Figure 1: PolyQ-expanded Htt inhibits ERAD and causes protein and chaperone accumulation in the ERQC.

(a) The ERAD substrate H2a accumulates in HEK 293 cells expressing myc-tagged Htt96Q, but not in the control GFP-expressing cells. Post-transfection (p.t.) times are indicated. Blots representative of three independent experiments are shown. (b) Inhibition of H2a degradation on expression of Htt96Q, but not of Htt20Q is revealed by metabolic labelling with [³⁵S]-cysteine in striatal STHdh^Q7/7 cells. The graph shows percent of H2a in the chase relative to pulse (100%), as an average of three experiments±s.e. (*P-value 2 h=0.03, *P-value 5 h=0.05, Student’s t-test (unpaired, two-tailed)). The experiments were performed with Htt containing only exon 1 of a polyQ-expanded Htt, a commonly used model that recapitulates the aggregation-prone behaviour of the whole protein^34,35. Coexpression of Htt7Q in the striatal cells mimics the typical heterozygous expression of pathogenic Htt from one allele in HD patients. (c,d) Very strong accumulation of H2a-RFP (c) and of calnexin (d) in the ERQC in STHdh^Q7/7 cells, apparently correlated to Htt96Q-myc visible aggregate formation. Htt20Q-myc does not affect the distribution of H2a-RFP in the ER (c). Scale bar, 10 μm. The graphs show the percent of cells with H2a-RFP (c) or calnexin (d) accumulated in the juxtanuclear region (ERQC, black bars) and those showing a disperse ER pattern (white bars) in STHdh^Q7/7 cells with visibly aggregated versus soluble Htt96Q. Bars are averages of three independent experiments±s.e. (N>60 in each) (**P-value<0.005, Student’s t-test (unpaired, two-tailed)).