The protein huntingtin (htt), aggregated in neuronal nuclear inclusions, is pathognomonic of Huntington's disease (HD). Constructs, translated in vitro from the N terminus of htt, containing either polyQ23 from a normal individual, or polyQ41 or polyQ67 from an HD patient, were all soluble. Transglutaminase (TGase) crosslinked these proteins, and the aggregations did not have the staining properties of amyloid. More TGase-catalyzed aggregates formed when the polyglutamine domain of htt exceeded the pathologic threshold of polyQ36. Furthermore, shorter htt constructs, containing 135 aa or fewer, formed more aggregates than did larger htt constructs. TGase activity in the HD brain was increased compared with the control, with notable increases in cell nuclei. The increased TGase activity was brain specific. In lymphoblastoid cells from HD patients, TGase activity was decreased. TGase-mediated crosslinking of htt may be involved in the formation of the nonamyloidogenic nuclear inclusions found in the HD brain. The staining properties of nuclear inclusions in the HD brain revealed that they were not amyloid.The protein huntingtin (htt) contains a stretch of glutamines near its N terminus. When the length of the polyQ domain exceeds 36Q in htt, the lethal neurological disease called Huntington's disease (HD) occurs. Aggregated htt in the nuclei of neurons and in dystrophic neurites in the brain are the pathologic hallmarks of HD (1-3), although it remains unknown whether the aggregates are deleterious for neurons, or whether they represent an adaptive response to a desperate situation. Two hypotheses dominate our perception of how htt is aggregated: Perutz et al. (4) proposed that polyglutamine domains on neighboring proteins organize themselves into polar zippers. Such aggregations have the classic properties of amyloid (5, 6). Green proposed (7,8), in the second hypothesis, that TGase polymerizes htt.In Vitro Studies on Polar Zipper Formation with htt and PolyQ Peptides. Synthetic polyglutamine polymers, containing polyQ domains far shorter than the pathologic threshold of 36Q in HD, form polar zippers and aggregate in an aqueous medium (9). Polymerized htt is not seen in the brains of normal individuals with htt Ͻ Q36. The polar zipper hypothesis cannot easily explain this. Scherzinger et al. (1) reported that there is a threshold for polar zipper formation, and that aggregates form in vitro only when the polyQ domain is above 36Q. They showed that glutathione S-transferase (GST) fusion proteins from exon 1 of htt, with Q Ͼ 36, aggregate, but only after proteolytic cleavage of the GST domain (1).In Vitro Studies with htt and TGase. Kahlem et al. (8) studied guinea pig TGase and TGase isolated from rat brain; they showed that htt isolated from the brains of juvenile HD patients could be crosslinked in vitro into aggregates. To date, no one has reported on the activity of TGase in the HD brain, on the biophysical properties of the aggregates catalyzed by TGase, or on the optical properties of inclusions in...