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Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis
J Padmanabhan1, D S Park, L A Greene
1Department of Pathology and Center for Neurobiology and Behavior, Taub Center for Alzheimer's Disease Research, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Abstract:
Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentrations of KCl, but the underlying molecular mechanisms are not yet clear. Although caspases have been postulated to be involved in CGN cell death, inhibitors of caspases failed to prevent apoptosis under our culture conditions, suggesting an involvement of other molecules and pathways. We find that inhibitors of cyclin-dependent kinases--flavopiridol, olomoucine, and roscovitine--protect CGNs from KCl withdrawal-induced apoptosis, suggesting that cell cycle components play a significant role in the death of these neurons. Analysis of the different cell cycle regulatory elements in this model revealed that apoptosis is preceded by an increase in the level of cyclin E protein, with elevated nuclear levels of cyclin D1 and with enhanced activity of the cyclin D1- and E- associated kinases. In addition, there was a significant decrease in the level of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these changes, analysis of a major substrate of cyclin-activated cdks, retinoblastoma protein (Rb), showed an increase in the level of phosphorylated forms within 1 hr of KCl withdrawal. Moreover, the overall levels of Rb protein were significantly reduced within 6-12 hr of KCl withdrawal and did so by a caspase-independent mechanism. All of these responses were blocked by cdk inhibitors. These findings indicate that cdks act at an early step in the pathway by which KCl withdrawal induces apoptotic death of cerebellar granule cells and suggest that additional elements of the cell cycle machinery participate in this mechanism.
Insights
Cerebellar granule neurons undergo apoptosis when KCl is withdrawn. Cell cycle regulators, specifically cyclin-dependent kinases (CDKs), play a critical role in this caspase-independent cell death pathway.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Cerebellar granule neurons (CGNs) undergo apoptosis upon KCl withdrawal.
- The molecular mechanisms driving this neuronal apoptosis are not fully understood.
- Caspase involvement in CGN apoptosis remains unclear under specific culture conditions.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying KCl withdrawal-induced apoptosis in CGNs.
- To investigate the role of cell cycle components in CGN cell death.
- To determine if caspases are involved in this specific apoptotic pathway.
Main Methods:
- Utilized KCl withdrawal to induce apoptosis in cultured CGNs.
- Administered caspase inhibitors and cyclin-dependent kinase (CDK) inhibitors (flavopiridol, olomoucine, roscovitine).
- Analyzed levels and activity of cell cycle regulatory proteins including cyclin E, cyclin D1, p27, and retinoblastoma protein (Rb).
Main Results:
- Caspase inhibitors did not prevent apoptosis, suggesting a caspase-independent mechanism.
- CDK inhibitors protected CGNs from apoptosis.
- KCl withdrawal led to increased cyclin E, nuclear cyclin D1, and associated kinase activity.
- Decreased levels of CDK inhibitor p27 and increased phosphorylation of Rb were observed.
- Rb protein levels decreased significantly via a caspase-independent pathway, blocked by CDK inhibitors.
Conclusions:
- Cell cycle components, particularly CDKs, are crucial in the early stages of KCl withdrawal-induced CGN apoptosis.
- The cell death pathway is largely caspase-independent.
- Further investigation into cell cycle machinery's role in neuronal apoptosis is warranted.