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Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture
Published on: September 16, 2013
Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state
1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Abstract:
In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.
Insights
Two enzymes, caspase-activated DNase (CAD) and DNase gamma, drive neuronal apoptosis. CAD is active in proliferating cells, while DNase gamma is crucial during neuronal differentiation, indicating distinct roles in programmed cell death.
Area of Science:
- Molecular Biology
- Neuroscience
- Cell Biology
Background:
- Neuronal apoptosis involves DNA fragmentation.
- The roles of specific endonucleases in this process remain incompletely understood.
Purpose of the Study:
- To investigate the distinct roles of caspase-activated DNase (CAD) and DNase gamma in neuronal apoptosis.
- To determine how their activities correlate with the differentiation state of neuronal cells.
Main Methods:
- Utilized N1E-115 neuroblastoma and PC12 cells.
- Assessed endonuclease expression and activity during proliferation and differentiation.
- Employed staurosporine and NGF deprivation to induce apoptosis.
- Used antisense RNA to downregulate DNase gamma.
Main Results:
- CAD was highly expressed in proliferating N1E-115 cells; its inhibition blocked staurosporine-induced DNA fragmentation.
- During differentiation, CAD disappeared, and DNase gamma was induced in N1E-115 cells.
- DNase gamma induction and its role in apoptosis were confirmed in PC12 cells during NGF deprivation.
Conclusions:
- CAD and DNase gamma function as key DNA-fragmenting enzymes in neuronal apoptosis, with activity dependent on the cell's differentiation state.
- DNase gamma is implicated in natural apoptosis during nervous system development.
