Ubiquilin is found in htt inclusions and its levels decrease in the R6/2 model of HD.

<p>(A) Confocal staining of a 15-week old end-stage R6/2 brain section of the hippocampus showing staining with anti-ubiquilin (red), anti-htt EM48 (green) and DAPI (blue). The merged image of the red and green fluorescence images shows ubiquilin colocalizes with htt inclusions (arrows). Bar = 15 µm. (B) Equal amounts of protein in whole brain homogenates from three end-stage R6/2 mice and three 19 week-old WT mice were immunoblotted for ubiquilin and for actin. Note the decline in ubiquilin levels in end-stage R6/2 animals.</p

Restoration of ubiquilin levels in animals carrying the R6/2 transgene and accompanying changes in htt protein levels.

<p>(A) Immunoblots of lysates made from dissected brain regions of 9 week-old mice with different genotypes obtained after crossing UBQ1 transgenic mice with R6/2 mice and probed for ubiquilin (upper panel) and for actin (lower panel). Note successful overexpression of ubiquilin in animals carrying the ubiquilin-1 transgene. (B) Immunoblot analysis similar to A, but this time showing ubiquilin levels in different regions of the brain (Ce  =  cerebellum, BS  =  Brain stem, FC  =  frontal cortex, St  =  striatum, Hi  =  hippocampus, VC =  visual cortex). Note overexpression of ubiquilin in all tissues of transgenic mice carrying the ubiquilin-1 transgene. (C) Immunoblots of equal amounts of protein in lysates made from the striatum (St), hippocampus (Hi) and frontal cortex (FC) of 9 week-old mice from the same cross mentioned above (W = non-transgenic for UBQ1 or R6/6, U = UBQ1 transgenic, R = R6/2 transgenic, R/U = R6/2-UBQ1 double transgenic) and probed for ubiquilin or actin. (D) Immunoblots of brain lysates from end-stage mice showing R6/2-UBQ1 double transgenic mice have higher ubiquilin levels detected in the resolving gel than R6/2 animals, which correlated with decreased ubiquilin that was trapped in aggregates in the stacking gel. Also included is a lysate from a 19-week old WT animal (right lane). (E and F) Quantification of ubiquilin levels in 9-week (E) and end-stage or equivalent time-point animals (F) showing the amount of ubiquilin protein expression in animals with different genotypes used in our study. Note that ubiquilin levels are reduced by 70% in end-stage R6/2 animals compared to wild type age-matched controls (<i>p</i> = 0.0011). Furthermore, ubiquilin levels were fully restored in the brains of end-stage R6/2-UBQ1 double transgenic mice compared to R6/2 transgenic mice (<i>p</i> = 0.0017). (G) Immunoblot of same lysates shown in D with EM48 antibody showing ubiquilin overexpression in R6/2-UBQ1 double transgenic mice have reduced levels of soluble mutant htt protein accumulation compared to R6/2 animals. Note the WT lysate was loaded on the left lane. (H) Quantification of soluble htt protein shown in panel G.</p

Ubiquilin-1 overexpression modifies aggregate load in the hippocampus and cortex but not the striatum.

<p>(A) Representative fluorescence microscopy images of EM48 and DAPI stained cryostat sections of the CA1 region of the hippocampus in R6/2 transgenic and R6/2-UBQ1 double transgenic mouse at 6 weeks, 9 weeks and following end-stage euthanasia. Bar = 15 µm. (B) Similar to A, but showing representative sections from the dentate gyrus in end-stage mice. Bar = 15 µm. (C) Quantification of htt inclusions >0.5 µm in size in the CA1 region of the hippocampus at 6 weeks, 9 weeks and end-stage R6/2 and R6/2-UBQ1 double transgenic mice. The R6/2-UBQ1 double transgenic mice contained 22% fewer inclusions than R6/2 mice at 6 weeks (<i>p = </i>0.04), but not at the other times. (D) Quantification of htt inclusions >1 µm in size in the CA1 region of the hippocampus at 6 weeks, 9 weeks and end-stage R6/2 and R6/2-UBQ1 double transgenic mice. The R6/2-UBQ1 double transgenic mice had 40% fewer inclusions at 9 weeks compared to R6/2 transgenic mice (<i>p</i> = 0.027). (E and F) Similar to B and C, but showing htt inclusions in the cortex. R6/2-UBQ1 double transgenic mice had 8.5% fewer inclusions greater than 0.5 µm at the end-stage of disease. (G, H) Similar to E and F, but comparing inclusions in the striatum. There was no difference in the number of inclusions in the striatum between the two genotypes at any time point.</p

Generation of transgenic mice that overexpress human ubiquilin-1.

<p>(A) Schematic of the Thy1.2 expression construct used to generate ubiquilin-1 transgenic mice. Human ubiquilin-1 with an N-terminal FLAG tag was cloned in the appropriate orientation between the XhoI site of the Thy1.2 expression cassette. (B) Southern Blot of the first generation offspring of two founder mice (48 and 62). (C) Validation of a PCR genotyping protocol. Amplification of the transgene was only observed in mice that Southern blotting revealed to be positive. (D) Immunoblots of brain cortical lysates with an anti-FLAG antibody and for tubulin indicated that line 62 offspring express higher levels of FLAG-ubiquilin-1 than line 48. (E) Immunoblots of equal amounts of total brain lysates from 12 month-old WT mouse, 12 month-old Ubqln-1 48 transgenic mouse, 12 month-old Ubqln-1 62 transgenic mouse and end stage 15 week-old R6/2 transgenic mouse. The top panel was probed with a monoclonal anti-ubiquilin antibody (Invitrogen antibody clone 3D5E2) and the lower panel with a different monoclonal anti-ubiquilin antibody (Novus antibody clone 5F5). Note two immunoreactive ubiquilin bands are seen at ∼70 kDa and at ∼90 kDa, which we presume is a modified form of ubiquilin. Both blots were also probed for actin to ensure equal loading. (F) Cryostat sections of a Ubqln-1 62 transgenic mouse brain (a–f) and WT mouse brain (g-i) showing anti-FLAG antibody staining (Alexa 594, left panels) and corresponding DAPI staining (center panels) and the result of merging the fluorescent and DAPI signals (right hand panels). The brain sections shown are of the hippocampus (a–c and g–i) and cerebellum (d–f). Identical exposure settings were used for the left hand panels.</p