Inclusion body formation in oligodendroglial cells after treatment with PR-619, an inhibitor of deubiquitination

A pathological hallmark of many neurodegenerative diseases is the aggregation of proteins. Glial cytoplasmic inclusions (GCIs) originating in oligodendrocytes are characterized by the presence of ubiquitin, heat shock proteins (HSP), and the MAP tau. GCI formation may be linked to a failure of the ubiquitin proteasome system (UPS) and the autophagy pathway. The UPS involves the ubiquitination of proteins followed by proteasomal degradation. Deubiquitination of target proteins is performed by proteases called deubiquitinating proteins (DUBs). Inhibition of DUBs may lead to the dysregulation of homeostasis and have pathological consequences. To assess the effects of DUB-inhibition, we have used the oligodendroglial cell line, OLN-t40, stably expressing the longest human tau isoform. Cells were incubated with PR-619, a non-selective, reversible inhibitor of ubiquitin isopeptidases. Incubation with PR-619 led to morphological changes, the upregulation of HSP32, and to protein aggregates near the MTOC, containing ubiquitin, αB-crystallin, HSP70, LC3, a marker of autophagy, and p62. P62 may be a link between autophagy and the UPS. Furthermore, incubation with PR-619 affected the microtubule network and led to accumulation of punctuated tau around the MTOC. Thus, inhibition of DUB activity leads to a change of cell morphology and formation of protein aggregates resembling GCIs. DUB inhibitors provide a useful tool to eluciate the manifold mechanisms of DUB functions in cells.