The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates

A common feature of CAG-expansion neurodegenerative diseases is the presenc e of intranuclear aggregates in neuronal cells. We have used a synthetic fu sion protein containing at the NH2 terminus the influenza hemoagglutinin ep itope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG ) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polygluta mine tracts and on the levels of expression of the fusion protein. Moreover , transglutaminase overexpression caused an increase of insoluble aggregate s only in cells expressing the mutant expanded protein. Conversely, treatme nt of cells with cystamine, a transglutaminase inhibitor, reduced the perce ntage of aggregates. We found also that the inhibition of the proteasome ub iquitin-dependent degradation increased the formation of intranuclear aggre gates. These data suggest that length of polyglutamine tract, its expressio n, unbalance between cellular transglutaminase activity, and the ubiquitin- degradation pathway are key factors in the formation of intranuclear aggreg ates. (C) 1999 Academic Press.