| Abstract No.: | B-C2099 |
| Country: | Canada |
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| Title: | MECP2 REGULATION OF MYELIN GENE EXPRESSION |
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| Authors/Affiliations: | 1 Parvez Vora*; 2 Emma Frost;
1 Dept of Human Anatomy and Cell Science; Manitoba Institute of Child Health(MICH), University of Manitoba; 2 Dept.s Pathology; Human Anatomy and Cell Science; Biochemistry and Medical Genetics; MICH, U of Manitoba;Canada
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| Content: | Objective: To investigate myelin specific gene expression levels in the brain of a MeCP2null transgenic mouse.
Materials and Methods:
Quantitative RT-PCR-Total RNA was isolated from brains of female MeCP2null transgenic mice [1] and expression levels compared to age-matched wild-type controls. Gene expression in different areas of the brain was performed using a SYBRGreen RT-PCR kit and analysed using an ABI7500 real-time thermal cycler. For each sample in each run, there were 7 replicates. RNA levels were compared to the housekeeping gene, GAPDH.
Cell Isolation -Mixed glial cultures were prepared from P0 rat pup cortices as previously described [2]. OL progenitors (OP) were shaken free of the underlying astrocyte layer and microglial contamination was removed as previously described [3]. The astrocyte monolayer was shaken for further 24 hours to remove all remaining OP and microglia prior to use.
Western Blot -Cells were lysed in Laemmli buffer and boiled for 4 minutes prior to loading on an 8% polyacrylamide gel. Proteins were transferred to PVDH membranes, and probed with polyclonal anti-MeCP2 antibody. Bands were visualised using HRP conjugated secondary antibody, enhanced with ECL reagent, and exposed to X-Ray film.
Immunohistochemistry (IHC)-OP were cultured for 1-7 days in vitro, and fixed in 10% formalin. MeCP2 expression was visualised using a polyclonal anti-MECP2 antibody with FITC conjugated secondary antibody. Images were captured using Image Pro 7.0 via an inverted Olympus IX51 fluorescent microscope with RETIGA 2000RV monochrome camera attached to an imaging station.
Results: Both astrocytes and OP express MeCP2 protein, as assessed by WB and IHC. Further, in the MeCP2null mouse, the expression of the myelin-specific genes, myelin basic protein (MBP) and myelin associated glycoprotein (MAG) genes are significantly up-regulated (by 2.38 and 4.59 fold respectively), while proteolipid protein (PLP) is down-regulated to 0.45X the level in control brain .
Conclusion: The majority of classical RTT syndrome cases are caused by mutations in the MeCP2 gene [4]. The current research on molecular mechanisms underlying RTT focuses on neurons, while glial cells are widely considered to not express MeCP2. We show convincingly that OLs express MeCP2. The role of MeCP2 in OL is yet left to be elucidated. The OLs also express various myelin proteins like MBP, MAG and PLP before becoming mature myelinating OLs. These myelin associated proteins are structural components of myelin sheath. Our qRT-PCR results show significant dysregulation of these genes in the MeCP2null mouse. Such a result showing that the expression patterns of myelin genes are altered in the MeCP2null mice supports to our hypothesis that myelination is abnormal in the RTT. Funding provided by the Manitoba Institute of Child Health
REFERENCES:
[1] Shahbazian, M. D. et al. Hum. Mol. Genet. 2002, 11, 115-124.
[2] Frost, E. et al. Dev Neurosci 1996, 18, 266-73.
[3] Milner, R. et al. Development 1994, 120, 3497-506.
[4] Bienvenu, T. et al. Hum Mol Genet 2000, 9, 1377-84
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