Inhibition of nucleolar transcription as a trigger for neuronal apoptosis.
In post-mitotic neurons, the mechanisms of the apoptotic checkpoint that is activated by DNA damage remain unclear. Here we show that in cultured cortical neurons, the DNA damaging agent camptothecin (CPT) reduced transcription of rRNA and disrupted nucleolar staining for B23/nucleophosmin suggesting DNA damage-induced nucleolar stress. Although CPT activated the pro-apoptotic protein p53, the CPT-induced nucleolar stress was unaffected by p53 inhibition. In addition, brain-derived neurotrophic factor-mediated protection from CPT-induced apoptosis prevented neither nucleolar stress nor p53 activation. Therefore, inhibition of rRNA transcription might be upstream of the pro-apoptotic p53 activity. Indeed, short hairpin RNA-mediated inhibition of a RNA-Polymerase-I co-factor, transcription initiation factor IA, attenuated rRNA transcription causing nucleolar stress and p53-dependent neuronal apoptosis. The protein synthesis inhibitor cycloheximide blocked apoptosis that was induced by over-expressed shTIF-IA or active form of p53. Also, the general transcription inhibitor actinomycin D triggered nucleolar stress and activated p53. However, it did not induce apoptosis except at the low concentration of 0.05 microg/mL with stronger inhibitory activity against nucleolar than extranucleolar transcription. Hence, nucleolar stress-activated apoptosis requires extranucleolar transcription. This study identifies the nucleoli of post-mitotic neurons as sensors of DNA damage coupling reduced rRNA transcription to p53-mediated apoptosis that requires de novo expression of protein-coding genes. Thus, rDNA selectivity of DNA damage may determine its ability to induce neuronal apoptosis.
Kalita
Katarzyna
K
Department of Neurological Surgery, University of Louisville, Kentucky Spinal Cord Injury Research Center, Louisville, Kentucky 40292, USA.
Makonchuk
Denys
D
Gomes
Cynthia
C
Zheng
Jing-Juan
JJ
Hetman
Michal
M
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002166:Camptothecin / Q000633:toxicity; D002466:Cell Nucleolus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*
D034741:RNA, Small Interfering; D002166:Camptothecin