Correspondence to Patrizia Casaccia-Bonnefil: casaccpa{at}umdnj.edu
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Introduction |
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The premise of this study is that the progression along the OL lineage is a complex event characterized by global changes in gene expression, resulting in loss of precursor markers and differentiation inhibitors and acquisition of late differentiation markers, including enzymes for the synthesis of myelin lipids and myelin proteins, such as ceramide-galactosyl-transferase (CGT), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG). We had previously reported that global changes affecting deacetylation of nucleosomal histones were critical for OL differentiation in vitro (Marin-Husstege et al., 2002). Reversible acetylation of selected lysine residues in the conserved tails of nucleosomal core histone proteins represents an efficient way to regulate gene expression (for reviews see Strahl and Allis, 2000; Turner, 2000; Yoshida et al., 2003; Yang, 2004). In general, increased histone acetylation (hyperacetylation) is associated with increased transcriptional activity, whereas decreased acetylation (hypoacetylation or deacetylation) is associated with repression of gene expression (Forsberg and Bresnick, 2001; Wade 2001).
The removal of acetyl groups from lysine residues in the histone tails is performed by specific enzymes called histone deacetylases (HDACs) that can be broadly grouped into three major classes. Class I includes HDAC-1, -2, -3, and -8 and is composed of small proteins (377488 aa), sharing sequence homology to the yeast transcriptional regulator RPD3 (Bjerling et al., 2002), and a broad expression pattern. Class II includes HDAC-4, -5, -6, -7, and -9 and is composed of proteins of larger size (6691215 aa), sharing sequence homology with the yeast HDA1 (Fischle et al., 2002), and a restricted expression pattern (de Ruijter et al., 2003). Class III HDACs, the Sir2 family proteins, includes molecules that are sensitive to the redox state of the cell and are inhibited by a different category of pharmacological inhibitors (Grozinger et al., 2001) than the other two classes (Phiel et al., 2001; Gottlicher, 2004; Gurvich et al., 2004).
Because the acetylation state of nucleosomal histones modulates chromatin structure and epigenetically regulates gene expression, we hypothesized that this could be the global mechanism responsible for timing of OL progenitor differentiation in vivo. We addressed this question in the developing corpus callosum because timing of myelination of this region has been thoroughly characterized (Bjelke and Seiger, 1989; Hamano et al., 1996, 1998) and because of its functional relevance as the major myelinated fiber tract of the adult brain. The corpus callosum is composed of millions of fibers that need to be properly myelinated to allow communication between the two brain hemispheres. Myelination in this structure follows a precise timing, during the first two postnatal weeks of development (Bjelke and Seiger, 1989; Hamano et al., 1996, 1998), and a precise topology, starting at caudal levels and progressing rostrally (Smith, 1973) and starting laterally and proceeding medially (Smith, 1973).
In this study we asked whether deacetylation occurred in OL progenitors residing in the developing anterior corpus callosum and whether inhibition of this process in vivo would affect timing of myelination.
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Results |
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Inhibiton of HDAC activity prevents myelin gene expression in the developing corpus callosum only during a critical temporal window
To address the functional relevance of histone deacetylation on OL differentiation and myelination, we investigated the effect of in vivo administration of the pharmacological inhibitor of class I HDACs, valproic acid (VPA). The short-term experimental paradigm included three groups of neonatal pups receiving a 2-d regimen of VPA (300 mg/kg body weight) starting at distinct developmental time points (Fig. 6 A). The first group of neonatal rats (n = 12) was injected with PBS (n = 6) or VPA (n = 6) at p6 and p7 and then harvested at p8 (injection 1), at the beginning of myelination. The second group (n = 12) was injected at p9 and 10 and harvested at p11 (injection 2), whereas the third group (n = 12) was injected at p19 and 20 and harvested at p21 (injection 3), after myelination had ensued in the rostral corpus callosum.
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Decreased MAG and MBP immunoreactivity in the corpus callosum of VPA-treated pups was accompanied by a reduction in the number of CC1+ cells (Fig. 7, AE). This decrease was not due to a toxic effect of the pharmacological inhibitor because the total number of DAPI+ cells/mm2 was quite stable (x =1488 ± 49 in VPA injected and x =1563 ± 52 in PBS control) and because there was no difference in the number of TUNEL+ (unpublished data) apoptotic cells in the two groups. The reduced number of CC1+ cells was likely due to delayed differentiation, as indicated by the increased percentage of cells expressing the bipotential progenitor marker NG2+ in VPA-treated animals compared with controls (Fig. 7, FH). The increased progenitor number was not due to an effect of VPA on proliferation, because the number of proliferating NG2+ cells, identified by in vivo labeling with the thymidine analogue BrdU, was very similar in treated and control rats (Fig. 7, IM). The inhibitory effect of VPA on differentiation was also supported by the detection of PSA-NCAM+ precursors in cells in the subcortical white matter of treated animals (Fig. 7, NQ). Together, these data suggest that short-term inhibition of HDAC activity does not impair the ability of OL progenitors to exit the cell cycle, but arrests their differentiation at a stage characterized by the expression of early progenitor traits and lack of differentiation markers.
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After myelination onset, cells in the developing corpus callosum acquire more permanent changes in chromatin components and this renders them refractory to the effect of HDAC inhibitors
We have previously discussed that the effectiveness of VPA administration on modulating myelin gene expression in vivo was limited to a specific temporal window coincident with the onset of myelination. To understand the molecular mechanisms defining this developmental window regulated by HDAC activity, we assessed the presence of AcH3 after each protocol of VPA injection (Fig. 10 A). Administration of VPA during the first two postnatal weeks increased the levels of AcH3 without significantly affecting the acetylation state of other high molecular weight proteins (Fig. 10 A). In contrast, administration of VPA during the third postnatal week did not affect the levels of AcH3 (Fig. 10 A). Because protein acetylation is the result of the equilibrium between histone acetyltransferases (i.e., HATs, such as p300 and CBP) and HDACs (Lehrmann et al., 2002; Rouaux et al., 2003), we hypothesized that the lack of VPA in the third postnatal week was consequent to low levels of HATs. This hypothesis was confirmed by the detection of decreased protein levels of CBP and p300 during the third postnatal week of development (Fig. 10 B). The results obtained at p24 suggested that perhaps reversible acetylation was a mechanism of regulation of gene expression that was best suited to maintain a certain "plasticity" of gene expression during early developmental stages. At later developmental stages, however, it was likely that committed cells would adopt more stable mechanisms of regulation of gene expression that would guarantee the maintenance of the differentiated phenotype. Because histone deacetylation is often followed by the more stable methylation of lysine 9 in histone H3 (Honda et al., 1975; Eberharter and Becker, 2002; Boulias and Talianidis, 2004), we asked whether in the corpus callosum the global changes in gene expression initiated by histone deacetylation were also maintained by histone methylation and chromatin compaction. To test this possibility, we stained p5 and p24 brain sections with antibodies specific for methylated histone H3 and for HP1 , a protein that specifically binds to methylated lysine 9 on histone H3 (MeK9H3) and identifies the presence of compact chromatin (Bannister et al., 2001; Lachner et al., 2001). In agreement with our hypothesis, at p5 before the peak of myelination, OL progenitors did not show MeK9H3+ or HP1
immunoreactivity (Fig. 10, CF). In contrast, by p24 the majority of the cells in the corpus callosum were MeK9H3+/HP-1
+ (Fig. 10, GJ), thus confirming the acquisition of compact chromatin structure.
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Discussion |
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We now show that deacetylation of histone H3 is a critical mechanism for myelination onset in vivo because it is required for the down-regulation of differentiation inhibitors and early progenitor markers. Administration of the HDAC inhibitor VPA during the critical period of myelination onset inhibited the progression of OL progenitors along the lineage. This arrest in differentiation was characterized by timely exit from the cell cycle, persistence of progenitor traits and lack of late differentiation markers, thereby identifying histone deacetylation as the molecular link of the transition between cell cycle exit and differentiation onset.
Deacetylation of histone H3 during myelination onset was attributed to class I HDACs. The HDAC enzymatic activity in tissue extracts was selectively inhibited by the inhibitor TSA but not by the class III inhibitor sirtinol, and only the class I isoforms (HDAC-1, -2, -3, and -8) showed nuclear localization. Interestingly, HDAC-1 was predominantly detected in OL lineage cells throughout development, whereas HDAC-2, -3, and -8 were also found in neurons and astrocytes. Therefore class I HDACs were likely to be responsible for the removal of acetyl groups from histone H3 in OL lineage cells, whereas class II HDACs played a role in modulating acetylation of cytosolic substrates
Histone acetylation was previously shown to result from a balance between the addition (by HATs) and the removal (by HDACs) of acetyl group on specific lysine residues (Lehrmann et al., 2002; Rouaux et al., 2003) and to result in a transcriptionally active chromatin conformation (Eberharter and Becker, 2002), thus implying this secondary modification of histone as a reversible switch regulating gene expression. In agreement with this concept, HDAC inhibition in VPA-treated rats created a disequilibrium characterized by the predominance of HAT activity. The resulting increase of H3 acetylation was functionally associated with high levels of differentiation inhibitors and with the persistence of progenitor traits. Due to the reversible nature of this secondary modification of histone H3, we anticipated that upon interruption of VPA treatment the animals would resume a normal pattern of myelination. Consistent with this hypothesis, after only two days of recovery, progenitors rapidly down-regulated the inhibitors, up-regulated transcriptional activators, and expressed OL markers such as CC1 and MAG. In addition, some of the newly generated OL started to myelinate the callosal axons, as indicated by the presence of MAG immunoreactive fibers. These data support the idea that HDAC activity is necessary for the repression of genes inhibiting differentiation and are in agreement with results recently obtained in zebrafish (Cunliffe, 2004).
Our study also has important clinical implications because it suggests that treatment with VPA, a pharmacological agent currently used in the management of seizures, can negatively affect myelination in the corpus callosum if delivered during a critical temporal window of development. The inhibitory effect of VPA on myelination was observed only if the administration occurred during the first two postnatal weeks. At later time points, VPA administration did not affect myelin gene expression and OL differentiation, thus suggesting the existence of alternative mechanism of regulation of gene expression, occurring at later developmental stages. It has been reported that histone deacetylation is often followed by the more stable histone methylation (Eberharter and Becker, 2002; Boulias and Talianidis, 2004). Indeed, we demonstrated that the reversible deacetylation of lysine residues on histone H3observed in cells of the OL lineage during the first two weeks of developmentwas later replaced by the more stable methylation of lysine 9 in histone H3 and by the expression of the HP1 protein, a marker of chromatin compaction. Therefore, our results identified histone acetylation as a reversible mechanism regulating the expression of progenitor traits during early developmental stages, when progenitors showed a certain degree of "plasticity." At later developmental stages, however, the committed cells adopted more stable mechanisms of repression, dependent on histone methylation and HP1
binding. These changes defined the acquisition of compacted chromatin associated with decreased levels of differentiation inhibitors possibly to favor the maintenance of the differentiated phenotype.
The progressive compaction of chromatin during OL development was consistent with morphological studies on the ultrastructure of developing OL in the corpus callosum of neonatal rats (Mori and Leblond, 1970; Kozik, 1976; Sturrock, 1976). According to these studies, at p1 progenitors were identified by the presence of a pale nucleus with dispersed chromatin and abundant cytoplasm (Mori and Leblond, 1970; Sturrock, 1976). At p8 the appearance of the differentiating cells, called "oligodendroblasts," was characterized by a slightly higher electron density and by the presence of large conglomerates of nuclear chromatin in the inner part of the nuclear membrane (Kozik, 1976). Finally, around the third week of postnatal development, the nucleus of OL was characterized by the presence of very large chromatin aggregates and smaller granules scattered throughout (Mori and Leblond, 1970; Kozik, 1976; Sturrock, 1976). Our data provide a molecular explanation to this very well-characterized morphological profile because histone acetylation was consistent with the dispersed chromatin observed in progenitor cells, histone deacetylation correlated with the initiation of chromatin compaction observed around p8, and finally, histone methylation and HP1 binding were observed coincident with the report of chromatin condensation in mature OL.
In conclusion, this study provides evidence that epigenetic regulation of gene expression in cells of the OL lineage modulates timing of OL differentiation in the developing corpus callosum. Although the appearance of nuclear chromatin in the corpus callosum is a well-established ultrastructural criterion for identification of cells from the OL lineage (Mori and Leblond, 1970; Kozik, 1976; Sturrock, 1976; Imamoto et al., 1978), the functional relevance of chromatin compaction in progenitor differentiation has never been investigated. In this manuscript we describe the mechanisms responsible for the progressive compaction of chromatin observed during the maturation of progenitor cells into myelinating OLs. Further, we identify the biological significance of post-translational modifications of nucleosomal histones as a global event responsible for timing of OL differentiation and myelination of the developing corpus callosum.
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Materials and methods |
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PBS/VPA injection
The first group of neonatal pups subject to subcutaneous injection (injection protocol 1) consisted of p6 animals injected either with PBS (n = 6) or with VPA (300 mg/kg weight; n = 6). Each animal received a total of four injections, administered every 12 h for two consecutive days. Injected rats were then either killed on p8 and the brain tissues were dissected out and subject to total RNA extraction or protein lysis, or perfused with 4% PFA for coronal cryosections. The second group of neonatal pups (injection protocol 2), consisting of p9 animals, was subject to subcutaneous injection of PBS (n = 6) or VPA (n = 6), repeated every 12 h for 4 times, and was followed by tissue harvesting at p11. Brain tissue from these injected rats was subject to total RNA extraction or protein lysis. The third group (injection protocol 3), consisting of p19 pups, received subcutaneous injection of PBS (n = 6) or VPA (n = 6) every 12 h for 4 times followed by harvesting on p21. The anterior portion of the corpus callosum surrounding the bregma was carefully dissected and the extracted proteins were processed for Western blot analysis. The fourth group (injection protocol 4), consisting of p3 pups, received a subcutaneous injection of PBS (n = 6) or VPA (n = 6) every 12 h for 4 times, followed by tissue harvesting on p5. The long-term treatment consisted of 13 injections from p3 to p10. The toxicity of the treatment was monitored by daily recording of the body weight. For the recovery experiments, neonatal rats received the injections as described in protocol 1 and 4 (n = 6 for PBS and n = 6 for VPA), followed by a recovery period of three days before sacrifice.
Immunohistochemistry and analysis of corpus callosum and anterior commissure
Untreated neonatal rats (p1, p5, p8, p11, and p24) and PBS- or VPA-injected neonatal rats (see injection protocols) were anesthetized and then perfused with 4% PFA in 0.1 M phosphate buffer. The whole brains were removed from the skulls, post-fixed, then cryopreserved in 30% sucrose, embedded in OCT and sectioned coronally (20 µm). Frozen sections were first permeabilized with blocking buffer (0.1 M phosphate buffer, 5% normal goat serum [Vector Laboratories], and 0.5% Triton X-100). Note that for better staining with HDACs it was necessary to process the sections for antigen retrieval by incubation in citrate buffer (Poly Scientific), pH 6.8, at 65°C for 2 h before the blocking step. Incubation with primary antibodies against HDACs, myelin proteins, or other lineage progression markers was performed overnight at RT (see Antibodies section for sources and dilutions). The following day, after 1 h incubation with secondary antibodies, either directly conjugated to specific fluorochromes (whole Ig-Cy3, 1:200; Sigma-Aldrich) or biotinylated (RPN-1004, 1:200; Amersham Biosciences), the sections were counterstained with DAPI (1:1,000; Molecular Probes, Inc.) in the absence or presence of avidin-conjugated FITC or Texas red (RPN-1232 and RPN-1233, diluted 1:500; Amersham Biosciences). Stained sections were visualized using an inverted fluorescence microscope (DM RA; Leica) and confocal microscopy (LSM510 Meta confocal laser scanning microscope; Carl Zeiss MicroImaging, Inc.). The immunohistochemical analysis on the rostral corpus callosum at p5 was conducted on coronal sections corresponding to plates 155160 of the p0 images of the "Atlas of the Developing Rat Nervous System" (Paxinos et al., 1994), upon adjustments of the distance from bregma to lambda at p0 to the measured distance between these two sutures at p5. The anterior commissure was analyzed in sections corresponding to plates 156 and 157 of the same atlas (Paxinos et al., 1994). The immunohistochemical analysis of the anterior portion of the body of the corpus callosum at p24 was conducted posterior to the genu, on coronal sections corresponding to plates 1519 of the atlas "The Rat Brain in Stereotaxic Coordinates" (Paxinos and Watson, 1986). The anterior commissure was analyzed in coronal brain sections corresponding to plates 18 and 19 of the same atlas (Paxinos and Watson, 1986). The "medial corpus callosum" was defined as the region corresponding to 1 L, +1R of the same atlases. For the functional studies on PBS- or VPA-injected rats of different neonatal ages, the rostral corpus callosum and the anterior commissure were analyzed using similar criteria, adjusting the Atlas to the exact bregma to lambda distance at each developmental age (Paxinos and Watson, 1986; Paxinos et al., 1994).
Western blot analysis
Upon carefully removing the skin over the skull, the positions of the bregma and lambda sutures were marked on the brain surface. These positions were used as a reference point in dissecting out the same regions of corpus callosum from rats of different developing stages. A coronal slice around the bregma was dissected out, and the corpus callosum was excised under a dissection microscope (Nikon) and used for either protein or RNA extraction. Tissue lysates from freshly dissected corpus callosum were prepared by digestion in a buffer containing 50 mM Hepes, pH 7.0, 250 mM NaCl, 0.15% Nonidet P-40, 1 mM DTT, 1 mM EDTA, 0.01% PMSF, 1 mM aprotinin, and 1 mM leupeptin for 15 min on ice. This was followed by mechanical disruption via serial passages through a series of syringes equipped with different-sized needles (18G11/2, 22G11/2, and 26G3/8). Cell membranes were further disrupted by sonication on ice at the highest output (twice, 30 s each; cells were kept on ice for 1 min between each pulse). After high speed centrifuge, protein concentration was determined using the Bradford's method (Bio-Rad Laboratories protein assay) and equal amounts (100 µg) were loaded on SDS-PAGE for separation. Transfer of protein onto a 0.22-µm nitrocellulose membrane was conducted using a Bio-Rad Laboratories apparatus at 30 V for 1618 h in a transfer buffer containing 25 mM Tris base, 192 mM glycine, 20% (vol/vol) methanol, and 0.04% SDS, pH 8.3. Western blot analysis was performed as reported previously (Casaccia-Bonnefil et al., 1997, 1999) using the appropriate dilutions of primary and secondary antibodies (see Antibodies section for details). The immunoreactive bands were detected by ECL Plus Western Blotting Detection System (Amersham Biosciences). Equal protein loading was guaranteed by probing the blots with antibody against actin.
RT-PCR
Total RNA was isolated using RNeasy Mini kit (QIAGEN) from individually dissected rat corpus callosum. Total RNA (9 µg/each sample) was used in 40 µl of reverse transcription reaction. The PCR was performed in a 20-µl reaction mixture containing 2 µl cDNA as template and 0.1 µM specific oligonucleotide primer pair. Cycle parameters were 30 s at 94°C, 30 s at 50°C, and 1.5 min at 72°C for 25 cycles. The following oligonucleotide primers were used: for rat cerebroside-galactosyl transferase (CGT) the forward primer was 5'-GGAGTGCTGTTGGAATAGCAA-3', and the reverse primer was 5'-CGTACTCCTAGAACACAGACTT-3'; for rat MAG, the forward primer was 5'-CACCTCGAGTCGCCTTTGCCATCCTGATT-3', and the reverse primer was 5'-TCTCCATGGCCTTGACTCGGATTTCTGCATAC-3'; for rat MBP, the forward primer was 5'-ATGGCATCACAGAAGAGACC-3', and the reverse primer was 5'-CATGGGAGATCCAGAGCGGC-3'; for rat Sox10, the forward primer was 5'-GGAGCAAGACCTATCAGAGGT-3', and the reverse primer was 5'-CAAAGGTCTCCATGTTGGACA-3'; for rat Notch-1, the forward primer was 5'-CAACGGCACTGAAGCCTGTGT-3', and the reverse primer was 5'-GCACAGTCATCAATGTTGTCA-3'; for rat nestin, the forward primer was 5'-TGCAGCCACTGAGGTATCTG-3', and the reverse primer was 5'-CAGTTCCCACTCCTGTGGTT-3'; for rat tenascin, the forward primer was 5'-AACAGGTCTCAGAGAGGCCA-3', and the reverse primer was 5'-CTTCTCTGCGGTCTCCAAAC-3'; for rat Jagged-1, the forward primer was 5'-AACAGAACACAGGGATTGCC-3', and the reverse primer was 5'-CTTGCCCTCGTAGTCCTCAG-3'; for rat actin, the forward primer was 5'-TGGAATCCTGTGGCATCC-3', and the reverse primer was 5'-TCGTACTCCTGCTTGCTG-3'.
Total HDAC enzymatic activity measurement
HDAC activity was measured by using HDAC Activity Assay/Drug Discovery Kit (BIOMOL Research Laboratories, Inc.). Experimental procedures were designed and performed according to the protocol provided within the kit. In brief, tissue lysates from the rat corpus callosum (prepared according to the same procedure described in Western blot analysis section) were used as sources for HDAC activity. Sample lysates containing 100 µg protein were added to a 96-well plate in 25 µl HDAC assay buffer (BIOMOL Research Laboratories, Inc.). A fluorimetric acetylated substrate was added and the reaction was allowed occurring at RT for 1 h, then incubated with developer for 1015 min. Enzymatic activity was evaluated in a microtiter platereading fluorimeter (excitation = 360 nm, detection of emitted light = 460 nm). HeLa nuclear extract (KI-140) was used as positive control.
BrdU incorporation in vivo and TUNEL
Neonatal rat pups received 10 mg/Kg BrdU injection 1 h before sacrifice. After perfusion and cryopreservation, brains were sectioned and stained with cellular markers. For immunolabeling with anti-BrdU antibodies, after the completion of the first staining, the cells were then treated with 2N HCl for 10 min at 37°C in order to denature DNA, followed by equilibration in 0.1 M sodium borate, pH 8.6, for 10 min. The primary antibody for anti-BrdU (DakoCytomation) was used at 1:100 dilution in PGBA containing 0.5% Triton X-100 for at least 3 h at RT, followed by Cy3-conjugated whole-Ig secondary antiserum. Cells were then counterstained with DAPI for nuclei visualization. The identification of apoptotic cells was performed using the ApopTag plus kit from CHEMICON International on cryosections, following the manufacturer's instructions.
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Acknowledgments |
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Submitted: 15 December 2004
Accepted: 18 April 2005
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References |
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