p40.05) (Fig. 6A and F).

3.

Discussion

In the present study we aimed to determine whether epigenetic perturbations occur in white matter following chronic cerebral hypoperfusion in mice. On the basis of previous studies linking cerebral ischemia with alterations in DNA methylation (Endres et al., 2000; Westberry et al., 2008), we hypothesized that chronic cerebral hypoperfusion might result in changes in the production of 5hmC in the brain as a result of alterations in 5mC and/ or impaired expression of TET oxygenases due to chronic mild reductions in brain oxygen levels. Therefore, we immunohistochemically examined 5mC, 5hmC and one of the TET family members-TET2,

Fig. 3 – 5mC, 5hmC and TET2 immunoreactivity in the cerebral cortex of sham (A–C) and chronically hypoperfused (A1–C1) mice. 5mC and 5hmC were detected in the cellular nuclei as demonstrated by immunocolocalization with ssDNA (A–A1; B–B1) in sham (A–B) and hypoperfused (A1–B1) mice. However, TET2 was predominantly observed in the cytoplasm of cortical neurons under both normal (C) and chronically hypoperfused (C1) conditions. One month after chronic cerebral hypoperfusion there were significant increases in the proportion of TET2 positive cells (po0.05) (C2) in the absence of significant alterations in methylation (A2) and hydroxymethylation (B2) in the cerebral cortex (p40.05). A schematic representation of a coronal brain section at bregma—2.12 with the cerebral cortex delineated in grey (D). Scale bar equals 20 lm.

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Fig. 4 – Representative images to illustrate CC1 positive mature oligodendrocytes (A–A1), NG2 positive OPCs (B–B1) and Iba1 immunoreactive microglia (C–C1) in the corpus callosum of sham (A–C) and chronically hypoperfused (A1–C1) mice. Following chronic cerebral hypoperfusion significant increases in the proportion of NG2 (B2) (po0.01) and Iba1 (C2) (po0.05), but not CC1 (A2) (p40.05) immunoreactive cells were detected in the corpus callosum. Schematic representation of a coronal brain section at bregma—2.12 with the corpus callosum delineated in pink (D). Scale bar equals 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5 – Representative images of in vitro 5hmC immunochemical distribution in oligodendroglia at different stages of maturation (A–A2), nonactivated and IFNγ/ LPS activated microglia (B–B1). 5hmC decreased with oligodendroglia maturation from 0 to 6 days in vitro (DIV) culture (A–A2), whereas it was high in both non-activated and activated microglia (B–B1). Scale bar equals 20 lm.

for which antibody probes were available in white and grey matter areas of sham and chronically hypoperfused mice one month after surgery. Our decision to employ immunohistochemistry for the evaluation of epigenetic marks in the hypoperfused brain was dictated by previous studies where this methodological approach has been shown to be sensitive to regional and cellular variations in 5mC and 5hmC distribution in the mammalian CNS (Ruzov et al., 2011; Chouliaras et al., 2013; Coppieters et al., 2014). Since the major pathological changes in this animal model occur in white matter tracts one month after surgery (Shibata et al., 2004, 2007; Nishio et al., 2010; Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014; supplementary materials, Fig. S1.2), the expectations at the outset of this study were that epigenetic alterations might appear in hypoperfused white matter areas. However, to rule out potential epigenetic changes in the hypoperfused grey matter, we also examined cortical and subcortical areas known to be susceptible to blood flow alterations (i.e. cerebral cortex, CA1 region of the hippocampus). Our findings suggest that 5hmC, an epigenetic DNA modification, was significantly and specifically up-regulated in the corpus collosum one month after cerebral hypoperfusion in mice. In other white (the external capsule, internal capsule) and grey (cortex, CA1) matter regions of interest, 5hmC distribution was not significantly altered by cerebral hypoperfusion by this time. The reasons for this differential regional regulation of 5hmC after chronic cerebral hypoperfusion remain unknown, but major pathological changes (e.g. myelin break down, axonal injury, increased inflammation) occur in the corpus callosum in this animal model (Coltman

et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014; Supplementary materials S1.1; Fig. S1.1) suggesting potential associations between increased levels of 5hmC and neuropathology. On the basis of our present data, we could only speculate as to the exact functional significance of the observed regional differences in 5hmC distribution in the hypoperfused white matter. Although axonal injury, myelin pathology and inflammation were observed in both white matter tracts one month following hypoperfusion, the total number of cells as accounted by ssDNA, DAPI and H&E staining in our study showed an absence of significant group differences between sham and hypoperfused mice, suggesting an overall absence of major cellular death at this postsurgery time point. These findings confirm previous studies on chronically hypoperfused mice where an absence of cell loss has been reported one month after surgery (Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014). Therefore, the observed increases in 5hmC are most likely associated with cell survival by promoting transcriptional activation, gene expression, repair of damaged DNA, and/ or proliferation/ migration of progenitor cells (Cannon et al., 1988; Ito et al., 2010; Surani and Hajkova, 2010; Ficz et al., 2011; Jin et al., 2011; Colquitt et al., 2013; Otani et al., 2013; Navarro et al., 2014). OPCs proliferation, migration, differentiation and functional integration into the demyelinated CNS is observed in human patients exhibiting white matter pathology and animal models of these conditions (Levine and Reynolds, 1999; Nait-Oumesmar et al., 1999; Ishii et al., 2001; Chang et al., 2002). A recent publication from our group demonstrated a

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decrease in OPCs and mature oligodendrocytes numbers during the initial days following injury and restoration of their respective pools a month after microcoils application (McQueen et al., 2014). Additionally, BrdU labeling revealed increased numbers of proliferating cells following chronic cerebral hypoperfusion and the differentiation of some BrdU positive cells into oligodendrocytes. In the present study we observed significant increases in the proportions of NG2 positive OPCs in the hypoperfused white matter one month after hypoperfusion. In accordance with our previous findings the proportions of CC1 positive mature oligodendrocytes were not altered at the same post-surgery time point. Although our in vivo correlation analysis, failed to reveal significant associations between the oligodendroglial lineage and 5hmC, our in vitro studies suggest that 5hmC is highly enriched in OPCs and decreases with their maturation. No alterations were observed with microglial activation. Additionally, we observed dynamic changes in TET protein levels with oligodendroglial maturation and oxidative stress in vitro, suggesting important developmental and environment-dependent epigenetic alterations in these cells. Similar results were observed in vivo where alterations in TET1, TET2, and TET3 proteins accompanied oligodendroglial maturation in the developing CNS (Zhao et al., 2014). Differences between OPCs and mature oligodendrocytes were reported for other epigenetic marks such as HDAC1—a known repressor of transcriptional activation (Liu and Casaccia, 2010) which is increased in the mature cells and associated with their more compact chromatin structure. The epigenetic alterations accompanying oligodendroglial development might be associated with functional differences between progenitor and mature cells such as the production and maintenance of myelin. These epigenetic processes might be additionally altered by environmental factors such as the occurrence of neuropathology. In the present study, we observed in vivo and in vitro discrepancies as to 5hmC content in OPCs. Specifically, although OPCs exhibited a high 5hmC content in vitro, our in vivo correlation analysis failed to show significant associations between 5hmC and NG2 positive OPCs in the corpus callosum. These results could be explained by the more complex microenvironment in the living animal and the examined post-surgery time point. In the hypoperfused CNS, the NG2 positive OPCs are subjected to a combination of harmful agents (increased oxidative, inflammatory, excitotoxic stimuli) which could differentially affect their epigenome including their 5hmC content. Furthermore, considering that cerebral hypoperfusion is a chronic condition characterized by the progressive, accumulative

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development of neuropathology (Nishio et al., 2010; McQueen et al., 2014), it is possible that alterations in the epigenome follow a strict spatio-temporal pattern in different cell lineages. Previous findings from our group (McQueen et al., 2014) suggest specific temporal dynamics in the oligodendroglial lineage in the hypoperfused corpus callosum. Therefore, it is possible that associations between NG2 positive OPCs and 5hmC positive cells exist, but at earlier time points following hypoperfusion in this animal model when dynamic changes in this cell population occur. In vitro, NG2 protein levels (a specific marker for OPCs) were higher in OPCs than in mature oligodendrocytes cultured under both normal and oxidative conditions. Interestingly, in vitro we observed significant increases in NG2 protein in mature oligodendrocytes subjected to an oxidative challenge. On the basis of previous studies (Matés et al., 2008; Shen et al., 2008b; Liu and Casaccia, 2010; Selvaraj et al., 2010; Gu et al., 2011; Richard et al., 2011; Doege et al., 2012) we believe that the observed up-regulation of this protein might be associated with active reprogramming of adult oligodendrocytes under pathological conditions. Future experimental work should elucidate the epigenetic phenomena governing potential reprogramming in oligodendrocytes in the injured CNS. PLP protein levels (a specific marker for mature oligodendrocytes) were higher in mature oligodendrocytes than in OPCs in vitro. Oxidative stress did not seem to influence on PLP protein content. Inflammatory microglia increase in response to injury to the CNS participating in the formation of a glial scar (Kreutzberg, 1996). These cells are also a major player in demyelinating disorders such as multiple sclerosis where they contribute to the observed pathological changes in white matter (Fischer et al., 2009). Increases in inflammatory cells are observed in white and grey matter areas in elderly people and aged animals (Sloane et al., 1999; Ye and Johnson, 1999; Conde and Streit, 2006; Fernando et al., 2006). Similarly, following chronic cerebral hypoperfusion, increases in inflammatory microglia have been evidenced in white matter areas (Shibata et al., 2004; Coltman et al., 2011; Holland et al., 2011; Kitamura et al., 2012). The present study confirms these previous observations demonstrating significant increases in the proportion of microglia in the hypoperfused white matter (the corpus callosum) where a significant association with 5hmC was detected. Hydroxymethylation is understood to be an integral step in active demethylation (reviewed in Kohli and Zhang, 2013) and previous studies on a mouse model of traumatic brain injury have shown active DNA demethylation in subpopulations of activated microglia and macrophages (Zhang et al., 2007). Microglia are unlikely to be the only cell

Fig. 6 – Western blot analysis demonstrated dynamic changes in TET proteins with oligodendroglial maturation and staurosporine-induced oxidative stress in vitro. TET1 protein levels decreased significantly in mature oligodendrocytes (po0.001) and increased with staurosporine treatment (po0.01) (A and B); TET2 was unaltered during oligodendroglial maturation (p40.05), but showed significant decreases under oxidative conditions with more pronounced dynamics in OPCs than in mature oligodendrocytes (po0.001) (A and C); TET3 protein levels were significantly higher in mature oligodendrocytes than in OPCs (po0.001) and exhibited development-specific response to oxidative stress being significantly increased in OPCs and significantly reduced in mature oligodendrocytes (po0.001) (A and D); OPCs cultured in the presence and absence of staurosporine had a higher NG2 protein content than mature oligodendrocytes under the same conditions (po0.001) Interestingly, oxidative stress was associated with up-regulation in NG2 protein levels only in mature oligodendrocytes (po0.01) (A and E); PLP protein levels were significantly higher in mature oligodendrocytes than in OPCs cultured under both normal and oxidative conditions (po0.001) (A and F).

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type accounting for the up-regulation of 5hmC in the hypoperfused corpus callosum as the difference between sham and hypoperfused animals in the proportion of 5hmC positive cells (0.5 vs. 0.84) was greater than for Iba1 positive microglia (0.08 vs. 0.14). Although the increase in the proportion of NG2 positive OPCs between sham and hypoperfused corpus callosum (0.16 vs. 0.42) could account for the observed differences in 5hmC, our study did not look at astroglial cells whose proportions are known to increase in the presence of CNS pathology (Volterra and Meldolesi, 2005). Cell-specific variations in genomic 5hmC content between different glial and neuronal lineages are likely. Hypermethylation of the GFAP promoter is associated with predominantly neuronal cell fate, whereas hypomethyaltion of the glial fibrillary acidic protein (GFAP) promoter triggers glial differentiation (Liu et al., 2007; Okada et al., 2008). Additionally, HDAC, a marker of gene silencing, is highly increased in mature oligodendrocytes, but not in neurons (MartinHusstege et al., 2002; Liu et al., 2007). 5hmC has also been reported to increase with neuronal maturation during CNS development (Colquitt et al., 2013). Furthermore, Mellén et al., 2012 demonstrated that the genomic distribution of 5hmC varies among differentiated neuronal cell types in the adult cerebellum associated with cell-type specific gene expression. In development of the human frontal cortex and retina, 5hmC has been implicated in promotion of transcription of genes associated with cell death (Jin et al., 2011; Rhee et al., 2012). Further studies are required to understand the functional significance of 5hmC dynamics in the hypoperfused white matter reported in the present study. The observed increases in 5hmC in the hypoperfused corpus callosum were not accompanied by alterations in 5mC and TET2 in the same white matter tract. In the mouse significant increases in hydroxymethylation in grey matter have been observed with ageing in the absence of alterations in methylation and TETs mRNA expression (Chen et al., 2012). 5mC and 5hmC are thought to antagonistically regulate gene silencing/transcription (Ficz et al., 2011; Jin et al., 2011) and might fulfill different functions. CpG islands are almost devoid of 5mC, but they are highly enriched in 5hmC (Ficz et al., 2011). Furthermore, transcriptional repressors known to bind methylated DNA such as MBD1, MBD2, and MBD4 do not recognize 5hmC (Jin et al., 2010). This apparently is not the case for the methyl-CpG-binding protein 2 in the brain (Mellén et al., 2012). In the present study we also observed significant increases in the proportion of TET2 positive cells in the hypoperfused cortex where an absence of overt pathology is evidenced one month after chronic cerebral hypoperfusion in mice (Shibata et al., 2004, 2007; Nishio et al., 2010; Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014 supplementary materials, Fig. S1.2). The more pronounced susceptibility of the cerebral white matter to chronic cerebral hypoperfusion could be explained by (1) its limited cerebrovascular irrigation (Nonaka et al., 2003; Thomas et al., 2014) and (2) the high vulnerability of the oligodendroglial cells to hypoxic-ischemic events (Dewar et al., 2003). The cerebral white matter is characterized by arterial end and border zones with very limited blood irrigation even under normal physiological conditions (Nonaka et al., 2003; Thomas et al.,

2014). Therefore, under mild chronic cerebral hypoperfusion, the white matter is subjected to a more severe glucose and oxygen deprivation than the highly vascularized cortical grey matter. This is associated with the development of selective white matter pathology in this animal model (Shibata et al., 2004, 2007; Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014) compared with alternative in vivo models where more severe reductions in the cerebral blood flow lead to the occurrence of mixed grey and white matter abnormalities (Pappas et al., 1996; Abraham and Lazar, 2000; Wakita et al., 2002; Farkas et al., 2004, 2007; Otori et al., 2003; Tomimoto et al., 2003). Further support comes from a recently developed rat model where chronic cerebral hypoperfusion is induced by the bilateral application of an ameroid constrictor device to the common carotids allowing regulated blood flow reductions associated with the development of white matter pathology in the absence of grey matter ischemia (Kitamura et al., 2012). Additionally, at the cellular level, the oligodendrocytesone of the major cell types present in the cerebral white matter are extremely vulnerable to oxidative, excitotoxic and inflammatory agents associated with chronic cerebral hypoperfusion due to: (1) their low anti-oxidative defenses (e.g. low gluthatione levels) (Yonezawa et al., 1996); (2) their high iron content (Thorburne and Juurlink, 1996); and (3) the expression of glutamatergic NMDA, AMPA/kainite receptors) (Karadottir and Attwell, 2007; Bakiri et al., 2009) mediating excitotoxic damage and receptors for inflammatory cytokines and interleukins (Otero and Merrill, 1994; Dopp et al., 1997) which are increased following hypoperfusion in mice (Shibata et al., 2004; Coltman et al., 2011; Holland et al., 2011). The molecular mechanisms and functional significance of the presently observed increases in the proportion of TET2 positive cells in the cerebral cortex remain unclear and clarifying their potential role is beyond the scope of our study. However, it is possible that TET2 up-regulation in the cerebral cortex is necessary for the survival program of neurons under mild hypoxic conditions protecting these cells from harmful agents. Due to the cytoplasmic localization of this protein in cells composing grey matter areas (predominantly neurons), its function is most likely trophic and/or antioxidative rather than gene regulatory. It is possible that higher levels of TET2 exert neuroprotective effects due to the absence of overt neuronal loss in this animal model one month after surgery. Alternatively, TET2 up-regulation in the hypoperfused grey matter might be an early sign of the subsequent neuronal cell loss occurring months after the initial microcoils surgery (Nishio et al., 2010) as observed for 5hmC during retinal development (Rhee et al., 2012). In summary, we report significant increases in 5hmC levels in the hypoperfused corpus callosum one month after microcoils surgery in mice. Our findings suggest that 5hmC may be associated with microglia infiltration and recruitment of OPCs to the hypoperfused white matter. Future experimental work focusing on the functional significance of this newly discovered epigenetic modification and its underlying cellular and molecular pathways in the healthy and hypoperfused brain will shed new light into the molecular mechanisms underlying the development of white matter

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pathology opening new therapeutic avenues for treatment of age-related cognitive decline.

4.

Experimental procedures

4.1.

Ethics statement

All animal procedures were authorised under a UK Home Office approved project licence (number 60/3722) held by Prof. K. Horsburgh. This licence was approved by the University of Edinburgh’s Ethical Review Committee and the Home Office, and adhered to regulations specified in the Animals (Scientific Procedures) Act (1986).

4.2.

Animals and surgery

Male C57Bl/6J mice (2973 g, 5 month old, purchased from Charles River, UK) were anesthetized with 5% isoflurane, followed by 1.2 to 1.6% in oxygen enriched air. The animals were randomly allocated to a sham (n ¼8) or a hypoperfused (n¼ 9) group. Chronic cerebral hypoperfusion was induced as previously described by introducing wire microcoils (0.18 mm internal diameter; Sawane Spring Co. Japan) around the common carotid arteries to induce luminal narrowing (Coltman et al., 2011; Holland et al., 2011; Reimer et al., 2011; McQueen et al., 2014). To minimize cerebral blood flow changes as a result of microcoil placement, a 30-min rest period was given between microcoils implantation during which the animal was placed in an incubator at 32 1C. Sham animals underwent identical surgical interventions with the omission of microcoils placement. Food and water were provided ad libitum during the entire experimental period.

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100 mM Tris–HCl (pH 8.5) for 10 min, followed by a standard immunostaining protocol. The samples were incubated with rat monoclonal anti-5hmC (Diagenode, Liege, Belgium, 1:1000 dilution) and mouse monoclonal anti-5mC (clone 33D3, Eurogentec, Southampton, UK, 1:100 dilution) primary antibodies overnight at 4 1C. An anti-single stranded DNA (ssDNA) antibody (Zymo research, Atlanta, USA, 1:200 dilution) was used for genomic DNA staining to correlate with 5mC and 5hmC. Peroxidase-conjugated anti-rat secondary antibody (DAKO, Cambridgeshire, UK, 1:200 dilution) and the tyramide signal enhancement system (Perkin Elmer, Massachusetts, USA) were employed for 5mC and 5hmC detection. ssDNA was visualized using donkey anti-rabbit Alexa 555 secondary antibody (Invitrogen, Paisley, UK, 1:200 dilution).

4.5. Immunohistochemical detection of TET2 and glial-specific markers

All animals were perfusion-fixed with 0.9% heparinized saline followed by 4% paraformaldehyde buffer one month post-surgery. Brains were paraffin embedded and 6 mm thick coronal brain sections were serially microtome cut. All analysis was performed at a neuroanatomical level of— 2.12 mm from bregma (Franklin and Paxinos, 1997). Examined regions of interest included white matter tracts (i.e. corpus callosum, external capsule, internal capsule) and control grey matter regions (i.e. the cerebral cortex, and CA1 region of the hippocampus) known to be susceptible to cerebral blood flow alterations. Prior to histology and immunohistochemistry, the brain sections were de-waxed using standard procedures. White and grey matter integrity were evaluated as previously described (Coltman et al., 2011; Holland et al., 2011; Supplementary materials—Fig. S1.1; Fig. S1.2; Table S1).

TET2 as well as adenomatous polyposis coli-CC1 (mature oligodendrocytes), chondroitin sulfate proteoglycan-NG2 (OPCs) and ionized calcium binding antigen 1-Iba1 (microglia) positive cells were examined by means of a standard immunohistochemical procedure. Brain sections were rehydrated using decreasing degrees of ethanol and the endogenous peroxidase activity was blocked by incubation with 0.5% H2O2 in methanol at room temperature for 30 min. Antigen retrieval was applied for CC1, NG2, and Iba1 immunostaining. This involved an additional incubation step in citric acid (pH 6.0) twice for 5 min in a 700 W microwave oven operating at its highest power setting, with citric acid replenished between incubations. After a blocking step with 10% serum for 1 h at room temperature, the samples were incubated with the respective primary antibodies at 4 1C overnight. The primary antibodies were rabbit polyclonal anti-TET2 (Santa Cruz, Wembley, UK, 1:50 dilution), mouse monoclonal antiCC1 (Calbiochem, Gibbstown, USA, 1:50 dilution), rabbit polyclonal anti-NG2 (Millipore, Watford, UK, 1:50 dilution) and rabbit polyclonal anti-Iba1 (Biocare Medical, 1:750 dilution). Following this, either horse anti-rabbit (for Iba1 detection) (Invitrogen, Paisley, UK, 1:100 dilution) or donkey anti-rabbit (for TET2 and NG2 detection) (Invitrogen, Paisley, UK, 1:200 dilution) Alexa 555-conjugated secondary antibodies were applied for 1 h at room temperature. Iba1 immunoreactivity was revealed by 2 min incubation with 3,3-diaminobenzide tetrachloride solution (Vector laboratories, Peterborough, UK) and haemotoxylin was used as a counterstain. Chicken antimouse secondary antibody (DAKO, Cambridgeshire, UK, 1:200 dilution) in combination with tyramide signal enhancement kit (Perkin Elmer, Massachusetts, USA) were used for CC1 detection. DAPI (1:1000) was used as a nuclear counterstain for CC1, NG2, and TET2.

4.4.

4.6.

4.3.

Histology

Immunohistochemical detection of 5hmC and 5mC

5mC and 5hmC immunohistochemistry was performed as previously described (Ruzov et al., 2011). Briefly, tissue sections were permeabilized with phosphate buffer saline (PBS) containing 0.5% Triton X-100 for 15 min. For 5mC and 5hmC staining, permeabilized tissue sections were incubated in 4 N hydrochloric acid (HCl) for 1 h at 37 1C and then neutralized in

Imaging procedures

Fluorescent immunostained samples (5mC, 5hmC, TET2, CC1, NG2) were imaged on an Axiovert (Carl Zeiss) fluorescence detecting microscope. Fluorescent images were generated using a lens magnification  40 and Axiovison software (Carl Zeiss). Five random fields were taken at the same excitation rate from each region of interest, for each marker. For the

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Iba1 immunostained samples, a light microscopy was applied using the same Axiovert microscope and imaging system.

4.7. In situ immunohistochemical evaluation of epigenetic and glial-specific markers For each marker and region of interest, the number of marker positive and total number of cells were manually counted from images taken from five random visual fields. Regional marker positive cell proportions were calculated by dividing the average number of marker positive cells by the average total number of cells. For 5mC and 5hmC, the total number of cells was determined by counting anti-ssDNA immunostained genomic DNA positive cells. For TET2, CC1, and NG2, the total number of cells was determined by counting DAPI stained cell nuclei. For Iba1, the total number of cells was determined by counting haemotoxylin stained cell nuclei. Since co-immunostaining of 5mC and 5hmC with TET2 or respective neuronal and glial-specific markers was technically impossible due to incompatibility of the corresponding antigen retrieval methods with reliable detection of modified forms of cytosine to identify the cell types enriched in these modifications, the regional localization of the cells (white vs. grey matter) and the relative size of their nuclei were taken as indicatives of the cellular identity (glia vs. neurons).

4.8.

In vitro oligodendroglial and microglial culture

OPCs were isolated from the cerebral cortices of mice pups (postnatal days 0–2). Cortices were separated from the rest of the brain under a dissecting microscope and subsequently digested with papain solution (1 ml DMEM (Invitrogen); 40 μl papain (Worthington Biochemical); 25 μl L-cysteine solution (Sigma); 25 μl DNase I type IV (Sigma)) at 37 1C for 10 min. Following this, cells were further mechanically isolated from the surrounding epithelia using a sterile filter. Cell pellets were collected by centrifugation at 1000 rpm for 5 min. This step was repeated three times and after each centrifugation cells were resuspended in a fresh 10% DMEM. Cells were finally seeded onto a poly-D-lysine coated flasks and cultured in SATO medium composed of DMEM supplemented with 4.5 g/l glutamine (Sigma), 1% penicillin and streptomycin (Invitrogen), 10% horse serum (Invitrogen), 10 mg/ml insulin (Sigma), 10 mg/ml transferrin (Sigma), 10 ng/ml fibroblast growth factor (FGF2) (Peprotech), and 10 ng/ml platelet derived growth factor (PDGF) (Peprotech). Cells were incubated at 37 1C, 7.5% CO2 for 3 days prior to the first media change. Subsequently, the media was changed every other day. Differentiation was achieved by removal of the growth factors (FGF2 and PDGF) from the SATO medium. The cells were cultured for additional 0, 2, 6 DIV to reach a certain maturation stage. For some experiments OPCs (2 DIV) and mature oligodendrocytes (6 DIV) were treated with vehicle vs. 20 nM Staurosporine (Cell Signaling) for 48 h to induce oxidative stress. Microglia cells were isolated from the cerebral cortices of Sprague Dawley rat pups (postnatal day 0–2). Following removal of meninges, cortices were mechanically minced and then enzymatically digested for 45 min at 37 1C in a solution containing 30 μ/ml papain (Worthington Biochemical

Corporation, USA), 24 mg/ml cysteine (Sigma) and 4 mg/ml DNaseI type IV (Sigma). The cell suspension was incubated with 10% DMEM (with addition of 4.5 g/l glucose, L-glutamine, and pyruvate; (all purchased from GIBCO) supplemented with 10% fetal calf serum (Invitrogen), and 1% penicillin/streptomycin (Sigma) and centrifuged at 1000 rpm for 5 min. After centrifugation the cells were resuspended in 10% DMEM and triturated with 19 and 23 gauge syringe needles. Cells were plated on poly-D-lysine (Sigma) coated plastic flasks and grown as mixed glial cultures for 10 days in 10% DMEM with media changed every 2–3 days. Microglia cells were isolated by collecting the floating fraction in the flasks following 1 h incubation on a rotary shaker at 37 1C at 250 rpm. Cells were plated on poly-D-lysine coated 16-well glass chamber slides (Lab-TEK, USA) at 50 000 cells per well in 10% DMEM. Microglia cells were either left untreated or activated by overnight treatment with 20 ng/ml IFNγ (Sigma) and 100 ng/ml LPS (Sigma). All in vitro experiments were performed in triplicates.

4.9.

Immunocytochemical detection of 5mC and 5hmC

Prior to immunocytochemistry oligodendroglial and microglial cells were fixed in 4% paraformaldehyde at room temperature for 20 min. Following three PBS washes for 5 min each, cell membranes were permeabilized with PBS containing 0.5% Triton X-100 for 15 min. For 5mC and 5hmC staining, permeabilized cells were incubated in 4 N HCl for 1 h at 37 1C and then neutralized in 100 mM Tris–HCl (pH 8.5) for 10 min, followed by a standard immunostaining protocol. Cell samples were incubated with rat monoclonal anti-5hmC (Diagenode, Liege, Belgium, 1:1000 dilution) and mouse monoclonal anti-5mC (clone 33D3, Eurogentec, Southampton, UK, 1:100 dilution) primary antibodies overnight at 4 1C. An anti-single stranded DNA (ssDNA) antibody (Zymo research, Atlanta, USA, 1:200 dilution) was used for genomic DNA staining to correlate with 5mC and 5hmC. Peroxidaseconjugated anti-rat secondary antibody (DAKO, Cambridgeshire, UK, 1:200 dilution) and the tyramide signal enhancement system (Perkin Elmer, Massachusetts, USA) were employed for 5mC and 5hmC detection. ssDNA was visualized using donkey anti-rabbit Alexa 555 secondary antibody (Invitrogen, Paisley, UK, 1:200 dilution). Immunostained cell samples were imaged as described in Section 4.6.

4.10.

Western blot analysis

Cells were lysed in cold 500 ml RIPA buffer (pH 7.5, 1% NP-40, 1% sodium-deoxycholate, 0.1% SDS, 0.15 M NaCl, 2 mM EDTA, and 0.01 M sodium phosphate) supplemented with protease (Roche) and phosphatase (Sigma) inhibitors. Protein lysates were centrifuged at 4 1C at 13.2 rpm for 10 min and the supernatant was collected. Protein concentration was determined by the Lowery assay (Pierce) and measured using Life Science UV/ VIS DU 530 Spectrophotometer (Beckman, USA). Protein samples were resolved on 8% (TET1, TET2, TET3) and 12% (PLP, NG-2) SDS-PAGE gels and transferred onto nictrocellulose membranes that were subsequently blocked with 5% milk in TBS-T buffer (20 mM Tris, 137 mM NaCl, 0.1%

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Tween) for 30 min prior to an overnight incubation in the respective primary antibody at 4 1C: goat polyclonal antiTET1, rabbit polyclonal anti-TET2, rabbit polyclonal antiTET3 (all TET antibodies were purchased from Santa Cruz Biotechnology; 1: 200 dilution), mouse monoclonal anti-PLP (Thermo Scientific, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, 1:200 dilution) and mouse monoclonal anti-β-actin (Cell Signaling, 1: 5000 dilution). Subsequently, the membranes were washed three times in TBS-T for 5 min/wash and the corresponding secondary HRP antibody (Jackson Laboratory, 1:5000 dilution) was applied for 1 h at room temperature. Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and densitometrically analyzed with One Quantity software (Bio-Rad). The level of protein expression was normalized to β-actin and represented as normalized densitometric intensity.

4.11.

Statistics

The statistical analysis was performed using GraphPad Prism (version 5.4) software. The regional group average marker positive cells, total number of cells, and the proportion of marker positive cells were statistically compared using an unpaired t-test. To account for potential associations among examined epigenetic markers in white matter, the regional proportions of 5hmC positive cells were correlated with the regional proportions of 5mC and TET2 positive cells in the corpus callosum (where significant increases in the proportion of 5hmC positive cells were observed following hypoperfusion) using a parametric Pearson’s correlation analysis. To determine the potential cellular basis underlying the observed 5hmC dynamics in white matter, the proportion of 5hmC positive cells in the corpus callosum was correlated with the proportion of mature oligodendrocytes (CC1), OPCs (NG2), and microglia (Iba1) in the same white matter tract by using a parametric Pearson’s correlation analysis. Western blot data was analyzed by means of one-way ANOVA followed by the Bonferroni correction post-hoc test in the case of significance. Significant group differences and correlations were reported for po0.05.

Author contributions Study concept and design: YT, KH, PDS. Surgeries: CG. Acquisition of data: YT. Analysis and interpretation of data: YT, AR, PDS. Drafting of the manuscript: YT, PDS. Critical revision of the manuscript for important intellectual content: YT, AR, CG, KH, PDS. Study supervision: KH, PDS.

Conflict of interest The authors declare no conflict of interest.

Acknowledgments We thank Anna Williams and Veronique Miron for providing oligodendroglial and microglial preparations. This study was funded by the support of Age UK (“The Disconnected Mind” project) to KH, the EUFP7 Grant (201619) to PDS. YT acknowledges the College of Medicine and Veterinary Medicine, the University of Edinburgh for her studentship.

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2014.09.060.

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