(A) Schematic representation of polyQ expanded mutant Htt-Ex1 variants (mHtt-Ex1) used in this study. mHtt-Ex1 contains N17 (red), an expanded polyQ tract (grey), the proline-rich domain ('PRD', blue), as well as a short, 10-amino acid C terminal tail ('C', white). Variants were generated by deleting the regions flanking the polyQ domain. An additional C-terminal S tag (not shown) was used for immunoblot detection of recombinantly produced mHtt. For recombinant expression of all mHtt variants, a 51-mer polyQ tract was used. (B) Kinetics of formation of SDS-insoluble, heat-stable aggregates for the mHtt-Ex1 variants as measured by the filter trap assay. Aggregation of purified recombinant mHtt-Ex1 variants from (A) was initiated by cleavage of a solubilizing N-terminal GST tag by TEV protease. All aggregation reactions were performed at a concentration of 3 µM. Data is representative of at least three independent experiments. (C) Rate of accumulation of amyloid aggregates for the mHtt variants measured by the ThioflavinT fluorescence assay. All variants were aggregated at a concentration of 3 µM. (D) Normalized curves of ThioflavinT amyloid aggregation kinetics from (C) used to compare kinetic rates. Variants without the N17 region (∆N, ∆N∆P) form amyloids much more slowly than variants with the N17 region (Ex1, ∆P). (E) Fluorescence images of each of the mHtt mutants transfected into the ST14a striatal neuron-derived cell line. mHtt variants were constructed similar as in (A) with a 51-mer polyQ length. Instead of a N-terminal GST and C-terminal S-tag, constructs had only a C-terminal GFP tag. Images were taken 48 hr post transfection. Scale bar is 20 µm. (F) Percentage of transfected cells containing aggregates for each of the Htt mutants as shown in (E). Similar to as seen in vitro deletion of the N17 region leads to overall less aggregation while deletion of the PRD leads to overall more aggregation. Data are mean ± SEM of three independent experiments counting at least 150 cells. *p <0.05, **p<0.005, ***p<0.001. (G) Summary model for how the N17 and PRD regions contribute to mHtt aggregation propensity.