PSA knockdown and inhibition increase aggregation and toxicity in cells expressing normal and polyglutamine-expanded huntingtin exon 1. (A) HeLa cells were treated with PSA siRNA and then transfected with plasmids encoding exon 1 of huntingtin with polyglutamine tract length of 74, tagged with EGFP (htt-exon-1-GFP). The proportion of transfected (GFP-positive) cells containing GFP aggregates was measured (black bars) and cellular toxicity was assessed by measuring the proportion of transfected cells with apoptotic nuclei, visualized by DAPI staining (grey bars). Aggregation and toxicity were increased by the knockdown of PSA. Data shown are normalized to control levels of aggregation and cell death and combined from four independent experiments, each performed in triplicate. Error bars represent SEM, as for all graphs in this study. In control conditions, the proportion of cells with GFP-positive macroaggregates and apoptotic nuclei were typically 6 and 4%, respectively. ***P < 0.001, *P < 0.05 by odds ratio. (B) Aggregation was also measured by filter trap assay in HEK 293T cells using htt-exon-1-Q103-GFP. Lysates from cells treated with PSA siRNA contained more insoluble aggregates trapped on the filter membrane as shown by the increase in GFP staining. (C) Effect of PSA knockdown on aggregation of htt-exon-1-GFP with a polyglutamine repeat length of 23 was measured, exactly as in (A); data are shown normalized to control. The proportions of control cells with macroaggregates and apoptotic nuclei were 2 and 1%, respectively. ***P < 0.001 (D–F). Small molecular inhibitors of PSA also increased aggregation of polyglutamine proteins with a range of repeat lengths. 293A cells were transfected with htt-exon-1-GFP with 23Q (D), 41Q (E) or 74Q (F). PSA was inhibited with PAQ-22 (dark grey lines) or bestatin (light grey lines) to either 50 or 80% of its endogenous activity: 50% inhibition: 0.05 μm bestatin, 0.12 μm PAQ-22; 80% inhibition: 0.5 μm bestatin, 1 μm PAQ-22. The number of transfected cells containing GFP-positive aggregates was counted at the time points indicated. ***P < 0.001, *P < 0.05 by two-way ANOVA followed by post hoc Fisher's LSD test. (G) In intact WT MEF cells, 80% of Ala-amc cleavage was inhibited by minimal effective concentration of both PAQ (10 μm) and bestatin (5 μm). In PSA−/− MEF cells, PAQ and bestatin inhibit only 10 and 20% of the residual Ala-amc cleavage activity, which must be by other aminopeptidases. **P = 0.001. (H) Bestatin (0.5 μm) treatment increased aggregation of HA-tagged huntingtin exon 1 with 74Q in HeLa cells, but not following knockdown of PSA by siRNA. *P < 0.05, ns, not significant by odds ratio.

Effect of PSA overexpression and knockdown on the formation of polyglutamine aggregates in mouse muscle. (A) Cross-sections of mouse tibialis anterior muscle electroporated with GFP or huntingtin exon 1 with 23, 41, 74Q fused to GFP (Htt23Q, Htt41Q, Ht74Q). One week after electroporation, the formation of inclusions was observed in all fibres electroporated with Htt41Q and Htt74Q. Fibres electroporated with GFP or Htt23Q showed a diffuse fluorescence and no inclusions. Scale bar represents 100 μm. (B) PSA overexpression decreased the number and size of polyglutamine inclusions found in tibialis anterior muscle 1 week after electroporation with expanded huntingtin exon 1 (74Q) and PSA. Note that Htt74Q is seen as red (fused to mCherry) in these images. Graph shows inclusion number and size in fibres where PSA (blue bars) or GFP (green bars) is overexpressed. (C–E) Reduction in the expression of PSA by electroporation of RNAi increased the number and size of polyglutamine inclusions in mouse skeletal muscle electroporated with expanded huntingtin exon 1. (C) Tibialis anterior muscle 1 week after electroporation with Htt74Q and a plasmid expressing either a scrambled RNAi, or an RNAi targeting PSA. Reduction of PSA expression by RNAi increased the number and size of polyglutamine inclusions formed by expanded huntingtin exon 1 (74Q). (D) Electroporation of expanded huntingtin exon 1 (74Q) to tibialis anterior muscles of PSA+/− heterozygous mice led to more inclusions than in muscle from WT (PSA+/+) mice, 1 week after electroporation. These inclusions also seemed larger in PSA+/− mice, but this effect did not reach statistical significance (P = 0.09). (E) Electroporation of expanded huntingtin exon 1 (41Q) in PSA+/− heterozygous mice led the formation of more, larger inclusions than in muscle from WT (PSA+/+) mice 1 week after electroporation. Scale bar (B–E) represents 50 μm. *P < 0.05 by t-test.

PSA inhibition enhances aggregate formation in cells expressing other aggregate-prone proteins (ataxin-3, mutant SOD1, mutant α-synuclein). (A and B) PSA inhibition increased aggregate formation in 293A cells expressing full-length ataxin-3 GFP with 28Q (A) and 84Q (B). The proportion of transfected cells with macroaggregates was also increased by inhibition of PSA in cells transfected with mutant SOD1 GFP G37R (C) or G93A (D). ***P < 0.001, **P < 0.01, *P < 0.05 by t-test. (E and F) Clearance of mutant α-synuclein (A53T) was slowed by inhibition of PSA. Stable inducible PC12 cells were treated with doxycycline to induce expression of mutant α-synuclein, and subsequently expression was turned off by removal of doxycycline from the medium. When PSA inhibitors were added after transgene expression was switched off, more α-synuclein remained present, indicating a decreased clearance. Levels of α-synuclein remaining after 16 h were assayed by western blot, a representative image is shown in (E). Densitometric quantification of this triplicate experiment is shown in (F). **P < 0.01, *P < 0.05, by ANOVA.

PSA overexpression reduces toxicity and aggregate content in cells expressing expanded polyQ huntingtin exon 1 and expanded polyglutamine tracts. (A) Effect of PSA overexpression on aggregate content in SK-N-SH cells expressing htt-exon-1-Q74-GFP. The proportions of cells with GFP-positive macroaggregates and apoptotic nuclei were decreased by the overexpression of PSA, when assayed as in Figure 1A and C. Data shown are normalized to control for each experiment and the average of data from four independent experiments carried out in triplicate are shown. In control conditions, typically around 30% of transfected cells contained macroaggregates and 10% had apoptotic nuclei. ***P < 0.001 by odds ratio. (B) Overexpression of PSA in 293T cells also decreased levels of insoluble aggregates of htt-exon-1-Q103-GFP, as measured by filter-trap assay. Q103 aggregates were visualized using anti-GFP antibody. (C) To mimic the polyQ sequences that may be released by the proteasome, we used a GFP–ubiquitin–polyQ construct (23). Upon expression, the GFP–UB and the polyQ peptide are efficiently cleaved by deubiquitinating enzyme(s), leading to the release of a polyQ polypeptide. (D) Levels of soluble and insoluble polyglutamine peptides were also decreased by overexpression of PSA in HEK293T cells transfected with GFP–UB–polyQ containing 65 or 112 glutamines. Levels of both soluble polyQ peptides (upper panel, arrows mark monomeric polyQ peptides and arrowheads mark high molecular weight oligomeric structures) and insoluble aggregates (lower panel) detected by an anti-polyglutamine antibody decreased, when PSA-GFP was co-transfected. Actin levels are shown as a loading control.

Effect of PSA overexpression and knockdown in Drosophila. (A) PSA expression levels alter lifespan in flies expressing htt-exon1 with 93 polyglutamine repeats in the brain. Knockdown of PSA expression levels by expression of two different PSA RNAi constructs enhanced toxicity (elav-Gal4 Q93 PSA-RNAi1 and elav-Gal4 Q93 PSA-RNAi2), compared with flies expressing mutant huntingtin alone (elav-Gal4 Q93). Overexpress­ion of PSA (elav-Gal4 Q93 PSA) extended lifespan relative to flies expressing Q93 alone. Graph shows Kaplan–Meier survival curves, P < 0.0001 in both cases. Expression of PSA RNAi or PSA in flies expressing WT huntingtin (elav-Gal4 Q20) did not affect lifespan (Supplementary Material, Fig. S3). (B–E) Degeneration was also decreased by overexpression of PSA. Overexpression of PSA in the WT fly eye using the glass promoter (gl-PSA) caused a slight decrease in rhabdomere number compared with flies expressing Q23 alone. However, when co-expressed with GMR-Q120, PSA protected against the neurodegeneration seen in these flies (B). (C) Expression of PSA RNAi enhanced neurodegeneration in flies expressing GMR-Q120, using two RNAi lines (elav-Gal4 GMR-Q120 PSA-RNAi1 and elav-Gal4 GMR-Q120 PSA-RNAi2). Expression of PSA RNAi in flies expressing Q23 did not affect rhabdomere number (Supplementary Material, Fig. S3), *P < 0.02, 2 tailed t-test. (D) Examples of rhabdomeres in flies expressing GMR-Q120 with or without PSA (gl-PSA). (E) Examples of rhabdomeres in flies expressing mutant huntingtin GMR-Q120 when PSA is downregulated using two RNAi lines (PSA-RNAi1 and PSA-RNAi2).

PSA activity enhances protein degradation by the autophagic/lysosomal pathway. (A–C) Effect of PSA modulation on different proteolytic pathways. Degradation of long-lived protein was measured in 293A cells. Proteolysis rates were measured in the presence of inhibitors of proteasomes, lysosomes or autophagy to determine flux through each of these pathways in cells starved of serum and amino acids. Proteasomes, lysosomes and autophagy inhibitors were velcade/bortezomib 1 µm, chloroquine 50 µm and 3-MA 10 mm, respectively. (A) PSA overexpression increased total, lysosomal and autophagy-dependent proteolysis. (B) PSA knockdown by RNAi decreased total and lysosomal proteolysis. (C) PSA inhibitors reduced total and lysosomal proteolysis. ***P < 0.0001. (D) Inhibiting autophagy in SK-N-SH cells expressing Q74 huntingtin exon-1 GFP using 3-MA, reduced the protective effect of PSA overexpression on aggregate content. Co-expression of PSA with htt-exon-1-GFP-74Q reduced the percentage of transfected cells with aggregates (grey bars). However, in the presence of 3-MA, added immediately after transfection, the reduction in aggregation caused by PSA expression was decreased (black bars). Control values with and without 3-MA are set to 100% in each case to allow ease of comparison, although Q74 aggregation was higher in the presence of 3-MA. Actual mean values were 24% of cells having aggregates in control and 30% in 3-MA treated cells. (E) Inhibitors of PSA caused an increase in the proportion of cells expressing htt-exon-1-GFP-Q74 with aggregates (grey bars), but if 3-MA was added at the same time as these inhibitors they did not increase the aggregate content (black bars). Actual mean values for aggregation were 19% in control cells and 32% in 3-MA treated cells. **P < 0.01, ***P < 0.001, ns, not significant, by odds ratio, relative to control. (F–K) Effect of PSA modulation on LC3-II levels. (F) Treatment of PC12 cells with PSA inhibitors also resulted in a decrease in LC3-II levels, a marker for autophagosomes. The western blot shown is a representative data set, with actin shown as a loading control. Quantification of data obtained in triplicate samples is shown in (G). (H and I) In HeLa cells treated with PSA siRNA, LC3-II levels were decreased, whereas cells overexpressing PSA showed increased levels of LC3-II (J and K). (I) and (K) show densitometric quantification of data from four experiments carried out in triplicate. LC3-II level changes were seen in the presence (grey bars) and absence (black bars) of bafilomycin A1, a lysosomal proton pump inhibitor. LC3-II levels are shown relative to actin, as a loading control in each case. Values are normalized to control, in the presence or absence of bafilomycin, to allow for comparison between different gels, although the levels of LC3-II were higher with bafilomycin than without. *P < 0.05, **P < 0.01.