Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process

Abstract

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.

FIG. 1.

The 26S proteasome activity decreases and ubiquitinated proteins accumulate with age. (A) Proteasome activity in wild-type flies decreased with age. The proteasome activity in wild-type fly heads was measured by hydrolysis of Suc-LLVY-AMC (chymotrypsin-like activity) on the indicated day posteclosion. (B) Accumulation of ubiquitinated proteins with age in wild-type flies. Wild-type fly head extracts were immunoblotted with antiubiquitin (anti-Ub) or anti-β-tubulin antibodies on the indicated day posteclosion. (C) The 26S proteasome activity in aged flies was significantly lower than that in young flies. Extracts of day 5 or day 30 flies were fractionated by glycerol density gradient centrifugation, and the Suc-LLVY-AMC hydrolysis activities were measured. The experiments were repeated three times. (D) Suc-LLVY-AMC hydrolysis activities of the same lysates shown in panel C were measured with the addition of 0.01% SDS [SDS(+)], a potent artificial activator of the 20S proteasome. The peptidase activity of the 20S proteasome did not significantly change between day 5 and day 30. (E) Immunodetection of the Rpn11 or the α2 subunit revealed a decrease in the level of this subunit with age in the 26S-containing fractions. Immunoblot analysis shows the even fractions probed with antibodies against Rpn11 and α2. (F) Fractions (fr.) 13 to 20 (19S or 20S included) or fractions 23 to 28 (26S included) shown in panel E were pooled and immunoblotted with antibodies against Rpn11 or α2. (G) The protein level of the 26S proteasome is lower in the aged fly than in the young fly. The amount of Rpn11 protein (the amount of the band indicated on panel F by a line) was determined by densitometric analysis. The graph shows the relative ratio of the amounts of Rpn11 in fractions 23 to 28 (26S included) at day 30 to that at day 5 from three individual experiments.

FIG. 2.

Identification of Rpn11 as a suppressor of age-related polyglutamine-induced progressive neurodegeneration. (A to H) Light photomicrographs of fly eyes expressing an expanded polyglutamine protein product of MJDtr-Q78, along with the indicated transgene. (A and B) Flies of genotype w; GMR-GAL4/UAS-LacZ; UAS-MJDtr-Q78/+ are shown at day 2 (A) and day 15 (B) posteclosion. These flies show eye degeneration, with a progressive loss of external pigment. (C to H) The progressive pigment loss phenotype was suppressed in flies expressing both MJDtr-Q78 and DANC (C and D), GS13423 (E and F), or Rpn11 (G and H). The fly genotypes shown are w; GMR-GAL4/GS13423; UAS-MJDtr-Q78/+ (E and F) and w; GMR-GAL4/UAS-Rpn11; UAS-MJDtr-Q78/+ (G and H). (I) The genomic structures around the DANC and GS13423 alleles are shown. (J and K) The DANC and hs-GAL4 flies (J) and the GS13423 and hs-GAL4 flies (K) were heat shocked (hs+) or not (hs−), and the expression levels of the surrounding genes, as well as that of GAPDH, were analyzed by RT-PCR. Note that rpn11 was upregulated in a GAL4-dependent manner. (L) Schematic of a single Drosophila ommatidium showing the regular trapezoidal arrangement of seven visible rhabdomeres within the photoreceptor neuron. (M) Representative semithin sections are shown of compound eyes from wild-type flies 2 days after eclosion. Normal ommatidia contain seven visible rhabdomeres at a given plane of the section. A representative single ommatidium is circled. (N to Q) Semithin sections of compound eyes from flies expressing GMR-Huntingtin 120Q, with LacZ as a control (N and O), or with Rpn11 (P and Q). When Rpn11 was coexpressed with Htt, the age-related loss of rhabdomeres was significantly improved (N to Q). The following genotypes are shown: w; UAS-LacZ/GMR-GAL4; GMR-Huntingtin120Q/+ (N and O) and w; UAS-Rpn11/GMR-GAL4; GMR-Huntingtin120Q/+ (P and Q). (R) Quantification of the number of rhabdomeres per ommatidium. More than 100 ommatidia per eye section were counted, and at least four eyes were sectioned for each fly line. The mean number of rhabdomeres per ommatidium ± SD for the control flies was 6.7 ± 0.5; that for the Htt plus control LacZ flies on day 15 was 5.2 ± 0.9; and that for the Htt plus Rpn11 flies on day 15 was 5.9 ± 0.6. Differences between the Htt plus control LacZ flies on day 15 and the Htt plus Rpn11 flies on day 15 are significant: P < 0.0001 (Student's t test).

FIG. 3.

Overexpression of Rpn11 ameliorates the toxicity caused by expanded polyglutamine. (A) The coexpression of Rpn11 with MJDtr-Q78 (MJD+Rpn11) significantly decreased the polyglutamine-induced aggregation. The expanded polyglutamine protein in flies ran as an SDS-insoluble complex in the stacking gel and at the top of the separating gel and was detected by immunoblot analysis with an anti-HA antibody, with which MJDtr-Q78 was tagged. Fly heads from each line of the indicated genotype were subjected to immunoblotting with an anti-HA antibody and an anti-β-tubulin antibody as a loading control. Fly genotypes were w; GMR-GAL4/UAS-LacZ, w; GMR-GAL4/UAS-LacZ; UAS-MJDtr-Q78/+ and w; GMR-GAL4/UAS-Rpn11; UAS-MJDtr-Q78/+. (B) The overexpression of Rpn11 after eclosion suppressed the aggregation induced by the eye-specific expression of the polyglutamine protein. Fly genotypes were w; tub-GAL80^ts/UAS-LacZ; GMR-GAL4/UAS-MJDtr-Q78/+ and w; tub-GAL80^ts/UAS-Rpn11; GMR-GAL4,UAS-MJDtr-Q78/+. Graphs show the ratio of HA to β-tubulin (A and B). The relative amounts of each protein were determined by densitometric analysis. For the anti-HA antibody, the band indicated on the figure by a line was analyzed. (C) The coexpression of Rpn11 and MJDtr-Q78 in adult flies partially suppressed the short life span of MJDtr-Q78 flies (the mean life span ± SD for MJD flies was 12.3 ± 0.1 days, n = 640; that for MJD plus Rpn11 flies was 13.8 ± 0.16 days, n = 260; log-rank test, P < 0.0001). Fly genotypes were w; tub-GAL80^ts/+; da-GAL4/UAS-MJDtr-Q78 and w; tub-GAL80^ts/UAS-Rpn11; da-GAL4/UAS-MJDtr-Q78.

FIG. 4.

Overexpression of Rpn11 suppresses the age-related reduction of the 26S proteasome activity. (A) Proteasome activity in whole Drosophila with overexpression or knockdown of rpn11 after eclosion. Fly genotypes were w; tub-GAL80^ts/UAS-LacZ; da-GAL4/+, w; tub-GAL80^ts/UAS-Rpn11; da-GAL4/+, and w; tub-GAL80^ts/+; da-GAL4/UAS-Rpn11IR. (B) The peptidase activity in the 26S proteasome fraction of 20-day-old flies expressing Rpn11 was significantly higher than that of control flies. Extracts of flies overexpressing LacZ or Rpn11 at day 20 were fractionated, and the Suc-LLVY-AMC hydrolysis activities were measured. The experiments were repeated three times. (C) The peptidase activity of the 20S proteasome in flies expressing LacZ was not different from that of flies expressing Rpn11. Suc-LLVY-AMC hydrolysis activities of the lysates used for panel B were measured with the addition of 0.01% SDS [SDS(+)]. (D) The amount of Rpn11 in the 26S proteasome fraction was significantly greater in flies overexpressing Rpn11 at day 20 than that in control flies. Immunoblot analysis for the even fractions shown in panel B was performed with anti-Rpn11 or anti-α2 antibody. (E) Fractions (fr.) 13 to 20 or fractions 23 to 28 in panel D were pooled and immunoblotted with anti-Rpn11 or anti-α2. (F) The amount of Rpn11 in the 26S fractions was significantly greater in flies overexpressing Rpn11. The amount of Rpn11 protein (the amount of the band shown in panel E by a line) was determined by densitometric analysis. The graph shows the ratio of the amount of Rpn11 in fractions 23 to 28 (26S included) in flies expressing Rpn11 at day20 to the amount of Rpn11 in fractions 23 to 28 in flies expressing LacZ at day 20 from three individual experiments.

FIG. 5.

Rpn11 is required to suppress the age-dependent accumulation of ubiquitinated proteins and to extend the life span. (A) The accumulation of ubiquitinated proteins with age in the adult body was suppressed by the overexpression of Rpn11 after eclosion and enhanced by the knockdown of rpn11. Whole flies from each line of the indicated genotype at the indicated day posteclosion were subjected to immunoblotting with antiubiquitin (anti-Ub), anti-Rpn11, and anti-β-tubulin antibodies. Fly genotypes were w; tub-GAL80^ts/UAS-LacZ; da-GAL4/+, w; tub-GAL80^ts/UAS-Rpn11; da-GAL4/+, and w; tub-GAL80^ts/+; da-GAL4/UAS-Rpn11IR. Numbers at left are molecular masses (in kDa). (B) The overexpression of Rpn11 significantly extended the life span compared with that of control flies (the mean life span ± SD of control flies was 27.2 ± 0.56 days, n = 220; and for Rpn11-overexpressing animals was 36.8 ± 0.53 days, n = 240; log-rank test, P < 0.0001). On the other hand, the ubiquitous expression of Rpn11IR in adult flies reduced their life span (the mean life span ± SD for control flies was 27.2 ± 0.56 days, n = 220; and for the rpn11 knockdown line was 11.1 ± 0.18, n = 320; log-rank test, P < 0.0001). Fly genotypes were w; tub-GAL80^ts/UAS-GFP; da-GAL4/+, w; tub-GAL80^ts/UAS-Rpn11; da-GAL4/+, and w; tub-GAL80^ts/+; da-GAL4/UAS-Rpn11IR. (C) Knockdown of rpn11 led to a decrease in the 26S proteasome activity. Extracts of flies expressing LacZ or Rpn11IR ubiquitously for 15 days after eclosion were fractionated by 8 to 32% glycerol gradient centrifugation. Genotypes shown are w; tub-GAL80^ts/UAS-LacZ; da-GAL4/+ and w; tub-GAL80^ts/+; da-GAL4/UAS-Rpn11IR. (D) Immunodetection of Rpn11 or α2 revealed that the knockdown of rpn11 inhibited the assembly of the 26S proteasome. The levels of Rpn11 and α2 in the fractions containing the 26S proteasome (fractions 24 to 30) were significantly decreased. Immunoblot analysis was performed for each fraction, using antibodies against Rpn11 or α2.

FIG. 6.

Knocking down rpn11 enhances the toxicity of expanded polyglutamine and causes the age-related onset of a neurodegenerative phenotype. (A to F) Knocking down rpn11 clearly enhances the phenotype of polyglutamine-induced neurodegeneration. Light microscopy (A to D) and semithin-section (E and F) images of the compound eyes are shown. The following genotypes are shown: w; GMR-GAL4/UAS-LacZ; UAS-MJDtr-Q78/+ (A, B) and w; GMR-GAL4/+; UAS-MJDtr-Q78/UAS-Rpn11IR (C and D), w; UAS-LacZ/GMR-GAL4; GMR-Huntingtin120Q/+ (E), and w; GMR-GAL4/+; GMR-Huntingtin120Q/UAS-Rpn11IR (F). (G to I) Polyglutamine aggregation was significantly enhanced when rpn11 was knocked down. Eye imaginal discs of wandering third-instar larvae were immunostained with anti-ELAV (green) and anti-HA (magenta), which MJDtr-Q78 was tagged with. ELAV is expressed in all photoreceptor neurons. The following genotypes are shown: w; GMR-GAL4/UAS-LacZ (G), w; GMR-GAL4/UAS-LacZ; UAS-MJDtr-Q78/+ (H), and w; GMR-GAL4/+; UAS-MJDtr-Q78/UAS-Rpn11IR (I). (J to O) Knocking down rpn11 caused the severe neural degeneration. Light microscopy (J to M) and semithin-section (N and O) images of fly eyes expressing Rpn11IR (L, M, and O) or control protein (J, K, and N) are shown. The following genotypes are shown: w; GMR-GAL4/UAS-LacZIR at day 1 (J) and day 10 (K and N), respectively, and w; GMR-GAL4/UAS-Rpn11IR at day 1 (L) and day 10 (M and O), respectively. (P) The Rpn11IR-induced neural degeneration is progressive. Quantification of the appearance of black dots on the surface of the eye with age is shown. A weak or strong phenotype is categorized as the appearance of one black dot or the appearance of more than two black dots, respectively. Numbers of individuals counted are shown at the right. Progressive phenotypes are seen in flies in which rpn11 was knocked down.