Figure 3. ATXN1 oligomer complexes from Atxn1^154Q/+ mouse cerebellum induce the formation of new ATXN1 oligomers.

(A) Histological analysis in Atxn1^154Q/+ brain sections reveals the regional presence of oligomers in PCs stained with calbindin (top panel) and oligomers stained with F11G3 (bottom panel) in degenerating PCs (bottom right panel) but not surviving PCs (bottom left panel). (B) Cell-based seeding assay: mRFP-ATXN1(82Q) cells were incubated with the indicated fractions (Frac 7–14) and the oligomeric and fibrillar inclusions were quantified. *p < 0.05. **p < 0.01. (C) Immunostaining for oligomers (F11G3, green) of mRFP-ATXN1(82Q) cells following treatment with fraction 11. Arrowheads denote internalized oligomers. Scale bar 15 μm. (D) Immunostaining for ATXN1 (11750, green signal) of mRFP-ATXN1(82Q) after treatment with fraction 11 from Atxn1^154Q/+ cerebellum (top row) demonstrated that exogenous ATXN1 (green signal) is internalized into the cells. Cells treated with fractions 11 from wild-type mouse cerebellum (bottom row) did not show internalized ATXN1 entities. Scale bar 15 μm. (E) Quantification of the percentage of mRFP-ATXN1(82Q) cells with oligomeric (blue bars) or fibrillar (red bars) inclusions following treatment of fraction 11 pre-incubated with the indicated antibodies *p < 0.05.

DOI: http://dx.doi.org/10.7554/eLife.07558.011

Figure 3—figure supplement 1. ATXN1 oligomers are detected in mRFP-ATXN1(82Q) cells.

Immunostaining for oligomers (F11G3, green) in a stable cell line expressing mRFP-ATXN1(82Q) (top panels) or a similar stable cell line expressing 82Q ATXN1 with the S776A mutation (bottom panels). The S776A mutation prevents formation of Nis and oligomers. Scale bar 15 μm.

DOI: http://dx.doi.org/10.7554/eLife.07558.012

Figure 3—figure supplement 2. Anti-oligomer antibody F11G3 does not get internalized into the cells.

(A) Daoy mRFP-ATXN1(82Q) stable cell lines were treated with SEC fraction 11 from Atxn1^154Q/+ cerebellum pre-incubated with F11G3 for 10 hr. After fixation cells were stained with a secondary anti-mouse IgM alexa 488 (green signal). No intracellular signal was observed in any cell. Low extracellular signal was observed (arrowhead). Scale bar 15 μm. (B) Cell media was analyzed by western blot using secondary anti-mouse IgM. Heavy and light chains from IgM were observed in treated samples but not in untreated control.

DOI: http://dx.doi.org/10.7554/eLife.07558.013