Effect of dystrophin antisense oligonucleotides on cultured human neurons.
Antisense oligonucleotides offer the potential to block the expression of specific molecules within the cell, thus providing a useful tool in cell function studies. In this paper, we tested the possibility to block dystrophin expression in in vitro cultured neurons with antisense oligonucleotides administration. Human fetal neuronal cultures were treated with different doses of antisense oligonucleotides against dystrophin, the protein coded by the Duchenne muscular dystrophy gene. Results showed that labelled oligonucleotides rapidly accumulated into cultured neurons, but were discarded 15-24 h after treatment. However, no effects could be observed until 3-4 days after treatment, when immunocytochemical staining for dystrophin was significantly decreased in treated neurons. This result was confirmed by polymerase chain reaction assay which showed a significantly lower expression of the dystrophin specific mRNA. Electron microscope observations confirmed that neurons were affected. Large inclusions or packed granules were detectable in their cytoplasm and in terminal endings. Neuronal nuclear membrane was sometimes shredded, so that nuclear shape was altered. These phenomena were dose-dependent, further substantiating the hypothesis of a specific effect of antisense treatment. This interpretation was supported by the absence of alterations when cultures were treated with mismatch or non specific antisenses. Since the function of dystrophin is still unknown, these data might help in understanding the role played by this protein in the developing brain.
Sogos
V
V
Department of Cytomorphology, School of Medicine, Cagliari, Italy.
Ennas
M G
MG
Mussini
I
I
Gremo
F
F
D002478:Cells, Cultured; D016189:Dystrophin / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology*; D016133:Polymerase Chain Reaction
D016189:Dystrophin; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense