title: Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain.
abstract: Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene.  MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2) that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A) cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.
authors.affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France ; Université de Toulouse, UPS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France.
authors.forename: Lara
authors.initials: L
authors.lastname: Kaddoum
authors.affiliation: INSERM U910, Unité de Génétique Médicale et Génomique Fonctionnelle, Faculté de Médecine de La Timone, Marseille, F-13385, France ; Aix-Marseille Université, Faculté de Médecine de La Timone, Marseille, F-13385, France ; Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Panayotis
authors.affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France ; Université de Toulouse, UPS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France.
authors.forename: Honoré
authors.initials: H
authors.lastname: Mazarguil
authors.affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France ; Université de Toulouse, UPS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Giglia-Mari
authors.affiliation: INSERM U910, Unité de Génétique Médicale et Génomique Fonctionnelle, Faculté de Médecine de La Timone, Marseille, F-13385, France ; Aix-Marseille Université, Faculté de Médecine de La Timone, Marseille, F-13385, France.
authors.forename: Jean Christophe
authors.initials: JC
authors.lastname: Roux
authors.affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France ; Université de Toulouse, UPS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, F-31077, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Joly