Comparison of MW8 and ubiquitin staining patterns.

<p>Higher magnification microscopy analysis was used in order to compare MW8 and ubiquitin staining patterns in single R6/2 neurons. In CA1, CA3 and cortex, MW8 labelling could be observed in cells before any aggregates were visible, a phenomenon that was not apparent with ubiquitin staining. The initial morphologies of MW8-positive aggregates were different from ubiquitin-positive inclusions, in that only nucleation centres of the aggregates were ubiquitinated and the MW8-immunolabelled Htt protein that was not localised to the nucleation centre did not appear to be ubiquitinated. In contrast, aggregates with clear nucleation centres were visible by 25 days in the striatum. And at the same time, larger punctate ubiquitin-labelled inclusions were already visible in STR neurones. Ubiquitinated inclusions were much bigger than MW8-positive aggregates and did not change much in size, even though Htt aggregates got bigger with time.</p

Thetiming of ubiquitination is dependent of cell soma size in R6/2 neurons.

<p>Regardless of whether or not huntingtin aggregates (visualized by staining with MW8 and ubiquitin antibodies) formed early or late, aggregates that formed in cells with small (DG neurons) and medium-sized (CA1 and STR) soma were found ubiquitinated within 2 to 3 days of initial aggregation. By contrast, there was typically a more than 3 days lag between initial aggregate formation and inclusion ubiquitination in cells with large soma (CA3). Scale bar  = 50 µm.</p

Aggregate appearance in CA1 and dentate gyrus (DG).

<p>In R6/2 hippocampus, Htt aggregates visualised by staining with the MW8 antibody could not be seen at 18 days of age but could be found in many neurons at 19 days in the CA1 (B, black arrowheads) and increased in number with time (C, black arrowheads). Cell distribution in this region is shown in a parallel section showed with a cresyl violet stain (A”–C”). In the DG, MW8-positive aggregates were absent up to 24 days, but were present at 26 days (F, black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–F’). Cell distribution in both regions was visualised with a cresyl violet (CV) stain (D”–F”). Scale bar  = 50 µm.</p

Aggregate appearance in different brain regions over time.

<p>Htt aggregates visualised by staining with the MW8 antibody (shown here at 14, 19, 22, 24, 26, 29, 41 and 63 days) in indisium griseum (IG), CA1, CA3, dentate gyrus (DG) and dorso-medial and ventro-lateral striatum (STR DM and STR VL) of R6/2 brains. A few aggregates were visualised in the IG as early as 14 days of age. In the hippocampus, inclusions were present at 19 days in CA1, whereas in the CA3 a few aggregates were visible at 29 days of age. In the dentate gyrus, MW8-positive aggregates were present at 26 days. In the striatum, Htt aggregates could be seen at 26 days of age in STR DM and STR VL. The number of aggregates in all the regions increased with time from the day of appearance. Scale bar  = 50 µm.</p

Inclusion appearance in different brain regions over time.

<p>Ubiquitinated Htt aggregates visualised by staining with the ubiquitin antibody (shown here at 14, 19, 22, 24, 26, 29, 41 and 63 days) in IG, CA1, CA3, DG and STR of R6/2 brains. A few inclusions are visible in the IG at 19 days of age. In the hippocampus, aggregates were present at 24 days in CA1, whereas in the CA3 a few aggregates were visible at 41 days of age. In the DG, a few ubiquitin-positive inclusions were present at 41 days. In the striatum, inclusions could be setected at 41 days of age in STR DM and STR VL. The number of aggregates in all the regions increased with time from the day of appearance. Scale bar  = 50 µm.</p