Activity-dependent changes in MeCP2 sub-nuclear localization

Malaika K. Singleton, B.A.¹, Dag H. Yasui, Ph.D.², Ynnez Gwye, B.S.², Karen N. Thatcher, B.S.³, Asmita Kumar, Ph.D.⁴, N. Carolyn Schanen, M.D., Ph.D.⁴, and Janine M. LaSalle, Ph.D.². (1) Neuroscience Graduate Group; Medical Microbiology and Immunology, University of California, Davis, 3424 Tupper Hall, One Shields Avenue, Davis, CA 95616, (2) Medical Microbiology and Immunology, University of California, Davis, 3424 Tupper Hall, One Shields Avenue, Davis, CA 95616, (3) Dept. Med Micro & Immunology, School of Medicine, University of California, Davis, University of California, Davis, Davis, CA 95616, (4) Biological Sciences, University of Delaware, Nemours Biomedical Research, 1600 Rockland Road, Room H3B-337, Wilmington, DE 19803

Background: Mutations in the methyl CpG binding protein 2 (MECP2) gene cause Rett Syndrome (RTT), a devastating autism-spectrum disorder that manifests almost exclusively in females and is characterized by a normal early development with a regression in development between the ages of 6-18 months of age. The gene for MeCP2 is located on the X-chromosome and is mutated in a number of neurodevelopmental disorders. Alternative splicing of Mecp2 exon 2 results in MeCP2 isoforms with different translational start sites and unique N-termini.

Objectives: The two MeCP2 isoforms (e1 and e2) have been investigated in mouse neuronal cultures using immunofluorescence (IF) and laser scanning cytometry (LSC) to detect changes in the expression level and localization of each isoform following potassium chloride (KCl) induced neuronal activity.

Methods: Primary neuronal cultures were established from embryonic (E14-E17) mice and cultured for 3-5 days in vitro in the presence or absence of 50 mM KCl from 10 minutes to 48hrs to observe short and long-term depolarization effects. Mouse 3T3 cells stably transfected with MECP2e1 or MECP2e2 were used to confirm isoform-specific antibodies. The co-localization of the MeCP2 isoforms in neurons was determined with isoform-specific antibodies and markers of different sub-nuclear locations by IF.

Results: The results showed that MeCP2 had a distinct staining pattern with MeCP2 e1 localized primarily in the nuclear matrix while MeCP2 e2 localized primarily to large nuclear heterochromatin in mouse brain and neuronal cultures. Results from the activity-dependent experiments showed that KCl treatment led to a redistribution of MeCP2 and a resulting increase in the fluorescent intensity of MeCP2 e1 and the nuclear matrix protein SFPQ/matrin 4 as detected by IF and LSC.

Conclusions: The dynamic changes in sub-nuclear localization of MeCP2 following neuronal stimulation may be important for regulation of downstream targets such as BDNF.

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