CELL CYCLE 2016, VOL. 15, NO. 20, 2780–2791 http://dx.doi.org/10.1080/15384101.2016.1220458
REPORT
Cyclin-dependent kinase inhibitor flavopiridol promotes remyelination in a cuprizone induced demyelination model Guiyun Mia, Yunyun Gaob, Shuai Liua, Enmao Yea, Yanyan Lia, Xiao Jina, Hongju Yanga, and Zheng Yanga a
Beijing Institute of Basic Medical Sciences, Haidian District, Beijing, China; bThe 89 Hospital of PLA, WeiFang City Shandong Province, China
ABSTRACT
ARTICLE HISTORY
The cuprizone (CPZ) model has been widely used for the studies of de-and remyelination. The CPZexposed mice show oligodendrocyte precursor cells (OPCs) increase and mature oligodendrocytes decrease, suggesting an imbalance between proliferation and differentiation of OPCs. In the first experiment of this study, we examined the expression of cell cycle related genes in brains of mice following CPZ administration for 5 weeks by means of microarray assay. In addition, we performed a double labeling of BrdU and Ki-67 to calculate cell cycle exit index in the mice. Our results showed that CPZ administration up-regulated the expression of 16 cell cycle related genes, but down-regulated the expression of only one in the prefrontal cortex (PFC) of mice compared to control group. The treatment inhibited potential precursor cells exit from cell cycle. In the second experiment, we evaluated effects of a CDK inhibitor flavopiridol (FLA) on CPZ-induced neuropathological changes and spatial working memory impairment in mice.FLA treatment for one week effectively attenuated the CPZ-induced increases in NG2 positive cells, microglia and astrocytes, alleviated the concurrent mature oligodendrocyte loss and myelin breakdown, and improved spatial working memory deficit in the CPZ-exposed mice. These results suggest that CPZ-induced neuropathological changes involve in dysregulation of cell cycle related genes. The therapeutic effects of FLA on CPZ-exposed mice may be related to its ability of cell cycle inhibition.
Received 11 May 2016 Revised 19 July 2016 Accepted 31 July 2016 KEYWORDS
astrocyte; cell cycle; cognition; cuprizone; flavopiridol; microglia; oligodendrocyte
Introduction The main function of oligodendrocytes is to provide support and insulation to axons. Demyelination impairs the neuron signal transmission and causes dysfunctions in sensation, movement, and cognition. Malfunction of oligodendrocyte development and demyelination have been considered as a pathological substrate for cognitive dysfunction inmultiple sclerosis (MS) and schizophrenia.1,2 Therefore, identifying and manipulating the factors regulating oligodendrocyteprecursor cells (OPCs) proliferation and differentiation are crucial to the elucidation of pathological mechanisms of demyelination diseases. The cuprizone (CPZ; biscyclohexanone oxalyldihydrazone) animal model has been widely used for the study of de-and remyelination. When given to young adult C57BL/6 mice (0.2 % in the diet), CPZ induces apoptosis of mature oligodendrocytes and demyelination accompanied by spatial working memory impairment in mice.3-6 Obvious demyelination was observed in the corpus callosum and the prefrontal cortex (PFC), external capsule, and hippocampus.7 The PFC and hippocampus are important brain regions for a large range of cognitive functions such as working memory. It is accepted that oxidative stress involves in the oligodendrocyte degeneration and myelin breakdown induced by CPZ, as evidenced by mitochondrial
dysfunction parameters, such as mitochondrial swelling, production of ROS (reactive oxygen species), and collapse of the mitochondrial membrane potential, in CPZ-treated mice.3-5,8 Moreover, certain antioxidants such as melatonin,9 quetiapine,10 and resveratrol,11 showed protective effects on the CPZ-induced oligodendrocyte loss and myelin breakdown in recent animal studies. Interestingly, oligodendrocyte loss and myelin breakdown were accompanied by increase in the number of OPCs labeled as NG2C (chondroitin sulfate proteoglycan) and PDGFRaC (platelet-derived growth factor a) cells in CPZexposed mice,12-14 suggesting the existence of an aberrant cell cycle regulation following CPZ administration. Cell cycle is divided into G0 (resting phase), G1 (preparing phase), S (DNA replication), G2 (gap between S and M), and M (cell division) phases. During development, after a limited number of dividing, OPCs need to exit from cell cycle and start the transcription of myelin related genes and finally differentiate into post-mitotic, mature oligodendrocytes.15,16 Cell cycle activation and failure of exit from cell cycle can keep the OPCs at a persistence of a proliferative state and lead to the differentiation deficit of OPCs.17 In addition, failure of exit from cell cycle can lead to oligodendrocyte apoptosis and hinder oligodendrocyte development and myelination.18,19 Based on the
CONTACT Zheng Yang [email protected]; Hongju Yang [email protected] Beijing Institute of Basic Medical Sciences, 27 Tai-Ping Road, Haidian District, Beijing, 100850, China. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis
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above knowledge and previous findings, we hypothesized that CPZ administration would change the expression of cell cycle related genes in subjects. To test this hypothesis, we conducted a microarray experiment to analyze the gene expression in PFC of mice after CPZ administration. In addition, we performed a double labeling of 5-Bromo-20 -deoxyuridine (BrdU) and Ki-67 (a marker of proliferation) to calculate the cell cycle exit index in the anterior subventricular zone (SVZ),where multi-potential precursor cells proliferate, to test whether the proliferation of mitotic cells was due to the failure of exit from cell cycle in the CPZ-exposed mice. It is known that the cyclin/CDK (cyclin-dependent kinase) complex functions as a regulatory subunit whose activity is required for cell cycle G1/Stransition and CDK inhibitors show neuroprotective effects in animal models by regulating the progression of the G1/S phase and the withdrawal from cell cycle.20-22 Flavopiridol(FLA) is a flavonoid alkaloid that blocks the activity of cyclin /CDK, either by forming an inactive complex or by acting as a competitive ligand for CDKs.23 It has been shown to reduce apoptosis of oligodendrocytes and promote remyelination in an animal model of spinal cord injury.24 Therefore, we examined effects of FLA on the OPCs proliferation and differentiation, and spatial working memory impairment in CPZ-exposed mice in the second experiment of this study.
Results CPZ administration upregulated the expression of cell cycle related genes To evaluate the effect of CPZ on cell cycle activity, we tested the expression of genes related to cell cycle in the PFC following CPZ administration for 5 weeks (timeline of experiment procedures are illustrated in Fig. 1). The Mouse Cell Cycle RT2 ProfilerTM PCR Arrays containing 86 probe sets that both positively and negatively regulate the cell cycle, the transitions between each of the phases, DNA replication, checkpoints and arrest were used. Of those, only probe sets with a difference of 1.2 folds or above in expression levels, and a p-value less than
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0.05 between control and CPZ groups were used for further analysis. Among 86 gene transcripts, 16 genes were upregulated and 1 gene was down regulated in the CPZ treated group compared to the control group. Literally, the upregulated cell cycle related gene expression in the PFC included cyclins A2, D1, and F, CDK5rap1(CDK5 regulatory subunit associated protein 1), cdc20, cdc25a, cdc7,CKS1b, E2f2, MCM2, MCM4, Hus1, Chek1, Didt3, p53, and SLFN1. The only downregulated gene was Mre11a (Table 1).
Cuprizone inhibited cell cycle exit Cell cycle exit is a prerequisite for oligodendrocyte differentiation. It is known that multipotential precursor cells in SVZ have the ability to differentiate into neurons, astrocytes, and oligodendrocytes.25 In the present study, we calculated the cell cycle exit index in the anterior SVZ using the antibody against BrdU to label a cohort of cells in S-phase, and the antibody against Ki-67 to label proliferating cells throughout the phases of the cell cycle except for G0. The BrdUC/Ki-67Cdouble labeled cells measured the proportion of cycling cells, whereas the BrdUC /Ki-67¡ cells indicated those that had exited from the cell cycle. The cell cycle exit index was calculated by dividing the number of BrdUC/Ki-67¡cells by the total population of BrdUC cells according the previous reports.26,27 Our result showed that CPZ administration caused a significant increase in the number of BrdUC/Ki67C cells (cycling cells) and decrease in BrdUC/Ki67¡ cells in the anterior SVZ compared to control group (Fig. 2), indicating that CPZ inhibited cell cycle exit of multi-potential precursor cells in the anterior SVZ. There was no difference in the numbers of BrdUC or Ki67C between control or CPZ groups.
Effect of FLA on CPZ-induced OPC proliferation and demylination CPZ-exposure for 5 weeks dramatically increased the number of NG2 positive cells (F (3, 22) D 26.87, P < 0.001, vs. the
Table 1. PCR array results following CPZ administration. Symbol Ccna2 Ccnd1 Ccnf Cdk5rap1 Cdc20 Cdc25a Cdc7 CKS1b E2f2 Mcm2 Mcm4 Hus1 Chek1 Ddit3 Trp53 Slfn1 Mre11a
Fold Change
p value
Description
Gene Name
Cyclin A2 Cyclin D1 Cyclin F CDK5 regulatory subunit associated protein 1 Cell division cycle 20 homolog A Cell division cycle 25 homolog A (S. pombe) Cell division cycle 7 (S. cerevisiae) Cell division cycle 20 homolog (S. cerevisiae) E2F transcription factor 2 Minichromosome maintenance deficient 2 mitotin (S. cerevisiae) Minichromosome maintenance deficient 4 homolog (S. cerevisiae) Hus1 homolog (S. pombe) Checkpoint kinase 1 homolog (S. pombe) DNA-damage inducible transcript 3 Transformation related protein 53 Schlafen 1 Meiotic recombination 11 homolog A
AA408589, Ccn-1, Ccn1, Ccna, CycA2, Cyca AI327039, CycD1, Cyl-1, PRAD1, bcl-1, cD1 CycF, Fbxo1 2310066P17Rik 2310042N09Rik, C87100, p55CDC D9Ertd393e AI597260, Cdc7l1, muCdc7 2310042N09Rik, C87100, p55CDC 9230110J10, E130207A07 AL033361, C80350, Mcmd, P1, p1.m
1.56 1.38 1.37 1.53 1.36 1.30 1.34 1.33 1.48 1.38
0.026 0.038 0.044 0.0066 0.0419 0.046 0.038 0.013 0.0072 0.0063
19G, AI325074, AU045576, Cdc21, Mcmd4, mKIAA4003, mcdc21 mHus1 C85740, Chk1, rad27 CHOP-10, CHOP10, chop, gadd153 Tp53, bbl, bfy, bhy, p44, p53 AV316259 Mre11, Mre11b
1.34
0.042
1.44 1.96 1.59 1.42 4.30 ¡1.20
0.0014 0.041 0.0032 0.0055 0.026 0.022
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Figure 1. Timeline of experimental procedures. (A) C57BL/6 mice were given 0.2% cuprizone-containing or normal diet for 5 weeks. On the last day, 3 mice from each group were sacrificed and their brains were used for microarray analysis. The remaining 5 of each group were intraperitoneally injected with a BrdU solution (50mg/kg) every 2 hours for 6 times on the same day. Twelve hours after the last injection, mice were sacrificed and their brain tissue were used for immunochemical staining. (B) C57BL/6 mice were given 0.2% cuprizone-containing or normal diet for 5 weeks. During the 5th week, FLA (5 mg/kg, ip) or vehicle (2.28% DMSO in saline) was administrated once daily. Mice were subjected to Y maze test in the subsequent 2 d. Twenty-four 24 hours after behavioral test, mice were sacrificed and their brain tissue was used for immunohistochemical staining.
control group), indicating an accelerated proliferation of the OPCs following CPZ administration. But, this increase was significantly alleviated in the mice co-administered with CPZ and FLA (P < 0.001, vs. the CPZ group), although FLA alone had no effect on the number of NG2 positive cells (Fig. 3). As expected, CPZ exposure caused evident myelin sheath disruption in the PFC. The optical density analysis of MBP (myelin basic protein) immune reactivity on the brain tissue sections revealed that FLA alone did not change MBP expression. But, administration of FLA during the last week of the 5-week CPZ exposure significantly ameliorated CPZ-induced reduction of MBP expression (P < 0.001, vs. the CPZ group) in the PFC (Fig. 4) and hippocampus (Supplement Figs S1&S2). Effect of FLA onCPZ-induced activation of astrocytes and microglia Immunohistochemical staining results revealed a robust increase in the number of glial fibrillary acidic protein (GFAP)positive cells in mice exposed to CPZ, indicating the activation of astrocytes [F(3,34) D 71.2, P < 0.001, vs. the control group] (Fig. 5).The number of Iba-1 positive cells was also increased in CPZ-treated mice [F(3,34) D 35.65, P < 0.001, vs. the control
group](Fig. 6). Importantly, administration of FLA significantly reduced the CPZ-induced astrocyte and microglial activation in PFC (GFAP: P < 0.001 vs. the CPZ group; Iba-1: P < 0.01 vs. the CPZ group) (Figs. 5 and 6) and the hippocampus (Supplement Figs. S3 & S4). Effects of FLA on CPZ-induced spatial working memory deficits The Y maze test showed that CPZ-exposure for 5 weeks decreased the spontaneous alternation compared to the control group[F (3,36) D 7.87, P < 0.05, vs. the control group). Intraperitoneal injection of FLA, however, significantly alleviated this CPZ-induced reduction of spontaneous alternation (P < 0.05, vs. the CPZ group), while FLA alone had no effect (Fig. 7).
Discussion Cuprizone induced cell cycle activation Cuprizone is a copper-chelating agent and has been shown to increase proliferation of OPCs, astrocytes, microglia and inhibit
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Figure 2. Effects of CPZ administration on cell cycle exit index. (A) Representative micrographs showing brain cells labeled by anti-BrdU and anti-Ki-67. Arrowheads point to BrdUC/Ki67¡ cells (cells that were BrdU positive but immunonegative for Ki-67). CC, corpus callosum; Str, striatum; lv, lateral ventricle. (B) Quantitative data showing proportions of cycling cells (quotient of BrdUC/Ki67C cells over the total number of BrdUC cells) in the anterior SVZ of mice. (C) Histogram representing the cell cycle exit index, which was calculated by dividing the number of BrdUC/Ki67¡ cells by the total number of BrdUC cells.
oligodendrocyte differentiation. Microarray assay in this study revealed that cyclins A2, D1, F, and CDK5rap1 were up-regulated in CPZ-exposed mice compared to the normal controls (Table 1). These findings are in line with the previous report of increased expression of cyclins D3 and E in CPZ-treated mice.28 These genes function as cell cycle positive regulators by promoting progress of the cell cycle to the next phase. For example, cyclin A2 joined the Cdk2 complex thus regulated the cell cycle G1/S transition and DNA synthesis.29 Moreover, knockout of Cdk2 promoted cell cycle exit and enhanced oligodendrocyte differentiation.30 CPZ-exposure also upregulated gene expression of cell cycle positive regulators, including cyclins, CDK5rap1, cdc20, cdc25a, cdc7, CKS1B, E2f2, MCM2, and MCM4. These effects may contribute to observed proliferation of OPCs, astrocyte and microglia in the CPZ-exposed mice. Indeed, Cdc20, cdc25a, cdc7 and CKS1B have been found to regulate the G1/S transition by releasing and activating the cyclin/Cdk complex and stimulating cell division.31,32 E2F family was shown to play a crucial role in controlling cell cycle and expression of E2F proteins during the G1/S transition.33 MCMs are essential factors for eukaryotic DNA replication.Of them, MCM2 is highly expressed in both proliferative and transformational phase and is considered a proliferative marker.34,35 Of the other changed genes in CPZ-treated mice, HUS1 and Chk1 encode components of cell cycle checkpoint complex able to cause cell cycle arrest in response to DNA repair and cell death thus preventing damaged cells from progressing through the cell cycle.36-38 Ddit 3 (also referred to Gadd153, Chop) is a DNA damage-inducible transcript 3 protein. It is able to induce cell cycle arrest and plays an essential role in
apoptosis and inflammation.39 The transcription factor p53 is able to induce cell cycle arrest and apoptosis in several cell types. Previous studies have demonstrated the expression of p53 was up-regulated during the first 2 to 3 weeks of CPZ exposure and this increase correlated with oligodendrocyte apoptosis and microglial activation. Moreover, p53 knock out or pharmacological inhibition of p53 decreased the extent of demyelination.40,41 SLFN1 has been shown to cause growth inhibition and a G1 cell cycle arrest in murine fibroblasts, while other reports suggested that SLFN1 was associated with immune response, but without anti-proliferative activities.42 The only one down-regulated gene in CPZ-exposed mice was Mre11a. Mre11 is believed to maintain genome stability and is essential for DNA repair by homologous recombination, in addition to its function as an important locus of checkpoint inhibition and stress relief.43,44 Thus, down regulation of Mre11a expression implies that CPZ-exposure may impair negative cell cycle regulation. Cuprizone inhibited cell cycle exit OPCs originate from SVZ, migrate into injury areas and finally differentiate into mature oligodendrocytesduring demyelination.45-47 Our results revealed a significant increase in number of BrdUC/Ki67C cells and reduction in BrdUC/ Ki67¡ cells in the anterior SVZ after CPZ exposure for 5 weeks, indicating that the treatment led to a failure of potential precursor cells exiting from cell cycle. This action may contribute to the CPZ-induced oligodendrocyte decrease due to cell death and deficient differentiation of OPCs. This view is in line with the report that maintaining a persistent state of mitotic cells in the cell cycle period led
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Figure 3. FLA alleviated the CPZ-induced increase in NG2 positive cells in PFC. Representative micrographs showed NG2 positive cells in the PFC of mouse from each group. CPZ-exposed mouse seemed to have the densest NG2 positive cells in the PFC among all groups. The bar equals 50 mm. The statistical analysis of quantitative data indicated that CPZ-exposure increased number of NG2 positive cells in the PFC as compared to other groups. No difference was found between any other 2 groups. Data (obtained from 4 animals in each group) were expressed as the mean § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
to apoptosis.17,18 Therefore, the cuprizone mouse model is suitable for us to explore the neurodevelopmental issues relevant to schizophrenia for which a neurodevelopmental hypothesis has been proposed and extensively studied.48 Flavopiridol alleviated NG2 cell proliferation and myelin breakdown induced by CPZ Previous studies have shown that the oligodendrocyte precursor NG2 cells originate from the SVZ and increase following demyelination.49-50 NG2 cells can keep self-renewing in cell cycle or exit from cell cycle to differentiate into mature oligodendrocytes or transform to astrocytes.51 In this study, CPZtreated mice showed many more NG2 cells, but decreased mature oligodendrocytes and MBP expression. This concurrent increase in NG2 cells and decrease in mature oligodendrocytes suggest a possibility that NG2 cells failed to differentiate into myelin forming cells in CPZ-exposed mice, although increased
NG2 cells may be viewed as a compensatory reaction of the body to oligodendrocyte loss and demyelination as explained in previous studies.52-54 In support of this view, CPZ administration significantly increased the number of BrdUC/NG2Ccells in the medial PFC, indicating that NG2 cells kept proliferating and failed to exit from cell cycle for differentiation in the CPZexposed mice.12 More significantly, the CDK inhibitor FLA administration alleviated the CPZ-induced increase in NG2 cells and myelin breakdown in the present study. This is in line with the findings in a previous study that FLA reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus after traumatic brain injury, while it reduced astroglial scar formation and microglial activation.21 Although we did not measure effects of FLA on cell cycle related genes in this study, the CPZ-induced cell cycle activation presented here and the ability of cell cycle inhibition of FLA shown in previous studies21,24,55 argue for a suggestion that FLA exerted its protective effects in the CPZ-exposed mice via its ability of cell cycle
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Figure 4. FLA alleviated the CPZ-induced demyelination in PFC. Representative micrographs showed MBP-immunostaining in the PFC of mouse from each group. CPZ-exposed mouse showed evident myelin breakdown in the PFC, but it was not so obvious in the mouse co-administer CPZ and FLA. The bar equals 50 mm. The optical density analysis confirmed the lowest optical density of MBP-immunostaining in the CPZ group. Differences between CPZCFLA and CON or FLA were also significant. Data (obtained from 4 animals in each group) were expressed as the means § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
inhibition. This suggestion encourages us to test the possible therapeutic effects of FLA on MS, a white matter disease for which no curative avenues are available now. Flavopiridol alleviated CPZ-induced activation of astrocytes and microglia Although the increased astrocytes and microglia in CPZtreated mice may be viewed as the responsive actions of the body to demyelination and oligodendrocyte loss, the activated astrocytes and microglia in turn may play certain roles in OPC proliferation, migration, and differentiation. They release pro-inflammatory cytokines and growth factors in the local sites. For example, astrocytes were shown to produce fibroblast growth factor 2 (FGF2) and plateletderived growth factor A (PDGF-A) during CPZ-induced demyelination.3,4,5 These growth factors promoted OPCs proliferation and inhibited OPCs differentiationby activating cell cycle.56 FGF2 even stimulated post-mitotic oligodendrocytes to
reenter the S-phase of the cell cycle, while FGF2 knockout enhanced OPCs differentiation and promoted oligodendrocyte repopulation in the demyelinated area following CPZ insult.57,58 Moreover, activation of microglia by CPZ led to increased growth factor production and secretion of pro-inflammatory cytokines such as TNFa (tumor necrosis factor a) and IFNg(interferon-g).3-5,59,60 The latter (IFN-g)was shown to inhibit cell cycle exit in oligodendrocyte progenitor cells and attenuate MBP expression.61 Taken together, these previous data point to an alternative view that activated astrocytes and microglia in brains of CPZ-treated mice may contribute to or exacerbate mature oligodendrocyte loss and myelin breakdown as shown in this study. It implies that antiinflammatory treatment or inhibiting astrocyte and microglia activation may be of help in improving the CPZinduced white matter damage. Indeed, minocycline, a tetracycline antibiotic with strong anti-inflammatory and antioxidant actions, has been proved to ameliorate the demyelinating effect of CPZ and increase remyelination by
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Figure 5. FLA alleviated the CPZ-induced increase in GFAP positive cells in PFC. Representative micrographs showed GFAP positive cells in the PFC of mouse from each group. CPZ-exposed mouse showed the densest GFAP positive cells in the PFC, but it was not so in the mouse co-administer CPZ and FLA. The bar equals 50 mm. The statistical analysis of the quantitative data confirmed the densest GFAP positive cells in the CPZ group. Differences between CPZCFLA and anyone of the other groups were also significant. Data (obtained from 4 animals in each group) were expressed as the means § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
inhibiting microglia activation and blocking inflammatory responses.62 Likewise, FLA in this study alleviated the astrocytes and microglia proliferation in CPZ-treated mice, in addition to ameliorating oligodendrocyte loss and myelin breakdown. The mechanism for this action of FLA may be related to its ability of cell cycle inhibition as discussed above. Flavopiridol alleviated the CPZ-induced spatial working memory impairment In accordance with previous studies,6,53,54 CPZ-exposed mice showed spatial working memory deficit in the present study. More significantly, FLA administered during the last week of CPZ exposure reversed the CPZ-induced reduction of spontaneous alternation of mice in the Y maze, in addition to ameliorating the CPZ-induced neuropathology as presented above.
Altogether, these data not only added supports for the claim that demyelination contributes to the cognitive and neuropsychiatric symptoms shown in MS and schizophrenic patients,63-65 but also provided a potential avenue for the treatment of these brain disorders. In conclusion, the results of the present study suggest that CPZ-induced neuropathological changes, including mature oligodendrocyte loss, myelin breakdown, and increases in NG2 cells, astrocytes and microglia, may involve in dysregulation of cell cycle related genes. FLA administration effectively alleviated the CPZ-induced neuropathological changes and spatial working memory deficit. The therapeutic effects of FLA on CPZ-exposed mice may be related to its ability of cell cycle inhibition. Further studies on this emerging research field may lead to a new effective avenue for the treatment of MS and schizophrenia, for which curative avenues are needed.
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Figure 6. FLA alleviated the CPZ-induced increase inIba-1positive cells in PFC. Representative micrographs showed Iba-1 positive cells in the PFCof mouse from each group. CPZ-exposed mouse showed the densest Iba-1positive cells in the PFC, but it was not so in the mouse co-administer CPZ and FLA. The bar equals 50 mm. The statistical analysis of the quantitative data confirmed the densest Iba-1 positive cells in the CPZ group. Differences between CPZCFLA and anyone of the other groups were also significant. Data (obtained from 4 animals in each group) were expressed as the mean § SEM. p < 0.001 compared with the control group, ##p < 0.01 compared with the CPZ group.
Materials and methods Animals
Figure 7. FLA alleviated the CPZ-induced spatial working memory impairment in mice. CPZ-exposed mice showed a lowest percentage of spontaneous alternation among all groups. Differences between CPZ C FLA and anyone of the other groups were also significant. Data (obtained from10 animals in each group) were expressed as mean § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
Male C57BL/6 mice (6–8 weeks old) were purchased from Vital River (Beijing, China). The animals were housed in groups of 4 to 5 in clear plastic cages with free accesses to water and food in a room with a climate-controlled environment on a standard 12 h light/dark cycle (lights on 8 a.m., lights off 8 p.m.). The mice were acclimated to the housing conditions and handled for 3 d before experiments. All experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The experimental procedures were approved by the Local Committee on Animal Care and Use. Cell cycle gene expression microarray Male C57BL/6 mice (6–8 weeks old) weregiven 0.2%(by weight) CPZ (Sigma-Aldrich, St. Louis, MO) mixed into a standard
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powdered rodent chow for 5 weeks, during which period agematched mice received the standard chow without CPZ as reported previously.6,7 Animals were sacrificed under a deep anesthesia at the end of 5th week. Total RNA in the PFC was isolated using Trizol (Invitrogen, Carlsbad, CA) and cleaned up with RNeasy Mini kit (Invitrogen). RNA from each group (n D 3) was pooled in an equimolar amount prior to hybridization with Mouse Cell Cycle RT2 ProfilerTM PCR Array (Qiagen GmbH, Hilton, GER; SABiosciences, Frederick, MD), which contains probes for detecting 86 transcripts. RNA isolation, assessing RNA yield and quality, DNA treatment, RNA cleanup, first strand cDNA synthesis, performing real-time PCR and data analysis were accomplished using standard procedures at Kangcheng Biotechnology Company (Shanghai, China).
Cell cycle exit index assay On the last day of CPZ-exposure and the same day for normal controls (as illustrated in Fig. 1A), mice were injected 5Bromo-20 -deoxyuridine (BrdU,50 mg/kg, intraperitoneal injection) (Sigma-Aldrich) once every 2 hrs in 12 hrs. Twelve hrs after the last injection, mice were deeply anesthetized with sodium pentobarbital and perfused through the ascending aorta with 0.01 M phosphate buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in PBS. The brains were removed and post-fixated overnight in the same fixative, followed by dehydration in 30% sucrose. Serial coronal sections (25 mm) of the brains were used for immunofluorescent staining. Free floating sections were pretreated with 10 mM sodium citrate buffer (pH 6) at 45 C for 30 min and transferred to 2N HCl for 30 min at 37 C. Then the sections were rinsed in 0.1 M boric acid (pH 8.5) for 10 min. After washing 3 times with PBS, sections were incubated for one hr at room temperature(22 C) with a blocking solution composed of 0.3% Triton X-100 and 4% BSA in PBS before they were incubatedwith rat monoclonal anti-BrdU (1:100; Abcam, UK) and rabbit polyclonal anti-Ki67 primary antibody (1:100; Millipore, Temecula, USA) in blocking solution for 24 hrs at 4 C. These sections were then washed and incubated with fluorochrome-conjugated secondary [Alexa Fluor 488 goat anti-rat IgG (HCL), Alexa Fluor 568 goat antirabbit IgG(HCL), 1:300; Life, Eugene, USA] for 2hrs at room temperature. Finally, tissue sections were washed in PBS and mounted using a fluorescent medium of DAPI (ZSGB-BIO, Beijing, China).
FLA administration and behavioral test Forty male C57BL/6 mice were divided into 4 groups: control (CNT), cuprizone (CPZ), Flavopiridol (FLA; Selleck, Shanghai, China) group and CPZCFLA groups. As illustrated in Fig. 1B, mice were given 0.2% CPZ-containing or normal diet for 5 weeks. During the5thweek, mice were subjected to intraperitoneal injection of FLA (5 mg/kg) or vehicle once daily for7 days. FLA was dissolved completely in DMSO. The concentrated solution was then diluted in sterile saline to a final concentration of 2.28% DMSO. Twenty-four hrs after the last FLA injection, animals were subjected to Y maze test.
Y maze test is a simple procedure measuring spatial working memory of rodents. The maze consisted of 3 arms of equal length(40 cm long £ 8 cm wide £ 20 cm high) with a 1208 angle between each 2 adjacent arms. Mouse was placed at the end of one arm of the maze and allowed to explore the instrument for 8 min. The total number and series of arm entries were recorded. Alternation was defined as entries into all 3 arms on consecutive occasions (e.g., ABCAB). The alternation rate was calculated using the following formula: Alternation rate (%) D 100 £ number of alternation / (number of total arm entries ¡ 2).
Immunohistochemical staining Serial coronal sections (20 mm) of the brains were used for immunohistochemical staining. Briefly, free floating sections were pretreated with 0.5% hydrogen peroxide in methanol for 20 min, then washed with PBS, and incubated for 1 hr at 22 C with a blocking solution composed of 1% Triton X–100 and 5% normal rabbit serum in PBS. Sections were then incubated with a primary antibody to MBP (1 : 200; Boster, Wuhan, China), GFAP (1 : 200; Boster), Iba-1(1 : 300, Wako, Japan), or NG2 (1 : 200; Millipore) in the blocking solution overnight. Antibody to NG2 was used to identify oligodendrocyte precursor cells and that to MBP was used to label myelin sheath. After rinsing in PBS, sections were incubated in biotinylated secondary antisera (1 : 200) for 2 hrs at 22 C. Following PBS rinses, the sections were incubated in an avidin–biotin–horseradish peroxidase complex (ABC; Boster) for 1 hr at 22 C. Finally, the antigen-antibody complexes were visualized using 0.025% 3, 3-diamino- benzidine (DAB, Sigma-Aldrich) as the chromogen.
Image analysis For quantitative analysis, 4 mice (brains) of each group were included. The examined brain regions included the hippocampus, PFC, and anterior SVZ. Three images (tissue sections) were digitally recorded for each region by Olympus BX–51 microscope equipped with a digital capture system. The ImagePro Plus software (Media Cybernetics, Inc., Silver Spring, MD) was used for analyzing the images. The same conditions were maintained during measurements of all the selected brain sections, and the values were read out automatically by the software. The data of astrocyte and microglia positive cells were expressed as the number/mm2. The data of optical density of MBP-immunostaining were expressed as integrated optical density (IOD), which equals to area times average optical density.
Statistical analysis The Student t-test was performed for the data of the first experiment. Two-way analysis of variance (ANOVA) was performed for the data of the second experiment followed by the Fisherprotected least-significant difference test for post hoc comparisons if appropriate. All data are given as the mean § SEM. P value < 0.05 was considered statistically significant.
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Abbreviations BrdU CDK CPZ FGF FLA GFAP IFN-g MBP MS OPCs PDGF PFC ROS SVZ TNFa
5-Bromo-20 -deoxyuridine cyclin-dependent kinase cuprizone fibroblast growth factor flavopiridol glial fibrillary acidic protein interferon-g myelin basic protein multiple sclerosis oligodendrocyte precursor cells Platelet-derived growth factor prefrontal cortex reactive oxygen species subventricular zone tumor necrosis factor a
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments We thank Dr. Gong Jing-Bo, professor Qian Ling-Jia for excellent technical support.
Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81473193).
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REPORT
Cyclin-dependent kinase inhibitor flavopiridol promotes remyelination in a cuprizone induced demyelination model Guiyun Mia, Yunyun Gaob, Shuai Liua, Enmao Yea, Yanyan Lia, Xiao Jina, Hongju Yanga, and Zheng Yanga a
Beijing Institute of Basic Medical Sciences, Haidian District, Beijing, China; bThe 89 Hospital of PLA, WeiFang City Shandong Province, China
ABSTRACT
ARTICLE HISTORY
The cuprizone (CPZ) model has been widely used for the studies of de-and remyelination. The CPZexposed mice show oligodendrocyte precursor cells (OPCs) increase and mature oligodendrocytes decrease, suggesting an imbalance between proliferation and differentiation of OPCs. In the first experiment of this study, we examined the expression of cell cycle related genes in brains of mice following CPZ administration for 5 weeks by means of microarray assay. In addition, we performed a double labeling of BrdU and Ki-67 to calculate cell cycle exit index in the mice. Our results showed that CPZ administration up-regulated the expression of 16 cell cycle related genes, but down-regulated the expression of only one in the prefrontal cortex (PFC) of mice compared to control group. The treatment inhibited potential precursor cells exit from cell cycle. In the second experiment, we evaluated effects of a CDK inhibitor flavopiridol (FLA) on CPZ-induced neuropathological changes and spatial working memory impairment in mice.FLA treatment for one week effectively attenuated the CPZ-induced increases in NG2 positive cells, microglia and astrocytes, alleviated the concurrent mature oligodendrocyte loss and myelin breakdown, and improved spatial working memory deficit in the CPZ-exposed mice. These results suggest that CPZ-induced neuropathological changes involve in dysregulation of cell cycle related genes. The therapeutic effects of FLA on CPZ-exposed mice may be related to its ability of cell cycle inhibition.
Received 11 May 2016 Revised 19 July 2016 Accepted 31 July 2016 KEYWORDS
astrocyte; cell cycle; cognition; cuprizone; flavopiridol; microglia; oligodendrocyte
Introduction The main function of oligodendrocytes is to provide support and insulation to axons. Demyelination impairs the neuron signal transmission and causes dysfunctions in sensation, movement, and cognition. Malfunction of oligodendrocyte development and demyelination have been considered as a pathological substrate for cognitive dysfunction inmultiple sclerosis (MS) and schizophrenia.1,2 Therefore, identifying and manipulating the factors regulating oligodendrocyteprecursor cells (OPCs) proliferation and differentiation are crucial to the elucidation of pathological mechanisms of demyelination diseases. The cuprizone (CPZ; biscyclohexanone oxalyldihydrazone) animal model has been widely used for the study of de-and remyelination. When given to young adult C57BL/6 mice (0.2 % in the diet), CPZ induces apoptosis of mature oligodendrocytes and demyelination accompanied by spatial working memory impairment in mice.3-6 Obvious demyelination was observed in the corpus callosum and the prefrontal cortex (PFC), external capsule, and hippocampus.7 The PFC and hippocampus are important brain regions for a large range of cognitive functions such as working memory. It is accepted that oxidative stress involves in the oligodendrocyte degeneration and myelin breakdown induced by CPZ, as evidenced by mitochondrial
dysfunction parameters, such as mitochondrial swelling, production of ROS (reactive oxygen species), and collapse of the mitochondrial membrane potential, in CPZ-treated mice.3-5,8 Moreover, certain antioxidants such as melatonin,9 quetiapine,10 and resveratrol,11 showed protective effects on the CPZ-induced oligodendrocyte loss and myelin breakdown in recent animal studies. Interestingly, oligodendrocyte loss and myelin breakdown were accompanied by increase in the number of OPCs labeled as NG2C (chondroitin sulfate proteoglycan) and PDGFRaC (platelet-derived growth factor a) cells in CPZexposed mice,12-14 suggesting the existence of an aberrant cell cycle regulation following CPZ administration. Cell cycle is divided into G0 (resting phase), G1 (preparing phase), S (DNA replication), G2 (gap between S and M), and M (cell division) phases. During development, after a limited number of dividing, OPCs need to exit from cell cycle and start the transcription of myelin related genes and finally differentiate into post-mitotic, mature oligodendrocytes.15,16 Cell cycle activation and failure of exit from cell cycle can keep the OPCs at a persistence of a proliferative state and lead to the differentiation deficit of OPCs.17 In addition, failure of exit from cell cycle can lead to oligodendrocyte apoptosis and hinder oligodendrocyte development and myelination.18,19 Based on the
CONTACT Zheng Yang [email protected]; Hongju Yang [email protected] Beijing Institute of Basic Medical Sciences, 27 Tai-Ping Road, Haidian District, Beijing, 100850, China. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis
CELL CYCLE
above knowledge and previous findings, we hypothesized that CPZ administration would change the expression of cell cycle related genes in subjects. To test this hypothesis, we conducted a microarray experiment to analyze the gene expression in PFC of mice after CPZ administration. In addition, we performed a double labeling of 5-Bromo-20 -deoxyuridine (BrdU) and Ki-67 (a marker of proliferation) to calculate the cell cycle exit index in the anterior subventricular zone (SVZ),where multi-potential precursor cells proliferate, to test whether the proliferation of mitotic cells was due to the failure of exit from cell cycle in the CPZ-exposed mice. It is known that the cyclin/CDK (cyclin-dependent kinase) complex functions as a regulatory subunit whose activity is required for cell cycle G1/Stransition and CDK inhibitors show neuroprotective effects in animal models by regulating the progression of the G1/S phase and the withdrawal from cell cycle.20-22 Flavopiridol(FLA) is a flavonoid alkaloid that blocks the activity of cyclin /CDK, either by forming an inactive complex or by acting as a competitive ligand for CDKs.23 It has been shown to reduce apoptosis of oligodendrocytes and promote remyelination in an animal model of spinal cord injury.24 Therefore, we examined effects of FLA on the OPCs proliferation and differentiation, and spatial working memory impairment in CPZ-exposed mice in the second experiment of this study.
Results CPZ administration upregulated the expression of cell cycle related genes To evaluate the effect of CPZ on cell cycle activity, we tested the expression of genes related to cell cycle in the PFC following CPZ administration for 5 weeks (timeline of experiment procedures are illustrated in Fig. 1). The Mouse Cell Cycle RT2 ProfilerTM PCR Arrays containing 86 probe sets that both positively and negatively regulate the cell cycle, the transitions between each of the phases, DNA replication, checkpoints and arrest were used. Of those, only probe sets with a difference of 1.2 folds or above in expression levels, and a p-value less than
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0.05 between control and CPZ groups were used for further analysis. Among 86 gene transcripts, 16 genes were upregulated and 1 gene was down regulated in the CPZ treated group compared to the control group. Literally, the upregulated cell cycle related gene expression in the PFC included cyclins A2, D1, and F, CDK5rap1(CDK5 regulatory subunit associated protein 1), cdc20, cdc25a, cdc7,CKS1b, E2f2, MCM2, MCM4, Hus1, Chek1, Didt3, p53, and SLFN1. The only downregulated gene was Mre11a (Table 1).
Cuprizone inhibited cell cycle exit Cell cycle exit is a prerequisite for oligodendrocyte differentiation. It is known that multipotential precursor cells in SVZ have the ability to differentiate into neurons, astrocytes, and oligodendrocytes.25 In the present study, we calculated the cell cycle exit index in the anterior SVZ using the antibody against BrdU to label a cohort of cells in S-phase, and the antibody against Ki-67 to label proliferating cells throughout the phases of the cell cycle except for G0. The BrdUC/Ki-67Cdouble labeled cells measured the proportion of cycling cells, whereas the BrdUC /Ki-67¡ cells indicated those that had exited from the cell cycle. The cell cycle exit index was calculated by dividing the number of BrdUC/Ki-67¡cells by the total population of BrdUC cells according the previous reports.26,27 Our result showed that CPZ administration caused a significant increase in the number of BrdUC/Ki67C cells (cycling cells) and decrease in BrdUC/Ki67¡ cells in the anterior SVZ compared to control group (Fig. 2), indicating that CPZ inhibited cell cycle exit of multi-potential precursor cells in the anterior SVZ. There was no difference in the numbers of BrdUC or Ki67C between control or CPZ groups.
Effect of FLA on CPZ-induced OPC proliferation and demylination CPZ-exposure for 5 weeks dramatically increased the number of NG2 positive cells (F (3, 22) D 26.87, P < 0.001, vs. the
Table 1. PCR array results following CPZ administration. Symbol Ccna2 Ccnd1 Ccnf Cdk5rap1 Cdc20 Cdc25a Cdc7 CKS1b E2f2 Mcm2 Mcm4 Hus1 Chek1 Ddit3 Trp53 Slfn1 Mre11a
Fold Change
p value
Description
Gene Name
Cyclin A2 Cyclin D1 Cyclin F CDK5 regulatory subunit associated protein 1 Cell division cycle 20 homolog A Cell division cycle 25 homolog A (S. pombe) Cell division cycle 7 (S. cerevisiae) Cell division cycle 20 homolog (S. cerevisiae) E2F transcription factor 2 Minichromosome maintenance deficient 2 mitotin (S. cerevisiae) Minichromosome maintenance deficient 4 homolog (S. cerevisiae) Hus1 homolog (S. pombe) Checkpoint kinase 1 homolog (S. pombe) DNA-damage inducible transcript 3 Transformation related protein 53 Schlafen 1 Meiotic recombination 11 homolog A
AA408589, Ccn-1, Ccn1, Ccna, CycA2, Cyca AI327039, CycD1, Cyl-1, PRAD1, bcl-1, cD1 CycF, Fbxo1 2310066P17Rik 2310042N09Rik, C87100, p55CDC D9Ertd393e AI597260, Cdc7l1, muCdc7 2310042N09Rik, C87100, p55CDC 9230110J10, E130207A07 AL033361, C80350, Mcmd, P1, p1.m
1.56 1.38 1.37 1.53 1.36 1.30 1.34 1.33 1.48 1.38
0.026 0.038 0.044 0.0066 0.0419 0.046 0.038 0.013 0.0072 0.0063
19G, AI325074, AU045576, Cdc21, Mcmd4, mKIAA4003, mcdc21 mHus1 C85740, Chk1, rad27 CHOP-10, CHOP10, chop, gadd153 Tp53, bbl, bfy, bhy, p44, p53 AV316259 Mre11, Mre11b
1.34
0.042
1.44 1.96 1.59 1.42 4.30 ¡1.20
0.0014 0.041 0.0032 0.0055 0.026 0.022
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Figure 1. Timeline of experimental procedures. (A) C57BL/6 mice were given 0.2% cuprizone-containing or normal diet for 5 weeks. On the last day, 3 mice from each group were sacrificed and their brains were used for microarray analysis. The remaining 5 of each group were intraperitoneally injected with a BrdU solution (50mg/kg) every 2 hours for 6 times on the same day. Twelve hours after the last injection, mice were sacrificed and their brain tissue were used for immunochemical staining. (B) C57BL/6 mice were given 0.2% cuprizone-containing or normal diet for 5 weeks. During the 5th week, FLA (5 mg/kg, ip) or vehicle (2.28% DMSO in saline) was administrated once daily. Mice were subjected to Y maze test in the subsequent 2 d. Twenty-four 24 hours after behavioral test, mice were sacrificed and their brain tissue was used for immunohistochemical staining.
control group), indicating an accelerated proliferation of the OPCs following CPZ administration. But, this increase was significantly alleviated in the mice co-administered with CPZ and FLA (P < 0.001, vs. the CPZ group), although FLA alone had no effect on the number of NG2 positive cells (Fig. 3). As expected, CPZ exposure caused evident myelin sheath disruption in the PFC. The optical density analysis of MBP (myelin basic protein) immune reactivity on the brain tissue sections revealed that FLA alone did not change MBP expression. But, administration of FLA during the last week of the 5-week CPZ exposure significantly ameliorated CPZ-induced reduction of MBP expression (P < 0.001, vs. the CPZ group) in the PFC (Fig. 4) and hippocampus (Supplement Figs S1&S2). Effect of FLA onCPZ-induced activation of astrocytes and microglia Immunohistochemical staining results revealed a robust increase in the number of glial fibrillary acidic protein (GFAP)positive cells in mice exposed to CPZ, indicating the activation of astrocytes [F(3,34) D 71.2, P < 0.001, vs. the control group] (Fig. 5).The number of Iba-1 positive cells was also increased in CPZ-treated mice [F(3,34) D 35.65, P < 0.001, vs. the control
group](Fig. 6). Importantly, administration of FLA significantly reduced the CPZ-induced astrocyte and microglial activation in PFC (GFAP: P < 0.001 vs. the CPZ group; Iba-1: P < 0.01 vs. the CPZ group) (Figs. 5 and 6) and the hippocampus (Supplement Figs. S3 & S4). Effects of FLA on CPZ-induced spatial working memory deficits The Y maze test showed that CPZ-exposure for 5 weeks decreased the spontaneous alternation compared to the control group[F (3,36) D 7.87, P < 0.05, vs. the control group). Intraperitoneal injection of FLA, however, significantly alleviated this CPZ-induced reduction of spontaneous alternation (P < 0.05, vs. the CPZ group), while FLA alone had no effect (Fig. 7).
Discussion Cuprizone induced cell cycle activation Cuprizone is a copper-chelating agent and has been shown to increase proliferation of OPCs, astrocytes, microglia and inhibit
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Figure 2. Effects of CPZ administration on cell cycle exit index. (A) Representative micrographs showing brain cells labeled by anti-BrdU and anti-Ki-67. Arrowheads point to BrdUC/Ki67¡ cells (cells that were BrdU positive but immunonegative for Ki-67). CC, corpus callosum; Str, striatum; lv, lateral ventricle. (B) Quantitative data showing proportions of cycling cells (quotient of BrdUC/Ki67C cells over the total number of BrdUC cells) in the anterior SVZ of mice. (C) Histogram representing the cell cycle exit index, which was calculated by dividing the number of BrdUC/Ki67¡ cells by the total number of BrdUC cells.
oligodendrocyte differentiation. Microarray assay in this study revealed that cyclins A2, D1, F, and CDK5rap1 were up-regulated in CPZ-exposed mice compared to the normal controls (Table 1). These findings are in line with the previous report of increased expression of cyclins D3 and E in CPZ-treated mice.28 These genes function as cell cycle positive regulators by promoting progress of the cell cycle to the next phase. For example, cyclin A2 joined the Cdk2 complex thus regulated the cell cycle G1/S transition and DNA synthesis.29 Moreover, knockout of Cdk2 promoted cell cycle exit and enhanced oligodendrocyte differentiation.30 CPZ-exposure also upregulated gene expression of cell cycle positive regulators, including cyclins, CDK5rap1, cdc20, cdc25a, cdc7, CKS1B, E2f2, MCM2, and MCM4. These effects may contribute to observed proliferation of OPCs, astrocyte and microglia in the CPZ-exposed mice. Indeed, Cdc20, cdc25a, cdc7 and CKS1B have been found to regulate the G1/S transition by releasing and activating the cyclin/Cdk complex and stimulating cell division.31,32 E2F family was shown to play a crucial role in controlling cell cycle and expression of E2F proteins during the G1/S transition.33 MCMs are essential factors for eukaryotic DNA replication.Of them, MCM2 is highly expressed in both proliferative and transformational phase and is considered a proliferative marker.34,35 Of the other changed genes in CPZ-treated mice, HUS1 and Chk1 encode components of cell cycle checkpoint complex able to cause cell cycle arrest in response to DNA repair and cell death thus preventing damaged cells from progressing through the cell cycle.36-38 Ddit 3 (also referred to Gadd153, Chop) is a DNA damage-inducible transcript 3 protein. It is able to induce cell cycle arrest and plays an essential role in
apoptosis and inflammation.39 The transcription factor p53 is able to induce cell cycle arrest and apoptosis in several cell types. Previous studies have demonstrated the expression of p53 was up-regulated during the first 2 to 3 weeks of CPZ exposure and this increase correlated with oligodendrocyte apoptosis and microglial activation. Moreover, p53 knock out or pharmacological inhibition of p53 decreased the extent of demyelination.40,41 SLFN1 has been shown to cause growth inhibition and a G1 cell cycle arrest in murine fibroblasts, while other reports suggested that SLFN1 was associated with immune response, but without anti-proliferative activities.42 The only one down-regulated gene in CPZ-exposed mice was Mre11a. Mre11 is believed to maintain genome stability and is essential for DNA repair by homologous recombination, in addition to its function as an important locus of checkpoint inhibition and stress relief.43,44 Thus, down regulation of Mre11a expression implies that CPZ-exposure may impair negative cell cycle regulation. Cuprizone inhibited cell cycle exit OPCs originate from SVZ, migrate into injury areas and finally differentiate into mature oligodendrocytesduring demyelination.45-47 Our results revealed a significant increase in number of BrdUC/Ki67C cells and reduction in BrdUC/ Ki67¡ cells in the anterior SVZ after CPZ exposure for 5 weeks, indicating that the treatment led to a failure of potential precursor cells exiting from cell cycle. This action may contribute to the CPZ-induced oligodendrocyte decrease due to cell death and deficient differentiation of OPCs. This view is in line with the report that maintaining a persistent state of mitotic cells in the cell cycle period led
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Figure 3. FLA alleviated the CPZ-induced increase in NG2 positive cells in PFC. Representative micrographs showed NG2 positive cells in the PFC of mouse from each group. CPZ-exposed mouse seemed to have the densest NG2 positive cells in the PFC among all groups. The bar equals 50 mm. The statistical analysis of quantitative data indicated that CPZ-exposure increased number of NG2 positive cells in the PFC as compared to other groups. No difference was found between any other 2 groups. Data (obtained from 4 animals in each group) were expressed as the mean § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
to apoptosis.17,18 Therefore, the cuprizone mouse model is suitable for us to explore the neurodevelopmental issues relevant to schizophrenia for which a neurodevelopmental hypothesis has been proposed and extensively studied.48 Flavopiridol alleviated NG2 cell proliferation and myelin breakdown induced by CPZ Previous studies have shown that the oligodendrocyte precursor NG2 cells originate from the SVZ and increase following demyelination.49-50 NG2 cells can keep self-renewing in cell cycle or exit from cell cycle to differentiate into mature oligodendrocytes or transform to astrocytes.51 In this study, CPZtreated mice showed many more NG2 cells, but decreased mature oligodendrocytes and MBP expression. This concurrent increase in NG2 cells and decrease in mature oligodendrocytes suggest a possibility that NG2 cells failed to differentiate into myelin forming cells in CPZ-exposed mice, although increased
NG2 cells may be viewed as a compensatory reaction of the body to oligodendrocyte loss and demyelination as explained in previous studies.52-54 In support of this view, CPZ administration significantly increased the number of BrdUC/NG2Ccells in the medial PFC, indicating that NG2 cells kept proliferating and failed to exit from cell cycle for differentiation in the CPZexposed mice.12 More significantly, the CDK inhibitor FLA administration alleviated the CPZ-induced increase in NG2 cells and myelin breakdown in the present study. This is in line with the findings in a previous study that FLA reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus after traumatic brain injury, while it reduced astroglial scar formation and microglial activation.21 Although we did not measure effects of FLA on cell cycle related genes in this study, the CPZ-induced cell cycle activation presented here and the ability of cell cycle inhibition of FLA shown in previous studies21,24,55 argue for a suggestion that FLA exerted its protective effects in the CPZ-exposed mice via its ability of cell cycle
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Figure 4. FLA alleviated the CPZ-induced demyelination in PFC. Representative micrographs showed MBP-immunostaining in the PFC of mouse from each group. CPZ-exposed mouse showed evident myelin breakdown in the PFC, but it was not so obvious in the mouse co-administer CPZ and FLA. The bar equals 50 mm. The optical density analysis confirmed the lowest optical density of MBP-immunostaining in the CPZ group. Differences between CPZCFLA and CON or FLA were also significant. Data (obtained from 4 animals in each group) were expressed as the means § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
inhibition. This suggestion encourages us to test the possible therapeutic effects of FLA on MS, a white matter disease for which no curative avenues are available now. Flavopiridol alleviated CPZ-induced activation of astrocytes and microglia Although the increased astrocytes and microglia in CPZtreated mice may be viewed as the responsive actions of the body to demyelination and oligodendrocyte loss, the activated astrocytes and microglia in turn may play certain roles in OPC proliferation, migration, and differentiation. They release pro-inflammatory cytokines and growth factors in the local sites. For example, astrocytes were shown to produce fibroblast growth factor 2 (FGF2) and plateletderived growth factor A (PDGF-A) during CPZ-induced demyelination.3,4,5 These growth factors promoted OPCs proliferation and inhibited OPCs differentiationby activating cell cycle.56 FGF2 even stimulated post-mitotic oligodendrocytes to
reenter the S-phase of the cell cycle, while FGF2 knockout enhanced OPCs differentiation and promoted oligodendrocyte repopulation in the demyelinated area following CPZ insult.57,58 Moreover, activation of microglia by CPZ led to increased growth factor production and secretion of pro-inflammatory cytokines such as TNFa (tumor necrosis factor a) and IFNg(interferon-g).3-5,59,60 The latter (IFN-g)was shown to inhibit cell cycle exit in oligodendrocyte progenitor cells and attenuate MBP expression.61 Taken together, these previous data point to an alternative view that activated astrocytes and microglia in brains of CPZ-treated mice may contribute to or exacerbate mature oligodendrocyte loss and myelin breakdown as shown in this study. It implies that antiinflammatory treatment or inhibiting astrocyte and microglia activation may be of help in improving the CPZinduced white matter damage. Indeed, minocycline, a tetracycline antibiotic with strong anti-inflammatory and antioxidant actions, has been proved to ameliorate the demyelinating effect of CPZ and increase remyelination by
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Figure 5. FLA alleviated the CPZ-induced increase in GFAP positive cells in PFC. Representative micrographs showed GFAP positive cells in the PFC of mouse from each group. CPZ-exposed mouse showed the densest GFAP positive cells in the PFC, but it was not so in the mouse co-administer CPZ and FLA. The bar equals 50 mm. The statistical analysis of the quantitative data confirmed the densest GFAP positive cells in the CPZ group. Differences between CPZCFLA and anyone of the other groups were also significant. Data (obtained from 4 animals in each group) were expressed as the means § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
inhibiting microglia activation and blocking inflammatory responses.62 Likewise, FLA in this study alleviated the astrocytes and microglia proliferation in CPZ-treated mice, in addition to ameliorating oligodendrocyte loss and myelin breakdown. The mechanism for this action of FLA may be related to its ability of cell cycle inhibition as discussed above. Flavopiridol alleviated the CPZ-induced spatial working memory impairment In accordance with previous studies,6,53,54 CPZ-exposed mice showed spatial working memory deficit in the present study. More significantly, FLA administered during the last week of CPZ exposure reversed the CPZ-induced reduction of spontaneous alternation of mice in the Y maze, in addition to ameliorating the CPZ-induced neuropathology as presented above.
Altogether, these data not only added supports for the claim that demyelination contributes to the cognitive and neuropsychiatric symptoms shown in MS and schizophrenic patients,63-65 but also provided a potential avenue for the treatment of these brain disorders. In conclusion, the results of the present study suggest that CPZ-induced neuropathological changes, including mature oligodendrocyte loss, myelin breakdown, and increases in NG2 cells, astrocytes and microglia, may involve in dysregulation of cell cycle related genes. FLA administration effectively alleviated the CPZ-induced neuropathological changes and spatial working memory deficit. The therapeutic effects of FLA on CPZ-exposed mice may be related to its ability of cell cycle inhibition. Further studies on this emerging research field may lead to a new effective avenue for the treatment of MS and schizophrenia, for which curative avenues are needed.
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Figure 6. FLA alleviated the CPZ-induced increase inIba-1positive cells in PFC. Representative micrographs showed Iba-1 positive cells in the PFCof mouse from each group. CPZ-exposed mouse showed the densest Iba-1positive cells in the PFC, but it was not so in the mouse co-administer CPZ and FLA. The bar equals 50 mm. The statistical analysis of the quantitative data confirmed the densest Iba-1 positive cells in the CPZ group. Differences between CPZCFLA and anyone of the other groups were also significant. Data (obtained from 4 animals in each group) were expressed as the mean § SEM. p < 0.001 compared with the control group, ##p < 0.01 compared with the CPZ group.
Materials and methods Animals
Figure 7. FLA alleviated the CPZ-induced spatial working memory impairment in mice. CPZ-exposed mice showed a lowest percentage of spontaneous alternation among all groups. Differences between CPZ C FLA and anyone of the other groups were also significant. Data (obtained from10 animals in each group) were expressed as mean § SEM. p < 0.001 compared with the control group, ###p < 0.001 compared with the CPZ group.
Male C57BL/6 mice (6–8 weeks old) were purchased from Vital River (Beijing, China). The animals were housed in groups of 4 to 5 in clear plastic cages with free accesses to water and food in a room with a climate-controlled environment on a standard 12 h light/dark cycle (lights on 8 a.m., lights off 8 p.m.). The mice were acclimated to the housing conditions and handled for 3 d before experiments. All experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The experimental procedures were approved by the Local Committee on Animal Care and Use. Cell cycle gene expression microarray Male C57BL/6 mice (6–8 weeks old) weregiven 0.2%(by weight) CPZ (Sigma-Aldrich, St. Louis, MO) mixed into a standard
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powdered rodent chow for 5 weeks, during which period agematched mice received the standard chow without CPZ as reported previously.6,7 Animals were sacrificed under a deep anesthesia at the end of 5th week. Total RNA in the PFC was isolated using Trizol (Invitrogen, Carlsbad, CA) and cleaned up with RNeasy Mini kit (Invitrogen). RNA from each group (n D 3) was pooled in an equimolar amount prior to hybridization with Mouse Cell Cycle RT2 ProfilerTM PCR Array (Qiagen GmbH, Hilton, GER; SABiosciences, Frederick, MD), which contains probes for detecting 86 transcripts. RNA isolation, assessing RNA yield and quality, DNA treatment, RNA cleanup, first strand cDNA synthesis, performing real-time PCR and data analysis were accomplished using standard procedures at Kangcheng Biotechnology Company (Shanghai, China).
Cell cycle exit index assay On the last day of CPZ-exposure and the same day for normal controls (as illustrated in Fig. 1A), mice were injected 5Bromo-20 -deoxyuridine (BrdU,50 mg/kg, intraperitoneal injection) (Sigma-Aldrich) once every 2 hrs in 12 hrs. Twelve hrs after the last injection, mice were deeply anesthetized with sodium pentobarbital and perfused through the ascending aorta with 0.01 M phosphate buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in PBS. The brains were removed and post-fixated overnight in the same fixative, followed by dehydration in 30% sucrose. Serial coronal sections (25 mm) of the brains were used for immunofluorescent staining. Free floating sections were pretreated with 10 mM sodium citrate buffer (pH 6) at 45 C for 30 min and transferred to 2N HCl for 30 min at 37 C. Then the sections were rinsed in 0.1 M boric acid (pH 8.5) for 10 min. After washing 3 times with PBS, sections were incubated for one hr at room temperature(22 C) with a blocking solution composed of 0.3% Triton X-100 and 4% BSA in PBS before they were incubatedwith rat monoclonal anti-BrdU (1:100; Abcam, UK) and rabbit polyclonal anti-Ki67 primary antibody (1:100; Millipore, Temecula, USA) in blocking solution for 24 hrs at 4 C. These sections were then washed and incubated with fluorochrome-conjugated secondary [Alexa Fluor 488 goat anti-rat IgG (HCL), Alexa Fluor 568 goat antirabbit IgG(HCL), 1:300; Life, Eugene, USA] for 2hrs at room temperature. Finally, tissue sections were washed in PBS and mounted using a fluorescent medium of DAPI (ZSGB-BIO, Beijing, China).
FLA administration and behavioral test Forty male C57BL/6 mice were divided into 4 groups: control (CNT), cuprizone (CPZ), Flavopiridol (FLA; Selleck, Shanghai, China) group and CPZCFLA groups. As illustrated in Fig. 1B, mice were given 0.2% CPZ-containing or normal diet for 5 weeks. During the5thweek, mice were subjected to intraperitoneal injection of FLA (5 mg/kg) or vehicle once daily for7 days. FLA was dissolved completely in DMSO. The concentrated solution was then diluted in sterile saline to a final concentration of 2.28% DMSO. Twenty-four hrs after the last FLA injection, animals were subjected to Y maze test.
Y maze test is a simple procedure measuring spatial working memory of rodents. The maze consisted of 3 arms of equal length(40 cm long £ 8 cm wide £ 20 cm high) with a 1208 angle between each 2 adjacent arms. Mouse was placed at the end of one arm of the maze and allowed to explore the instrument for 8 min. The total number and series of arm entries were recorded. Alternation was defined as entries into all 3 arms on consecutive occasions (e.g., ABCAB). The alternation rate was calculated using the following formula: Alternation rate (%) D 100 £ number of alternation / (number of total arm entries ¡ 2).
Immunohistochemical staining Serial coronal sections (20 mm) of the brains were used for immunohistochemical staining. Briefly, free floating sections were pretreated with 0.5% hydrogen peroxide in methanol for 20 min, then washed with PBS, and incubated for 1 hr at 22 C with a blocking solution composed of 1% Triton X–100 and 5% normal rabbit serum in PBS. Sections were then incubated with a primary antibody to MBP (1 : 200; Boster, Wuhan, China), GFAP (1 : 200; Boster), Iba-1(1 : 300, Wako, Japan), or NG2 (1 : 200; Millipore) in the blocking solution overnight. Antibody to NG2 was used to identify oligodendrocyte precursor cells and that to MBP was used to label myelin sheath. After rinsing in PBS, sections were incubated in biotinylated secondary antisera (1 : 200) for 2 hrs at 22 C. Following PBS rinses, the sections were incubated in an avidin–biotin–horseradish peroxidase complex (ABC; Boster) for 1 hr at 22 C. Finally, the antigen-antibody complexes were visualized using 0.025% 3, 3-diamino- benzidine (DAB, Sigma-Aldrich) as the chromogen.
Image analysis For quantitative analysis, 4 mice (brains) of each group were included. The examined brain regions included the hippocampus, PFC, and anterior SVZ. Three images (tissue sections) were digitally recorded for each region by Olympus BX–51 microscope equipped with a digital capture system. The ImagePro Plus software (Media Cybernetics, Inc., Silver Spring, MD) was used for analyzing the images. The same conditions were maintained during measurements of all the selected brain sections, and the values were read out automatically by the software. The data of astrocyte and microglia positive cells were expressed as the number/mm2. The data of optical density of MBP-immunostaining were expressed as integrated optical density (IOD), which equals to area times average optical density.
Statistical analysis The Student t-test was performed for the data of the first experiment. Two-way analysis of variance (ANOVA) was performed for the data of the second experiment followed by the Fisherprotected least-significant difference test for post hoc comparisons if appropriate. All data are given as the mean § SEM. P value < 0.05 was considered statistically significant.
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Abbreviations BrdU CDK CPZ FGF FLA GFAP IFN-g MBP MS OPCs PDGF PFC ROS SVZ TNFa
5-Bromo-20 -deoxyuridine cyclin-dependent kinase cuprizone fibroblast growth factor flavopiridol glial fibrillary acidic protein interferon-g myelin basic protein multiple sclerosis oligodendrocyte precursor cells Platelet-derived growth factor prefrontal cortex reactive oxygen species subventricular zone tumor necrosis factor a
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments We thank Dr. Gong Jing-Bo, professor Qian Ling-Jia for excellent technical support.
Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81473193).
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