Phosphorylation of myelin regulatory factor by PRKG 2 mediates demyelination in Huntington's disease
2020; Springer Nature; Volume: 21; Issue: 6 Linguagem: Inglês
10.15252/embr.201949783
ISSN1469-3178
AutoresPeng Yin, Qiong Liu, Yongcheng Pan, Weili Yang, Su Yang, Wenjie Wei, Xing-Xing Chen, Yan Hong, Dazhang Bai, Xiao‐Jiang Li, Shihua Li,
Tópico(s)Hereditary Neurological Disorders
ResumoArticle9 April 2020Open Access Source DataTransparent process Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease Peng Yin Corresponding Author [email protected] orcid.org/0000-0002-4811-6956 Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Qiong Liu Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Key Laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Yongcheng Pan Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Key Laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Weili Yang Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Su Yang Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Wenjie Wei Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xingxing Chen Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Department of Physiology and Pathophysiology, Brain and Cognition Research Institute, Medical College, Wuhan University of Science and Technology, Wuhan, China Search for more papers by this author Yan Hong Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dazhang Bai Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Xiao-Jiang Li Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Shihua Li Corresponding Author [email protected] orcid.org/0000-0003-1775-6536 Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Peng Yin Corresponding Author [email protected] orcid.org/0000-0002-4811-6956 Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Qiong Liu Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Key Laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Yongcheng Pan Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Key Laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Weili Yang Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Su Yang Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Wenjie Wei Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xingxing Chen Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Department of Physiology and Pathophysiology, Brain and Cognition Research Institute, Medical College, Wuhan University of Science and Technology, Wuhan, China Search for more papers by this author Yan Hong Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dazhang Bai Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Xiao-Jiang Li Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Shihua Li Corresponding Author [email protected] orcid.org/0000-0003-1775-6536 Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China Search for more papers by this author Author Information Peng Yin *,1,2, Qiong Liu2,3, Yongcheng Pan2,3, Weili Yang1, Su Yang1, Wenjie Wei2,4, Xingxing Chen2,5, Yan Hong2, Dazhang Bai1, Xiao-Jiang Li1 and Shihua Li *,1 1Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China 2Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA 3Key Laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, China 4Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 5Department of Physiology and Pathophysiology, Brain and Cognition Research Institute, Medical College, Wuhan University of Science and Technology, Wuhan, China *Corresponding author. Tel: +86 2085 222157; E-mail: [email protected] *Corresponding author. Tel: +86 1371 6952480; E-mail: [email protected] EMBO Rep (2020)21:e49783https://doi.org/10.15252/embr.201949783 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Demyelination is a common pathological feature of a large number of neurodegenerative diseases including multiple sclerosis and Huntington's disease (HD). Laquinimod (LAQ) has been found to have therapeutic effects on multiple sclerosis and HD. However, the mechanism underlying LAQ's therapeutic effects remains unknown. Using HD mice that selectively express mutant huntingtin in oligodendrocytes and show demyelination, we found that LAQ reduces the Ser259 phosphorylation on myelin regulatory factor (MYRF), an oligodendrocyte-specific transcription factor promoting the expression of myelin-associated genes. The reduced MYRF phosphorylation inhibits MYRF's binding to mutant huntingtin and increases the expression of myelin-associated genes. We also found that PRKG2, a cGMP-activated protein kinase subunit II, promotes the Ser259-MYRF phosphorylation and that knocking down PRKG2 increased myelin-associated protein's expression in HD mice. Our findings suggest that PRKG2-regulated phosphorylation of MYRF is involved in demyelination and can serve as a potential therapeutic target for reducing demyelination. Synopsis Laquinimod reduces Ser259 phosphorylation of myelin regulatory factor and its binding to mutant huntingtin, alleviating expression of myelin-associated genes and demyelination caused by mutant huntingtin in oligodendrocytes. Laquinimod alleviates the phenotypes and pathology of HD mice expressing mutant HTT in oligodendrocytes. Laquinimod inhibits the interaction of mutant HTT with MYRF by reducing the phosphorylation of MYRF and restores the expression of myelin genes. Reducing the phosphorylation of MYRF by knocking down PRKG2 increases the expression of myelin genes in HD mice. Introduction Myelination, a process during which myelin wraps around axons, is critical for the development and function of the nervous system, as myelination vitally protects axon integrity and electrically insulates axons to enable its rapid conduction of action potential 1, 2. Myelin is constituted of abundant myelin-associated proteins such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP), which are produced by oligodendrocytes 3. In disease conditions, demyelination occurs and disrupts the nerve connection, leading to a variety of neurological diseases including multiple sclerosis 4, 5. In Huntington's disease (HD), the mutant huntingtin (HTT) protein carries an expanded polyglutamine (polyQ) repeat in its N-terminal region, which promotes aggregate formation in aged neuronal and glial cells and causes progressive neurodegeneration and neurological symptoms 6-8. Growing evidence indicates that non-neuronal mutant HTT toxicity plays an important role in HD 6, 8, 9. For example, defect in oligodendrocyte-enriched white matter is a typical characteristic feature in the early stages of HD patients 10-12. Laquinimod (LAQ) was initially known as an immunomodulatory agent and was used to treat multiple sclerosis 13, 14 and alleviate demyelination in the mouse models of different diseases 15-18. LAQ has also been shown to improve behavioral phenotypes and white matter integrity in HD mice 19-21. The neuroprotective effect of LAQ is well supported by its therapeutic effects on animal models of neuroinflammatory diseases, such as the experimental autoimmune encephalomyelitis 13, 14, and is therefore thought to be due to an anti-inflammation effect 17, 22. However, the protective effect of LAQ on axons and myelination in HD mice was not found to associate with the anti-inflammatory effect 17, 22. Thus, the mechanism underlying the protective effects of LAQ remains unknown, and understanding this mechanism would be helpful for developing therapeutic strategies for brain diseases with demyelination. We previously established a HD mouse model (PLP-150Q) that selectively expresses mutant HTT in oligodendrocytes and displays severe demyelination 23. Thus, PLP-150Q mice allowed us to study the mechanism for demyelination. We found that LAQ could restore the expression of myelin-associated genes by reducing Ser259 phosphorylation in MYRF, an oligodendrocyte-specific transcription factor, and decreasing the subsequent binding of mutant HTT to MYRF. Increased Ser259 phosphorylation is accompanied by decreased expression of MBP in the brain tissues of HD patients. We also found that cGMP-activated protein kinase subunit II (PRKG2) phosphorylates Ser259-MYRF and that knocking down PRKG2 increased MBP expression in PLP-150Q mouse. Our findings suggest that phosphorylation of MYRF mediates demyelination and can serve as a potential therapeutic target for reducing demyelination in neurodegenerative diseases. Results LAQ upregulated the myelin gene expression at the transcriptional level We previously established transgenic mice that selectively express N-terminal HTT (1-212 aa) containing either 150Q (PLP-150Q) or 23Q (PLP-23Q) in oligodendrocytes under the control of the oligodendrocyte's specific proteolipid protein (PLP) promoter and found that PLP-150Q mice display robust demyelination 23. Since LAQ can alleviate demyelination in the mouse models of different diseases 15-18 and since PLP-150Q mice express mutant HTT only in oligodendrocytes, we wanted to use PLP-150Q mice to investigate the specific effect of LAQ on mutant HTT-mediated demyelination. Three-month-old PLP-150Q and PLP-23Q mice were orally administrated with LAQ at two doses (5 or 25 mg/kg/day) or with the same volume of purified water (Vehicle) for 2 months (Fig EV1A). Consistent with the previous reports of the therapeutic effects of LAQ on different HD mouse models 19-21, treatment with LAQ (5 mg or 25 mg/kg/day) improved the rotarod and balance beam performance of PLP-150Q mice as compared with PLP-150Q mice treated with vehicle control (Fig EV1B), but did not alleviate the body weight reduction and early death of PLP-150Q mice (Fig EV1C). Because PLP-150Q mice only express mutant HTT in oligodendrocytes, the protective effects of LAQ indicate that LAQ could improve the function of oligodendrocytes to reduce neurological symptoms of HD mice and motivated us to further explore the mechanistic action of LAQ on oligodendrocytes. Click here to expand this figure. Figure EV1. (Related to Fig 1). LAQ partially alleviated phenotypes in PLP-150Q mice Schematic of study design. PLP-150Q mice and the control PLP-23Q mice at the age of 3 months were orally administrated with LAQ (5 or 25 mg/kg/day as indicated in figure) for 2 months. The brain tissues were collected for pathological examination after behavioral studies. Rotarod performance and balance beam tests showed severe motor impairment in PLP-150Q mice. Administrating 5 or 25 mg/kg LAQ could alleviate the impairment. One-way ANOVA followed with Tukey's test. **P = 0.00735; *P = 0.04086; n = 12 mice per group for rotarod perform test and n = 6 mice per group for balance beam test. Data are presented as mean ± SEM. The PLP-150Q mice show reduced body weight and early death, which were not altered by the treatment with 5 or 25 mg/kg LAQ for 2 months. n = 12 mice each group, which were treated with LAQ at 3 months of age. Data are mean ± SEM. PLP-150Q/GFP mice show GFP labeling of oligodendrocyte processes, which are reduced in the corpus callosum when compared with PLP-23Q/GFP mice and were increased after 5 mg/kg LAQ treatment for 2 months. Quantitative analysis of the number of GFP-positive oligodendrocytes and process length was shown under the micrographs. n = 3 mice in each group. Process length was presented in a.u. One-way ANOVA followed with Tukey's test. ***P = 3.92 × 10−5; **P = 0.00187. Data were mean ± SEM. Scale bar: 40 μm. Download figure Download PowerPoint Transmission electron microscopy revealed that a number of degenerated axons, which appeared swollen and dark, were present in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment of 3-month-old PLP-150Q mice for 2 months, however, significantly improved the axon myelination (Fig 1A). Calculating g-ratios (the inner axonal diameter to the total outer diameter) verified that LAQ reduced this ratio or increased myelination of axons (vehicle: g = 0.7727 ± 0.0203 versus LAQ: g = 0.6418 ± 0.0191; *P < 0.05) (Fig 1B). We also crossed PLP-150Q mice to transgenic PLP-GFP mice and obtained PLP-150Q/PLP-GFP mice in which oligodendrocytes express both mutant HTT and GFP, allowing us to directly examine the integrity of GFP-labeled oligodendrocyte processes. We found that there was indeed reduced oligodendrocyte process length in PLP-150Q mice as compared with PLP-23Q mice, and LAQ (5 mg/kg) treatment for 2 months increased oligodendrocyte processes in their density and length in the corpus callosum of PLP-150Q mice (Fig EV1D). Interestingly, treatment with LAQ at 5 or 25 mg/kg could also restore the expression of myelin-related proteins in PLP-150Q mice, such as myelin basic protein (MBP), myelin-associated oligodendrocytic basic protein (MOBP), and myelin oligodendrocyte glycoprotein (MOG) (Fig 1C). However, in PLP-23Q mice, there were no significant changes of these myelin-associated proteins between the vehicle- and LAQ-treated groups (Fig EV2A), suggesting that LAQ selectively inhibited the toxicity of mutant HTT on myelin gene expression. Figure 1. LAQ increased the expression of myelin-associated proteins and reduced the binding of MYRF to mutant HTT in PLP-150Q mice Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2 μm (low magnification) and 0.5 μm (high magnification). G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G = 0.6418 ± 0.0191), compared with age-matched vehicle group (G = 0.7727 ± 0.0203). One-way ANOVA with Tukey's test. *P = 0.0166. At least 182 axons per genotype were examined from 3 mice in each group. Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months upregulates multiple myelin proteins (MBP, MOBP, and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP, or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANOVA followed with Tukey's test. MBP: ***P = 0.0009; MOG: ***P = 0.0003; MOBP: **P = 0.0067. Data are mean ± SEM Quantitative PCR of the transcript expression of myelin-associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P = 0.0007; MOG: ***P = 0.0009. Data are mean ± SEM (n = 3). The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P < 0.001; which was reversed by LAQ (5 μM) treatment, **P < 0.01. The ratios were obtained from three independent experiments. One-way ANOVA followed with Tukey's test. Data are mean ± SEM. Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from three independent experiments was shown on the right. One-way ANOVA followed with Tukey's test. **P = 0.009. Data are mean ± SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. (Related to Fig 1). Expression of the myelin-associated proteins in PLP-23Q mice treated with LAQ Western blotting showing the multiple myelin proteins (MBP, MOBP, and MOG) in the corpus callosum in PLP-23Q mice that were treated with LAQ (5 or 25 mg/kg) at 3 months of age for 2 months. Vinculin served as a loading control. Quantitative PCR analysis of myelin-associated gene (MBP and MOG) in the corpus callosum of LAQ (5 or 25 mg/kg)-treated PLP-23Q mice. n = 3 each group. Data are mean ± SEM. Western blotting with anti-HTT (EM48) showing the aggregated and soluble huntingtin in the corpus callosum in PLP-150Q mice that were treated with LAQ (5 mg/kg) at 3 months of age for 2 months. Beta-actin served as a loading control. Co-transfection of N-terminal MYRF (nMYRF) with N-terminal HTT containing 150Q or 23Q in HEK293 cells and immunoprecipitation of nMYRF. More HTT-150Q binds nMYRF than HTT-23Q. Five μM LAQ treatment could decrease the immunoprecipitated HTT. The ratio of precipitated HTT to input in transfected cells treated with DMSO or LAQ obtained from three independent experiments was shown on the right. One-way ANOVA followed with Tukey's test. ***P = 1.25 × 10−5; **P = 0.00552. Data are mean ± SEM. Download figure Download PowerPoint To explore whether myelin-associated genes are upregulated at the transcriptional level by LAQ, we used quantitative RT–PCR to examine the mRNA levels of myelin-associated genes. The results showed that, compared with PLP-23Q mouse, the levels of MBP and MOG transcripts were obviously decreased in PLP-150Q mice and were increased by LAQ (5 or 25 mg/kg) after treatment for 2 months (Figs 1D and EV2B). However, LAQ treatment did not alter the levels of aggregated and soluble huntingtin proteins when the antibodies to HTT (EM48) or polyglutamine repeats (1C2) were used to detect mutant HTT (Fig EV2C). These findings suggest that LAQ may antagonize the effect of soluble mutant HTT on suppressing the expression of myelin-associated genes. LAQ upregulated MBP transcription by dissociating MYRF from mutant HTT We have previously found that mutant HTT abnormally binds myelin regulatory factor (MYRF), an oligodendrocyte-specific transcription factor, to affect its critical function for the regulation of transcription of myelin-associated genes 23. It is known that N-terminal fragment of MYRF (nMYRF) is generated by cleavage of full-length MYRF in the cytoplasm and then moves into nucleus to activate myelin gene transcription in oligodendrocytes 24, 25. We then investigated whether LAQ influences the interaction of mutant HTT with MYRF to prevent its inhibitory effect on nMYRF. To address this issue, the MBP promoter was cloned into the reporter vector for a luciferase assay to examine its transcriptional activity on the basis of an earlier finding 26, and this reporter was co-transfected with mutant HTT in HEK293 cells to assess the effect of mutant HTT. Although nMYRF co-transfection could significantly promote the reporter activity, co-expression of mutant HTT decreased this activity, and this decrease could be partially reversed by treating the transfected cells with 5 μM LAQ for 12 h (Fig 1E). We then performed in vivo immunoprecipitation of transgenic mutant HTT and MYRF from the corpus callosum of PLP-150Q mice. N-terminal MYRF was co-precipitated with mutant HTT; however, LAQ (5 or 25 mg/kg) treatment obviously attenuated this interaction (Fig 1F). The inhibitory effect of LAQ was further tested by using transfected HEK293 cells, which confirmed that 5 μM LAQ also diminished the binding of transfected mutant HTT (150Q-HTT) to MYRF (Fig EV2D). LAQ dephosphorylated MYRF to affect its interaction with mutant HTT LAQ was found to regulate phosphorylation of various proteins 27, 28, and transgenic expression of mutant HTT could lead to the increased phosphorylation of several proteins 29-32. In PLP-150Q mice, we also found that expression of mutant HTT in oligodendrocytes resulted in the increased phosphor-NF-kB, -Akt or -JNK when compared with PLP-23Q mice and that 2-month treatment with LAQ (5 or 25 mg/kg) could significantly decrease the phosphorylation of these proteins in PLP-150Q mice (Fig 2A). Figure 2. LAQ dephosphorylated N-terminal MYRF at pS259 to affect its interaction with mutant HTT Western blotting of the phosphorylated NF-kB, NF-Akt, NF-JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANOVA with Tukey's test. NF-kB: ***P = 0.00067(left) and 0.00086(right); JNK: ***P = 0.00013(left) and 0.00047(right); Akt: **P = 0.0014 and ***P = 0.00012. Data are mean ± SEM. In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild-type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by Western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation. Co-immunoprecipitation of nMYRF and N-terminal HTT (1–212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent Western blotting experiments. One-way ANOVA with Tukey's test. ***P = 6.51 × 10−5. Data are mean ± SEM. Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow). Transcriptional activity of wild-type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wild-type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from three independent experiments. One-way ANOVA with Tukey's test. ***P < 0.001. Data are mean ± SEM. Co-transfection of wild-type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wild-type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from three independent experiments. One-way ANOVA with Tukey's test. ***P = 2.30 × 10−5 Data are mean ± SEM. Download figure Download PowerPoint The above findings led us to investigate whether MYRF phosphorylation is involved in its interaction with mutant HTT such that LAQ may regulate its interaction via altering phosphorylation to alleviate the inhibitory effect of mutant HTT on myelin gene expression. We then performed in vitro phosphorylation assay by incubating GST-nMYRF with lysates from the oligodendrocyte-enriched corpus callosum. The results revealed that the lysates from the PLP-150Q mouse brain yielded strong phosphor-serine modification signals on GST-nMYRF as compared with the lysates from the PLP-23Q mouse brain and that 5 mg/kg LAQ treatment eliminated this phosphorylation (Fig 2B, left panel). Furthermore, in the co-transfected HEK293 cells expressing nMYRF and mutant HTT, 5 μM LAQ treatment for 12 h or co-expression of the protein phosphatase, Λ-PPase, could attenuate the phosphorylation of the immunoprecipitated nMYRF and diminished its binding to mutant HTT (Fig 2C). Phosphorylation of Ser259 in MYRF is critical for its interaction with mutant HTT There are multiple amino acids in MYRF for potential phosphorylation (Fig EV3A). We immunoprecipitated nMYRF with mutant HTT in HEK293 cells and isolated the nMYRF band (arrow in Fig 2D) for mass spectrometry to uncover its post-translational modifications (PTMs). The result identified a highly enriched phosphorylation signal on serine 259 (S259) in nMYRF (Fig EV3B). Replacing Ser259 with alanine (S259A) or its adjacent Ser261 with alanine (S261A) confirmed that only S259A substitution prevented the phosphorylation by the PLP-150Q mouse brain lysate (Fig 2B, right panel and Fig EV3C). We also transfected S259A or S261A nMYRF with mutant HTT (amino acid 1-212 HTT-150Q) in HEK293 cells to examine their transcriptional activity on the MBP promoter using luciferase assay and the interactions of different MYRF forms with mutant HTT. Indeed, nMYRF with S259A, but not S261A, substitution produced much higher transcriptional activity than wild-type nMYRF when mutant HTT was present (Fig 2E). Consistently, in co-transfected MYRF and mutant HTT cells, the immunoprecipitated S259A MYRF displayed very weak phosphorylation and a marked reduction in association with mutant HTT, while S261A MYRF remained to be phosphorylated and bound more mutant HTT (Fig 2F). All these findings suggest that S259 in MYRF can be phosphorylated and that this phosphorylation increases its interaction with mutant HTT and reduces its transcriptional activity. Click here to expand this figure. Figure EV3. (Related to Figs 2 and 3). The anti-pMYRF (Ser259) antibody recognized phosphor-Serine 259 in N-terminal MYRF Predicted potential phosphorylation sites in mouse N-terminal MYRF (nMYRF). S259 (red) is a top candidate. Mass spectrometry of the immunoprecipitated MYRF revealed an enrichment phosphorylated signal at pS259. The sequences of mutant MYRF S259A and S261A verified Ser259 substitution. Western blotting of HEK293 cell expressing wild-type N-terminal MYRF (nMYRF), S259A or S261A cDNAs, and mouse tissues showing that replacing Ser259 with alanine can eliminate the labeling of MYRF by antibody to Ser259. The samples were also probed with antibody to total MYRF (Sigma, HAP018310) and pre-immune serum. endo-fMYRF: endogenous full-length MYRF; endo-nMYRF: endogenous N-terminal MYRF; trans-MYRF: transfected MYRF. The anti-MYRF (Sigma, HAP018310) immunohistochemical staining of the corpus callosum of PLP-23Q and PLP-150Q mice showing no significant effect on the total MYRF expression by LAQ (5 mg/kg) treatment. n = 3 mice in each group. Scale bar: 10 μm. Quantitative analysis of the MYRF-positive cells per field (40×) was presented on the right. Data were mean ± SEM. Download figure Download PowerPoint Increased MYRF phosphorylation in HD mouse an
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