Cathepsin B promotes Aβ proteotoxicity by modulating aging regulating mechanisms

Abstract

While the activities of certain proteases promote proteostasis and prevent neurodegeneration-associated phenotypes, the protease cathepsin B (CTSB) enhances proteotoxicity in Alzheimer's disease (AD) model mice, and its levels are elevated in brains of AD patients. How CTSB exacerbates the toxicity of the AD-causing Amyloid β (Aβ) peptide is controversial. Using an activity-based probe, aging-altering interventions and the nematode C. elegans, we discovered that the CTSB CPR-6 promotes Aβ proteotoxicity but mitigates the toxicity of polyQ stretches. While the knockdown of cpr-6 does not affect lifespan, it alleviates Aβ toxicity by reducing the expression of swsn-3 and elevating the level of the protein SMK-1, both involved in the regulation of aging. These observations unveil a mechanism by which CTSB aggravates Aβ-mediated toxicity, indicate that it plays opposing roles in the face of distinct proteotoxic insults and highlight the importance of tailoring specific remedies for distinct neurodegenerative disorders.

Fig. 1. Active CPR-6 binds GB123.

a An illustration of the activity-based probe GB123. This compound includes a Cy5 tag, a linker, and a warhead that covalently binds active CTSB. b GB123 is bound by a C. elegans protease. Pre-treatment with the competitive inhibitor GB111-NH₂ abolishes the signal. The pattern of bands this protease produces differs in homogenates of young (day 1) and post-reproductive animals (days 5 and 9 of adulthood). c The inhibition of CTSB by the inhibitor CA-074 but not of CTSL (by CAS 108005-94-3) reduces the binding of GB123 to the active protease (n = 1). d A directed RNAi screen indicates that the knockdown of two CTSBs, cpr-6 and cpr-9, but not of other cysteine proteases, abolishes GB123 signal (n = 3). e Highly specific RNAi constructs that target the 3’UTR of cpr-6 or of cpr-9, indicate that CPR-6 is the protease that binds GB123 (n = 2). f A cpr-6 deletion mutant exhibits remarkably reduced GB123 binding in different ages (lanes 3, 6 and 9). Similarly, reduced signal was observed when wild type worms were treated with cpr-6 RNAi (lanes 2, 5 and 8).

Fig. 2. Opposing effects of cpr-6 on Aβ- and polyQ35-YFP-mediated toxicity.

a, b The knockdown of cpr-6 by RNAi of the Vidal library (a) or the 3’UTR cpr-6 RNAi (b) protects Aβ worms from proteotoxicity as measured by the paralysis assay. n = 3, Bars represent average daily rates of paralysis of the population ± SEM. Statistical test used: unpaired, one-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. c, d. WB analysis using CL2006 worms and an Aβ antibody shows that cpr-6 RNAi treatment reduces Aβ aggregation in the worm debris (c) in a reproducible manner (d). Bars represent average signal intensity of 4 replicates ± SEM. Statistical test used: unpaired, two-tailed Student’s t-test. e A thrashing assay indicates that cpr-6 RNAi treatment exacerbates polyQ35-YFP proteotoxicity in day 6 old worms but not in day 4 old animals. In contrast, IIS reduction by daf-2 RNAi protects the worms in both ages. Bars represent median thrashing rate within the population ± 95% confidence intervals. Statistical test used: 2-way ANOVA (Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01). f–i The knockdown of cpr-6 results in an elevated polyQ35-YFP aggregation level as measured by native agarose gels (f), increased number of polyQ35-YFP containing foci, seen by fluorescent microscopy (g, scale bar 60μm), and measured by foci quantification (h) as well as by comparison of total fluorescence levels in untreated and cpr-6 3’UTR RNAi-treated animals (i). Bars represent average number of foci and average YFP fluorescence levels ± SEM. Statistical test used: unpaired, two-tailed Student’s t-test. j cpr-6 3’UTR RNAi does not affect lifespan of wild-type worms (strain N2, n = 3).

Fig. 3. CPR-6 activity is modified with aging by a temperature-sensitive modulator.

a An analysis of the age-dependent changes in CPR-6 activity patterns from embryonic development through larval development and adulthood (n = 3). b Mixing homogenates of L4 larvae (lane 3) and day 5 old worms (lane 4) shows changes in the intensities of the different bands. Bands 2 and 3 are largely intensified whereas bands 4 and 5 are greatly weakened (lane 5). These effects are abolished when the L4 homogenate was heat inactivated (lane 6) or when the homogenates were not incubated prior to the analysis (lane 7) (n = 3). c Different mixing ratios indicate that 25% of L4 homogenate is sufficient to greatly reduce the intensities of bands 4 and 5 (lane 7) (n = 2). d Protein identification by mass spectrometry indicates that CPR-6 is the sole protein that appears in all five bands. e–h Analysis of proteins that sediment with each CPR-6-containg band shows an enrichment of cytosolic and mitochondrial proteins in all bands. Interestingly, lysosomal proteins were less abundant.

Fig. 4. CPR-6 activity and aging are interrelated.

a, b The alteration of aging by knocking down daf-2 (a) or by bacterial deprivation (b) changes CPR-6 activity patterns in homogenates of day 2 adult worms (n = 3). c skn-1 is needed for the cpr-6 RNAi-mediated protection from Aβ proteotoxicity as the mix of skn-1 RNAi and EV bacteria (red line) and a concurrent knockdown of skn-1 and cpr-6 (dashed red line) similarly enhance the toxicity of Aβ. No such effect was seen when cpr-6 and daf-16 were concomitantly knocked down (dashed green line). d Three independent paralysis assays confirm the requirement of skn-1 for cpr-6 RNAi to counter Aβ proteotoxicity. Bars represent the average daily rates of paralysis in the three replicates ± SEM (n = 3). Statistical test used: unpaired, one-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). e RNA-seq experiment indicates that the knockdown of cpr-6 results in the elevated expression of 46 genes and reduced expression of 29 genes (log2 ≤ 0.58 or ≥0.58, p-value ≤ 0.05). f A heat map of prominently upregulated and downregulated genes. g The knockdown of swsn-3 by RNAi enhances paralysis of CL2006 worms and a concurrent knockdown of swsn-3 and of cpr-6 prevents cpr-6 RNAi from alleviating Aβ proteotoxicity. Bars represent the average rates of paralysis in the three replicates ± SEM. Statistical test used: unpaired, one-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). h Cellular localization analysis unveils that most genes whose expression levels are affected by cpr-6 RNAi encode membrane proteins.

Fig. 5. cpr-6 RNAi modulates the proteome of larvae and adult worms.

a, b Mass spectrometry indicates that the knockdown of cpr-6 by the 3’UTR RNAi leads to an increase in the levels of 77 proteins and to decreased levels of 52 proteins in L4 larvae. 204 proteins showed increased levels and 42 exhibited reduced amounts in day 5 adult worms upon the knockdown of cpr-6. c, d Clustering of the identified proteins by biological function shows an enrichment of proteins that are involved in splicing, nuclear import and organization in day 5 old worms. e The most prominent proteins that showed at least two-fold elevated (upper table) or decreased (lower table) levels in day 5 old CL2006 worms (p-value was calculated using T-test, 2 tails, unpaired). f A paralysis assay indicates that smk-1 is crucial for the cpr-6 RNAi-mediated protection from Aβ proteotoxicity. g Three independent repeats of the paralysis assay as in (f). Bars represent average daily rates of paralysis in the population ± SEM (n = 3). Statistical test used: unpaired, one-tailed Student’s t-test (*p < 0.05, **p < 0.01).

Fig. 6. A model.

The activity of CPR-6 (Cathepsin B) is modulated by aging (i). CPR-6 protects worms from the toxicity of polyQ35 stretches (ii) but exacerbates Aβ proteotoxicity. Accordingly, the knockdown of cpr-6 mitigates the toxicity of Aβ. The counter-proteotoxic effect of cpr-6 RNAi is linked with increased levels of SMK-1 protein (iii), which probably cooperates with SKN-1 (iv), and with reduced expression of the swsn-3 gene (v) which encodes for a chromatin modulator. SWSN-3 is predicted to be important for the proteostasis-maintaining activity of SKN-1 (vi) by modulating gene expression (vii). This modulation mitigates Aβ proteotoxicity (viii), possibly by the modification of nuclear import and organization.