Ubiquilin-1 protein levels are decreased in late onset AD patient brains. A, Western blot of ubiquilin-1 and α/β-tubulin levels in post-mortem cortical brain samples (50 μg of total protein/lane) of human patients (aged 72–85 years) arranged by Braak staging, as indicated. All of the samples were handled under identical conditions, and the blots were exposed simultaneously on the same film. The genotypes of each patient for ubiquilin-1 and APOE SNPs are indicated below each lane. H indicates heterozygosity for that SNP. SNPs highlighted in red indicate risk alleles. The rs2781002 risk allele has been proposed to be either C (5) or T (8) (in italics). B, quantification of ubiquilin-1 protein levels as a function of Braak stage, displayed as ratios of the intensities of the bands corresponding to ubiquilin-1 over α/β-tubulin, determined densitometrically for each sample (symbols) and their means ± S.E. (bars). The p values were determined by t test comparison with Stage I. Samples 6, 7, 16, 21, 22, and 24 have lower tubulin levels, which may be indicative of degradation. Excluding these samples from the analysis shows that all Braak stages analyzed (II–VI) were significantly different from Braak stage I. The Student's t test comparison between groups were as follows: stage I versus II, p < 0.05; I versus III, p < 0.01; I versus IV, p < 0.01; I versus V, 0.001; and I versus VI, p < 0.01).

A transient interaction exists between ubiquilin-1 and APP. A, histogram depicting interaction partners of ubiquilin-1 derived from a yeast two-hybrid screen, displayed as individual genes (italicized) or organized into functional classes. B, metal affinity pulldown (see “Experimental Procedures”) of APP from HeLa cells overexpressing c-Myc-tagged ubiquilin-1 and/or APP, as indicated. The cells were treated with or without DSP (Lomant's reagent) prior to lysis. Co-precipitating ubiquilin-1 was detected with an anti-c-Myc antibody. Mock cells were transfected with yellow fluorescent protein (YFP). The asterisk marks a nonspecific band. C, purified ubiquilin-1 binding to APP immunoprecipitated from rat brain with or without DSP cross-linking. No antibody (No Ab) and cytochrome c (Cyt C Ab) immunoprecipitations were used as controls. 0.25 μg of purified ubiquilin-1 was used as a loading control. Coomassie Blue staining of gels after transfer indicated that DSP treatment significantly inhibited transfer of proteins out of the gel (data not shown), and thus these lanes are underrepresented. The experiments in B and C were repeated three times with similar results.

Ubiquilin-1 exerts chaperone activity on APP in vitro and in live cells. A, inactivation kinetics of CS alone or in mixtures supplemented with purified Hsp90 or ubiquilin-1, as indicated, upon exposure to 43 °C. The inset shows the mean times (± S.E.) for half of the enzyme molecules to become inactivated (t½) from four separate experiments. B, aggregation kinetics monitored by light scattering at 420 nm of purified AICD alone or in combination with ubiquilin-1 upon exposure to 43 °C. AICD aggregated in every one of five separate trials, whereas AICD supplemented with ubiquilin did not demonstrate an increase in light scattering in four separate trials. C, dot blot analysis of in vitro aggregation mixtures of AICD with ubiquilin-1, AICD plus GST as a control, or AICD plus the UBL domain of ubiquilin upon exposure to 43 °C. Aliquots were removed at the indicated times, and insoluble material was sedimented by centrifugation. Equal amounts of total, soluble, and insoluble fractions were spotted onto a nitrocellulose membrane and probed with an anti-AICD antibody. D, AFM images of aliquots of total material from mixtures set up as in B after 48 h of incubation at 43 °C. Aggregates had a diameter of 46 ± 24 nm (n = 611 particles). E, representative images of the three patterns of APP and APP-GFP localization upon overexpression in differentiated PC12 cells observed by immunofluorescence (lower panels) and fluorescence (upper panels) microscopy, respectively. F, quantification of APP-GFP fluorescence patterns in differentiated PC12 cells co-transfected with APP-GFP and vector (pcDNA), or ubiquilin-1, as indicated. Total numbers of scored cells are displayed above each bar, and significant p values are indicated within the bar. In the inset, the effect of ubiquilin-1 expression on total APP-GFP accumulation is shown by Western blotting with anti-APP and anti-GFP antibodies. Tubulin is used as a loading control. G, quantification of APP-GFP fluorescence patterns in rat primary cortical neurons, as in F. H, quantification of APP-GFP fluorescence patterns in differentiated PC12 cells treated with RNAi oligonucleotides targeting ubiquilin-1 or control RNAi oligonucleotides (left panel) and Western blot confirming decreased levels of ubiquilin-1, but not ubiquilin-2 (asterisk), protein levels only in cells treated with ubiquilin-1 RNAi oligonucleotides (right panel). I, filter trap assay of total lysates from cells as in F, filtered through cellulose acetate membranes (0.22 μm) probed with an anti-APP antibody. The bottom panel is the quantification of APP levels normalized to cells not expressing ubiquilin-1 from three separate experiments. The p values in A, C, and I were determined by a Student's t test. The p values in F–H were determined by Fisher's exact test and are indicated within the bars.

Ubiquilin-1 reduces APP amyloidogenesis and APP-associated cell death. A, quantification of Aβ₄₂/Aβ₄₀ production in HeLa cells co-transfected with vector only (pcDNA), APP alone, APP and ubiquilin-1, or APP and UBL domain, as indicated. n.s., not significant relative to APP or APP plus ubiquilin-1. The data are pooled from three separate experiments. B, quantification of cell death determined by propidium iodide staining in HeLa cells transfected with the yellow fluorescent protein (YFP), APP, APP and ubiquilin-1, or APP and ubiquilin-1 RNAi oligonucleotides, as indicated. The n values are as follows: YFP, 6; APP, 4; APP/Ubiquilin-1, 4; APP/Ubiquilin-1 RNAi, 7; and Ubiquilin-1 RNAi, 4. C, representative photomicrographs of PI-stained cells in various experimental conditions stated above.