Tissue transglutaminase crosslinks ataxin-1: possible role in SCA1 pathogenesis

Abstract

Transglutaminase type 2 (TG2) has recently been implicated in crosslinking of mutant huntingtin protein into aggregates. Here we show that TG2 also crosslinks spinocerebellar ataxia-1 (SCA1) gene product ataxin-1. HeLa cell lysates expressing GFP tagged ataxin-1 with 2, 30 or 82 glutamines showed covalent crosslinking of ataxin-1 when incubated with exogenously added TG2. This crosslinking was inhibited by TG2 inhibitor cystamine. SCA1 transgenic mice which overexpress the mutant ataxin-1 in cerebellar Purkinje cells showed elevated nuclear TG2 in the absence of ataxin-1 nuclear aggregates. The addition of purified TG2 to the nuclear extracts or addition of SCA1 nuclear TG2 to GFP-Q82 HeLa cell lysates resulted in the formation of insoluble aggregates. These data indicate that ataxin-1 is a substrate of TG2. Further, in SCA1 TG2 may translocate to the nucleus in response to nuclear accumulation of mutant ataxin-1 at early stages of the disease.

Fig. 1

Western blots of HeLa cell lysates expressing ataxin-1 (Q2, 30 or 82)-GFP. (A and B) Time course crosslinking of ataxin -1-GFP by TG2. Immunoreactive bands were detected with anti-polyglutamine (A) and anti-GFP (B) antibodies. When indicated, 10 μl aliquots were removed and electrophoresed on a reducing 4–20% polyacrylamide gel. In controls (A) TG2 was omitted except ‘Enz’ (enzyme) lane. The reaction produced large multimers, which stayed on top of the gel. (C) Silver stained gel after 0 and 30 min incubations with TG2. (D and E) GFP immunoblots showing inhibition of ataxin-1 aggregate formation in the presence of various concentrations of TG2 inhibitor cystamine. In ‘E’ the reaction mixtures were incubated for 10 min with guinea pig TG2. Lysate prepared from HeLa cells transfected with GFP without extra Q tract (D) shows no crosslinking.

Fig. 2

Immunodetection of tissue transglutaminase type 2 (TG2) in cerebellar fractions in SCA1 transgenic and wildtype mice. (A and B) Western blots showing TG2 and calbindin D28k in cerebellar fractions in 4 (n = 4) and 6 (n = 3) weeks old wildtype and SCA1 heterozygous mice. Since calbindin D28k is expressed only in Purkinje cells of the cerebellum we used calbindin as an internal control. As reported previously [17], lower nuclear calbindin levels were observed in SCA1 mice as compared to wildtype animals. On the other hand, elevated levels of nuclear TG2 were seen in SCA1 mice. Figure A also shows altered nuclear and cytosolic distribution of TG2 in 4 weeks old SCA1 mice. TG2 appeared as a single band of about 75 kDa. (C) Immunoblot showing TG2 crosslinked nuclear proteins of 4 weeks old SCA1 and wildtype mice. The insoluble aggregates stayed on top of the gel. The crosslinked proteins were detected with anti-polyglutamine antibody. The reaction was catalyzed by exogenous guinea pig TG2 in the presence and absence of cystamine. (D) Endogenous TG2 in the nuclear fractions crosslinked Hela Q82-GFP (detected with anti-GFP antibody). The nuclear fractions were prepared from the cerebella of 5 weeks old SCA1 homozygous and wildtype mice. Equal amounts of proteins were used in all reactions. In SCA1 mice the intensity of immunoreactive aggregates was markedly higher than the age-matched wildtypes.