Fig 2: TGs preferentially cross-link mutant ATXN1 and regulate its solubility and oligomerization.(A) In vitro TG assay of ATXN1[82Q] with recombinant human TG2 under various conditions: with normal or heat-inactivated enzyme; with or without Ca2+ ion, pan-TG inhibitor cystamine (40 mM), or TG2-specific inhibitor LDN-27219 (2 mM). The proportion of ATXN1 high MW (HMW) species in each lane represented in bottom graph. Data shown as mean ± SD, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test. (B) In vitro TG assay of ATXN1[82Q], ATXN1[30Q], and ATXN1[2Q] with recombinant human TG2. Either 1× (+) dose or 2× (++) doses of ATXN1 was used for the reaction. The proportion of ATXN1 HMW species in lane 10–17 represented in bottom graph. Data shown as mean ± SD, ****P < 0.0001, 1-way ANOVA, post hoc Tukey’s test. (C) Western blot analysis of Triton X-100–soluble or –insoluble ATXN1 HMW species and monomer extracted from HEK293T cells overexpressing ATXN1[82Q] and TG5 or LacZ. Empty, empty vector; LacZ, overexpression control. Triton X-100–insoluble ATXN1 (HMW species + monomer) to Triton X-100–soluble ATXN1 (monomer) ratio in each overexpression group represented in bottom right graph. Asterisk indicates nonspecific bands. Data shown as mean ± SD, ***P < 0.001, ****P < 0.0001, 2-tailed t test for bottom right graph; 1-way ANOVA for other graphs with post hoc Dunnett’s test. (D) Western blot analysis of Triton X-100–soluble or –insoluble ATXN1 HMW species and monomer extracted from HEK293T cells overexpressing ATXN1[82Q] and shRNA against TG5. Triton X-100–insoluble ATXN1 (HMW species + monomer) to Triton X-100–soluble ATXN1 (monomer) ratio in each experimental group represented in middle right graph. Data shown as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test. (E) Western blot analysis of soluble ATXN1 oligomers in cerebellar lysate of WT mice and SCA1 mice expressing shTgm5 (same lysate in Figure 3D) using oligomer-specific F11G3 antibody. Data shown as mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test.

Fig 3: TG5 but not TG2 colocalizes with nuclear inclusions of ATXN1 in the pons of patients with SCA1.(A) Representative IF images of ATXN1 nuclear inclusions (NIs) in the MAP2+ neurons that have large nuclei in the control and SCA1 patient pons. Scale bar: 50 µm. (B) Representative IF images of TG5 (left) and TG2 (right) in the neurons that display ATXN1 NIs in the pons of patients with SCA1. Control pons were also stained for comparison. White dashed-line boxes in the top images are enlarged in the bottom. Scale bar: 25 µm in the top images; 10 µm in the bottom images. (C) Quantification of the proportion of ATXN1 NIs that colocalize with TG. Images were obtained from 3 controls and 3 patients with SCA1, and the proportion was calculated from 20–30 NIs in each individual. Data shown as mean ± SD, ****P < 0.0001, 2-tailed t test. (D) Representative IF images of TG5 (left) and TG2 (right) in the CALB1+ Purkinje cells in control and SCA1 patient cerebellum. White dashed-line boxes in the middle images are enlarged in the bottom. Scale bar: 50 µm in the top and middle images; 20 µm in the bottom images. (E) Quantification of the proportion of Purkinje cells with nuclear TG. Images were obtained from 3 controls and 3 patients with SCA1, and the proportion was calculated from 18–24 Purkinje cells in each individual. Data shown as mean ± SD, **P < 0.01, ***P < 0.001, 2-way ANOVA, post hoc Tukey’s test.