title: Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain.
abstract: BACKGROUND
  Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon.
  
  
  METHODS
  For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants.
  
  
  RESULTS
  We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression.
  
  
  CONCLUSIONS
  We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function.
authors.affiliation: Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University/Leiden University Medical Center, Leiden, The Netherlands. I.Zalachoras@lumc.nl
authors.forename: Ioannis
authors.initials: I
authors.lastname: Zalachoras
authors.forename: Gwendolynn
authors.initials: G
authors.lastname: Grootaers
authors.forename: Lisa T C M
authors.initials: LT
authors.lastname: van Weert
authors.forename: Yves
authors.initials: Y
authors.lastname: Aubert
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: de Kreij
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Datson
authors.forename: Willeke M C
authors.initials: WM
authors.lastname: van Roon-Mom
authors.forename: Annemieke
authors.initials: A
authors.lastname: Aartsma-Rus
authors.forename: Onno C
authors.initials: OC
authors.lastname: Meijer
mesh_terms: D000679:Amygdala / Q000187:drug effects* / Q000378:metabolism*; D000818:Animals; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D061346:Laser Capture Microdissection; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D056920:Nuclear Receptor Coactivator 1 / Q000235:genetics / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000187:drug effects; D013997:Time Factors
chemical_list: D005904:Glial Fibrillary Acidic Protein; D020319:Oligodeoxyribonucleotides, Antisense; D020033:Protein Isoforms; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1; D017493:Leukocyte Common Antigens; D010751:Phosphopyruvate Hydratase