Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis.

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.

Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis.

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.

NMDA receptor modulation by a conditioned medium derived from rat cerebellar granule cells.

Our previous studies have shown that the response to the excitotoxic action of glutamate by cultured cerebellar granule cells depends upon the cell density or the volume of medium in which they have been grown: the higher the cell density or the lower the volume, the higher the response to glutamate. We have hypothesized that this variable response is due to the formation in culture of a glutamate-sensitizing activity GSA more abundantly in conditioned medium derived from high-density or low-volume cultures than that present in low-density or high volume cultures and capable of restoring sensitivity in previously resistant granule cells. In order to elucidate the mechanism of action of glutamate-sensitizing activity, we measured the extent and function of NMDA receptors in low- and high-volume cultures and assessed the effect of glutamate-sensitizing activity on the same receptors. We found that under high-volume conditions the extent of MK-801 binding, the amount of NMDA receptor type 1, the currents evoked in whole cells after an NMDA pulse and the response of cultured cells to this ligand were markedly reduced compared with low-volume cultures. Addition of glutamate-sensitizing activity to high-volume cultures increased their glutamate sensitivity, the NMDA-evoked currents, the extent of MK-801 binding and the amount of NMDA receptor type 1 protein present. The corresponding mRNA transcripts, on the contrary, were unchanged in high-volume, low-volume and high-volume GSA-treated cultures.