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

Figure 1. ATXN1 oligomers are located in areas prone to SCA1 degeneration.

(A) Western blot analysis of soluble fractions from cerebella shows the existence of amyloid oligomers exclusively in Atxn1154Q/+ mice model but in neither wild-type nor Atxn1−/− controls. (B) Immunofluoresence studies of Atxn1154Q/+ brain sections using anti-Ataxin-1 (green) and F11G3 (red) confirms the ATXN1 identity of the amyloid oligomers. Scale bar = 15 μm. (C) Western blot using anti-ATXN1 antibody showed that the isolated oligomers (IP with F11G3), were indeed ATXN1 oligomers. These ATXN1 oligomers were IP’d exclusively from Atxn1154Q/+ mouse cerebellum and not from Atxn1−/− controls. (D) AFM analysis showing brain-derived ATXN1 oligomers IP from Atxn1154Q/+ mouse using F11G3. Scale bar 200 nm. (E) Double staining using the Purkinje cell (PC) marker, calbindin (green) and F11G3 (red) revealed that ATXN1 oligomers accumulate in PC dendrites before dendritic degeneration is observed (top panel). With progression of dendritic degeneration, ATXN1 oligomers tend to accumulate in both the cytoplasm and nucleus (middle and bottom panel). Scale bar 30 μm. No degeneration is represented by (−), a low level of degeneration is represented by (+), and advanced degeneration by (++). Mice analyzed were 28 weeks old. (F) Histological staining for ATXN1 (top panel) and oligomers (F11G3, bottom panel) in cerebellum and cortex of Atxn1154Q/+ (left panels) and WT mice (right panels). Scale bar 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.07558.003

Figure 1.

Figure 1—figure supplement 1. ATXN1 oligomers in SCA1 mouse.

Figure 1—figure supplement 1.

Whenever the antibody F11G3 was preincubated with the antigen Aβ or IAPP oligomers, no immunolabeling was observed in Atxn1154Q/+ sections that were previously positive for oligomers. Double immunofluorescence between anti-ATXN1 antibody (green) and F11G3 (red) preincubated with recombinant oligomers show the positive signal only for ATXN1. Double immunofluorescence between anti-ATXN1 antibody (green) and F11G3 (red) preincubated with recombinant monomers show positive signal for both antibodies. Scale bar 25 μm.
DOI: http://dx.doi.org/10.7554/eLife.07558.004
Figure 1—figure supplement 2. ATXN1 oligomers levels are higher in the cerebellum than in the cortex.

Figure 1—figure supplement 2.

Western blots using F11G3 confirmed higher levels of ATXN1 oligomers in the cerebellum (Cer) than in the cortex (Cx) in the Atxn1154Q/+ mouse. ATXN1 was detected using 11750 antibody; arrowhead corresponded to 154Q ATXN1 band. 2Q ATXN band (*) demonstrated equal amount of loading between the Cer and Cx sample.
DOI: http://dx.doi.org/10.7554/eLife.07558.005