San1p accelerates nuclear Htt degradation. A, rescue expression of wt San1p in san1Δ yeast cells reduces Htt-NLS. ADH1 promoter-driven wild-type San1p or its RING mutants (C257S and C279S) were cotransformed into san1Δ cells with GAL4-driven Htt-GFP-NES (left) or NLS (right) containing 25 or 97 glutamines (Q#). Normalized Htt-GFP expression was measured after 2% galactose induction for 4 h. Bars indicate ±S.E. *, p < 0.0001 versus cotransformation with empty vector (–), C257S, or C279S. B, San1p reduces NLS-Q72 aggregates in HeLa cells. FLAG-tagged San1p was expressed in NESQ72 or NLSQ72 cells. After 48 h, the amount of soluble GFP-Htt (Sol Htt) was measured by immunoblot (upper bands and graphs) and those of insoluble aggregates (Ag Htt) were measured by filter trap analysis (lower dots and graphs). The intensity (relative to empty vector (–)) of bands or filter-retained spots was quantified and plotted. Bars indicate ±S.E. *, p = 0.0016; **, p < 0.001 versus empty vector. C, effect of San1p overexpression on NESQ72 (left) or NLSQ72 (right) aggregation was assessed by IB frequency. Cells with IBs were counted (n > 100 from three independent trials) and quantified (top graphs). Black bars indicate ±S.E. Representative images from each experiment are shown at the bottom. Arrows indicate IBs; scale bar = 20 μm. *, p = 0.0108 versus C279S. D, San1p accelerates NLSQ46 degradation. wt or C279S San1p was overexpressed in NESQ46 or NLSQ46 cells for 48 h, and a pulse-chase experiment was performed. Band intensities from SDS-PAGE were normalized by the ratio to those at time = 0 and plotted onto semi-log charts below with fitted lines (wt: straight lines; C279S: broken lines). Bars indicate ±S.E. E, San1p overexpression rescues acute pQ toxicity. NES- or NLS-attached Htt-GFP (signal) with 25 or 97 glutamines (Q#) were transiently coexpressed with wt San1p or C279S mutant (San1p) in HeLa cells for 72 h. Cell viability was measured by colorimetric assay (relative to NESQ25 plus San1pwt; n = 3). Bars indicate ±S.E. *, p = 0.005511.

UHRF-2 shows specific E3 activity for nuclear pQ. A, UHRF-2 ubiquitylates nuclear Htt in NES- or NLS-stable HeLa cells. FLAG-tagged UHRF-1 (1), UHRF-2 (2), or empty vector (–) was cotransfected with HA-tagged ubiquitin to NES or NLS HeLa cells for 48 h. After an additional 4 h of 5 μm MG-132 treatment, cell lysates were immunoprecipitated by anti-GFP antibody and subjected to immunoblots by anti-HA antibody (upper panels). This was reblotted by anti-GFP antibody to show immunoprecipitated monomeric Htt-GFP (middle panels). Lower panels show UHRF-1 or UHRF-2 expression in pre-immunoprecipitated lysates. B, schematic diagram showing the wt and mutants of UHRF-2. The RING domain (black box) is at its carboxyl terminus, and one of the critical cysteine residues is at amino acid 735, which was mutated to disrupt ligase activity (C735S). ΔNLS is a nuclear localization signal-defective mutant, from which amino acids 602–693 (dotted box) were deleted. C, wt UHRF-2 decreases aggregate burden. NESQ72 (left) or NLSQ72 (right) cells were transfected with wt, C735S, or ΔNLS mutants of UHRF-2 and evaluated for filter-trapped Htt-GFP. The middle panels show expression of the UHRF-2s detected by anti-FLAG antibody (FLAG). Bars indicate ±S.E. *, p = 0.0149; **, p < 0.0001 versus wt. D, UHRF-2 accelerates Htt-GFP-Q72 degradation. wt, RING domain mutant (C735S), or ΔNLS UHRF-2 was overexpressed in NESQ72 or NLSQ72 cells and a pulse-chase experiment was performed after 48 h. Relative band intensities from SDS-PAGE were normalized to 100% at time = 0 and plotted onto semi-log charts with fitted lines (wt: straight line; C735S: broken line; ΔNLS: dotted line). Bars indicate ±S.E. E, UHRF-2 rescues pQ toxicity. Htt-GFP-NES or NLS with 25Q or 97Q was transiently coexpressed with wt or C735S UHRF-2 in 293T cells. After 48 h, cell viability was measured by colorimetric assay (relative to NES plus wt UHRF-2; n = 12). Bars indicate ±S.E.; *, p = <0.01; **, p < 0.001.

UHRF-2 contributes to nuclear Htt degradation. A, effect of UHRF-2 RNAi on HeLa cells was evaluated by quantitative RT-PCR and immunoblot. The quantitative RT-PCR results showed 74% knockdown of mRNA (upper graph: relative quantity shown below the graph), resulting in a 50% protein reduction (lower blot: measured intensity shown below the blot). Bars indicate 95% confidence level. B, UHRF-2 knockdown increases nuclear aggregates in HeLa stable cell lines. After 72 h of transfection, the amount of soluble GFP-Htt (Sol Htt) was measured by immunoblot analysis (upper bands and graphs), and those of insoluble aggregates (Ag Htt) were measured by filter trap analysis (lower dots and graphs). Bars indicate ±S.E.; *, p = 0.0289; **, p = 0.0174 versus control. C, UHRF-2 knockdown increases IB frequency in HeLa stable cells. Effect of UHRF-2 on NESQ72 (left) or NLSQ72 (right) aggregation was assessed by IB frequency after 72 h of transfection. Cells with IBs (representative field, GFP) were counted (n > 100 from three independent trials) and quantified (top). Black bars indicate ±S.E.; *, p < 0.0001 versus control siRNA. Representative images are shown at the bottom. Arrows indicate IBs; scale bars = 20 μm. D, UHRF-2 rescues nuclear pQ toxicity. NES- or NLS-attached Htt-GFP 97Q was cotransfected with UHRF-2 siRNA (upper panel) or short hairpin RNA plasmid (lower panel) to 293T cells for 48 h. The effect of UHRF-2 RNAi on pQ toxicity was measured by colorimetric assay (relative to NES plus control; n = 13). Bars indicate ±S.E. *, p < 0.001. E, nuclear E3s degrade DRPLA aggregates. San1p (left panel,*, p = 0.0341 versus wt), UHRF-2 (middle panel,*, p = 0.001; ** p = 0.0023 versus wt) or siRNA against UHRF-2 (right panel,*, p = 0.0144 versus control) were coexpressed with HA-tagged DRPLA proteins in HeLa cells. After 48 h of transfection, the aggregate burden was analyzed with filter trap analysis. Intensities (relative to wt or control) of filter-retained spots were quantified by image analysis (upper corresponding dots) and plotted (lower graphs). Bars indicate ±S.E.

Physiological role of UHRF-2 in pQ pathogenesis. A, fluorescence micrograph of Htt-NLS-Q97 IB surrounded by coexpressed FLAG-tagged UHRF-2 in HeLa cells. IBs are visualized by GFP fluorescence; UHRF-2 was visualized by anti-FLAG and Alexa 546 secondary antibody. Scale bar = 10 μm. Arrows indicate IB; arrowheads indicate neighboring cell nuclei expressing UHRF-2 alone. B, Htt-NLS-Q97 IB transiently expressed in HeLa cells is surrounded by endogenous UHRF-2. Scale bar = 10 μm. Arrows indicate IBs. C, ataxin-1-Q78 IBs transiently formed in HeLa cells colocalize with UHRF-2. IB was labeled with anti-Myc antibody and Alexa 488 secondary antibody. Scale bar = 2 μm. Arrows indicate IBs. D, DRPLAQ80 IBs transiently formed in HeLa cells are positive for UHRF-2. IBs are labeled with anti-FLAG antibody and Alexa 488 secondary antibody. Scale bar = 2 μm. Arrows indicate IBs. E, Htt inclusion is positive for UHRF-2 in vivo. Section from a 6-week-old R6/2 mouse was stained with anti-UHRF-2 antibody. Anti-UHRF-2 antibody stained the IBs. IBs are labeled with anti-ubiquitin antibody. Scale bar = 10 μm. F, DRPLA inclusion is positive for UHRF-2 in vivo. Section from a postmortem DRPLA patient was stained with anti-UHRF-2 antibody. Anti-UHRF-2 antibody stained the IB labeled with anti-ubiquitin antibody. Scale bar = 10 μm. G, UHRF-2 overexpression rescues pQ toxicity in primary cultured mouse neurons. Mouse primary cortical neurons were cotransfected with Htt-Q97 and UHRF-2. After 96 h, coverslips were stained with anti-MAP2 antibody to identify neurons. The shape of the neurons determined by neurite outgrowth was classified into three grades: healthy, fully neurite-grown cells were grouped as grade 1 (a representative cell is shown in the right upper panels); cells with moderately grown neurites were grade 2 (middle panels); cells with unhealthy appearance were grade 3 (lower panels). At least 100 cells from four different coverslips were counted for each transfection. Scale bars = 10 μm. Black bars indicate ±S.E.; *, p = 0.0020; **, p = 0.0407; ***, p = 0.0009 versus wt NLS. H, UHRF-2 knockdown enhances pQ toxicity in primary cultured mouse neurons. Mouse primary cortical neurons were cotransfected with Htt-Q97 and mUHRF-2 or control short hairpin RNA plasmid. After 96 h, coverslips were stained with anti-MAP2 antibody to identify neurons. Evaluation was carried out by the same method as in the overexpression experiment. At least 100 cells from three different coverslips were counted for each transfection. Black bars indicate S.E.; *, p = 0.0049; **, p = 0.0182; ***, p = 0.007 versus NLS RNAi (–).