| Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, caused by an unstable CAG repeat expansion in the open reading frame of the first exon of the gene encoding the huntingtin protein. Striatal and cortical perinuclear cytoplasmic aggregates and intranuclear inclusions of mutant huntingtin fragments are prominent neuropathological hallmarks of HD. Our hypothesis is that the enzyme transglutaminase cross-links huntingtin into aggregates and intranuclear inclusions in HD.; Using confocal microscopy and double-label immunofluorescence, we demonstrate 99% co-localization of transglutaminase-catalyzed ϵ-(γ-glutamyl) lysine cross-links with huntingtin in HD nuclei. Furthermore, transglutaminase 2 co-localizes with both huntingtin (>70% overall) and cross-links (>98% overall) in intranuclear inclusions. We also demonstrate that transglutaminase 2 cross-links huntingtin in cells in culture.; We found a significant increase in transglutaminase 2 mRNA above control levels in HD cortex (225% of controls) and HD striatum (399% of controls). Transglutaminase 1, 2 (full-length and short forms), and 3, cross-link huntingtin in HEK 293T cells co-transfected with huntingtin containing an expanded glutamine domain (htt-N63-148Q-myc). Cells transfected with transglutaminase 1, 2 (full-length and short forms), and 3, do not cross-link huntingtin with normal glutamine-lengths in cells in culture. Treatment of cells with the transglutaminase inhibitor cystamine, results in decreased cross-linking and aggregation of huntingtin, and increased viability of cells co-transfected with htt-N63-148Q-myc and transglutaminase 2, or vector.; Since calmodulin regulates cross-linking activity of transglutaminase in other systems, we examined whether it plays a role in regulating transglutaminase in HD. We observed co-localization of huntingtin with calmodulin (>98%), transglutaminase 2 with calmodulin (>99%), and the ϵ-(γ-glutamyl) lysine cross-link with calmodulin (100%), in HD cortical nuclei. Transglutaminase 2 and polyglutamine-expanded huntingtin co-immunoprecipitated with calmodulin, in the presence of transfected htt-N63-148Q-myc and transglutaminase 2, or vector. Treatment with w5-hydrochloride, a calmodulin inhibitor, results in a decrease in cross-linked and aggregated huntingtin in cells co-transfected with transglutaminase 2 or vector, and htt-N63-148Q-myc.; These studies support the hypothesis that transglutaminase cross-linking is involved in intranuclear inclusion formation. Specific transglutaminase inhibitors may be beneficial treatments to decrease neuronal cell death in HD and lead to an improved quality of life for HD patients. |
