Ldn 27219

For the TG assay of ATXN1[82Q], 1 μg of purified human ATXN1[82Q] was mixed with 250 ng of normal or boiled (95°C for 3 minutes) recombinant human TG2 (R&D Systems, 4376) with or without cystamine (Tocris, 4981, final concentration: 40 mM) or LDN-27219 (Tocris, 4602, final concentration: 2 mM) to inhibit Tg activity in TG assay buffer (25 mM Tris-HCl, 5 mM CaCl₂, pH 8) supplemented with protease inhibitor cocktail. For the TG assay of ATXN1 with different polyQ-length, 1 μg of purified human ATXN1[2Q], ATXN1[30Q], or ATXN1[82Q] was mixed with 250 ng of recombinant human TG2 in the TG assay buffer supplemented with protease inhibitor cocktail. Total volume of the reaction mixture was 50 μL, and the final concentration of urea was 300 mM. The reaction mixture was incubated at 30°C for an hour with gentle shaking. For terminating TG reaction, NuPAGE LDS sample buffer (4×) was added into reaction mixture and boiled at 95°C for 15 minutes. For Western blot, samples were cooled at room temperature, loaded onto 3%–8% Tris-acetate gels and steps were performed as described above.

iPSCs derived from patients with SCA1 were first induced into NPCs and differentiated into neurons (15 (link)). Briefly, NPCs were seeded in a Cultrex-coated T75 flask within NIM (1:1 mixture of DMEM/F12 and neurobasal with B27, N2, and GlutaMAX [2 mM]) supplemented with FGF2 (20 ng/mL) and incubated for 3 days. The cells were further incubated for 4 days within NIM without FGF2, and differentiated within NDM (Neurobasal with B27; GlutaMAX, 2 mM; BDNF, 20 ng/mL; GDNF, 10 ng/mL; NT-3, 10 ng/mL; db-cAMP, 100 μM; and ascorbic acid, 200 μM) for 14 days with exchange of media every 48 hours. The differentiated neurons were then passaged with trypsin into 24-well plates. To transduce neurons, virus was added into the media on the next day of passaging, and neurons were incubated for 9 days with full exchange of media in every 3 days. For inhibitor treatment, neurons were treated with LDN-27219 (Tocris, 4602) at 72 hours after passaging and incubated for 3 days.