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. 2015 May 19;4:e07558. doi: 10.7554/eLife.07558

Figure 2. ATXN1 oligomers form high molecular weight complexes that correlate with motor impairment.

(A) Western blot of cerebellar lysates of 8-, 18- and 28-week old Atxn1154Q/+ mice using F11G3. Right panels indicate cropped regions of the blot with exposure adjusted to permit comparative quantification among mice of different ages. (B) Correlation plots between the different molecular weight oligomers (Figure 1A) and the latency to fall in the rotarod assay in Atxn1154Q/+ mice. Each black dot corresponds to one Atxn1154Q/+ mouse from 1A. (C) Size exclusion chromatography (SEC) from Atxn1154Q/+ mouse cerebellum probed for oligomers (F11G3, left panel) and ATXN1 (11750, right panel). Note that top panels reveal a higher exposure than the bottom panels. In corresponded to Input before fractionation. (D) Representative atomic force microscopy (AFM) pictures from fractions 7, 8, 11 and 12 (Scale bar 140 nm). (E) MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay performed on cerebellar granule neurons. Cells grown 7 days in vitro (DIV) were treated with different fractions (labeled below the x-axis) and viability was measured 18 hr following treatment (black bars). Gray bars denote viability following incubation of fraction 11 previously co-incubated with the indicated antibodies. *p < 0.05.

DOI: http://dx.doi.org/10.7554/eLife.07558.006

Figure 2.

Figure 2—figure supplement 1. Correlation between the levels of 154Q or 2Q ATXN1 and the latency to fall from the rotarod.

Figure 2—figure supplement 1.

(A) A slight decrease in the level of 154Q ATXN1 was observed with age in Atxn1154Q/+ samples, but no changes were observed in the levels of 2Q ATXN1. The antibody 11750 was used to detect ATXN1 in western blot. (B) No correlation was observed between the levels of 154Q or 2Q ATXN1 and the latency to fall from the rotarod.
DOI: http://dx.doi.org/10.7554/eLife.07558.007
Figure 2—figure supplement 2. ATXN1 oligomers characterization by hydrophobic interaction column.

Figure 2—figure supplement 2.

Fraction 11 of the SEC from Atxn1154Q/+ mouse cerebellum was passed through a hydrophobic interaction column (HIC). ATXN1 oligomer complexes were detected in fractions with high levels of exposed hydrophobic domains. F11 corresponded to Fraction 11 input; FT corresponded to the HIC flow through; (−) and (+) indicates lower and higher levels of hydrophobic fractions, respectively. Oligomers were detected with F11G3 and ATXN1 was detected with 11750.
DOI: http://dx.doi.org/10.7554/eLife.07558.008
Figure 2—figure supplement 3. ATXN1 oligomers from Atxn1154Q/+ are highly stable.

Figure 2—figure supplement 3.

Dot-blot probed with F11G3 of SEC fractions from Atxn1154Q/+ cerebellum incubated at 37°C for 12, 24 and 48 hr.
DOI: http://dx.doi.org/10.7554/eLife.07558.009
Figure 2—figure supplement 4. ATXN1 oligomeric complex induce cellular toxicity.

Figure 2—figure supplement 4.

Cellular staining of CGPC cells after fraction 11 treatment using Tuj1 antibody: the ATXN1 oligomer complex induces degeneration in cells.
DOI: http://dx.doi.org/10.7554/eLife.07558.010