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Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis
N Canu1, C Barbato, M T Ciotti
1Dipartimento di Neuroscienze, Facoltà di Medicina e Chirurgia, Università di Tor Vergata, 00133 Roma, Italia. nadia@biocell.irmkant.rm.cnr.it
Abstract:
We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsin-like activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic accumulation of ubiquitinated proteins and decline of proteasome function are reversed by the pan-caspase inhibitor Z-VAD-fmk. The decline in proteasome function is accompanied by, and likely attributable to, a marked and progressive decline of deubiquitinating activities. The finding that the proteasomes are early involved in apoptosis and that ubiquitinated proteins accumulate during this process prospect granule neurons as a model system aimed at correlating these events with neurodegenerative diseases.
Insights
The ubiquitin proteolytic system plays a critical role in programmed cell death of cerebellar neurons. Proteasome dysfunction and protein accumulation occur during apoptosis, offering insights into neurodegenerative diseases.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- The ubiquitin proteolytic system regulates protein degradation.
- Programmed cell death (apoptosis) involves complex cellular pathways.
- Cerebellar granule neurons are vulnerable to specific environmental changes.
Purpose of the Study:
- To investigate the role of the ubiquitin proteolytic system in cerebellar granule neuron death induced by low extracellular potassium.
- To determine the temporal relationship between proteasome function and apoptosis.
- To explore the link between proteasome dysfunction and neurodegenerative diseases.
Main Methods:
- Induction of apoptosis in cerebellar granule neurons by reducing extracellular potassium.
- Administration of proteasome inhibitors at different time points.
- Assay of caspase-3 activity and tau protein processing.
- Measurement of proteasome and deubiquitinating enzyme activities.
- Analysis of ubiquitinated protein levels.
Main Results:
- Proteasome inhibitors blocked apoptosis when given early but not late.
- Proteasomes act upstream of caspase activation in this model.
- Apoptosis progression led to proteasome dysfunction and ubiquitinated protein accumulation.
- Decline in deubiquitinating activities contributed to proteasome impairment.
- Pan-caspase inhibition reversed proteasome dysfunction and protein accumulation.
Conclusions:
- The ubiquitin proteolytic system is crucial in early apoptosis of cerebellar neurons.
- Dysfunctional proteasomes and protein aggregation characterize later stages of apoptosis.
- Cerebellar granule neurons serve as a model to study proteasome involvement in neurodegeneration.