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. 2020 Jul 6;12(1):80.
doi: 10.1186/s13195-020-00649-8.

Cathepsin D regulates cerebral Aβ42/40 ratios via differential degradation of Aβ42 and Aβ40

Caitlin N Suire  1   2 , Samer O Abdul-Hay  3 , Tomoko Sahara  3 , Dongcheul Kang  3 , Monica K Brizuela  1 , Paul Saftig  4 , Dennis W Dickson  3 , Terrone L Rosenberry  3 , Malcolm A Leissring  5   6
Affiliations

Cathepsin D regulates cerebral Aβ42/40 ratios via differential degradation of Aβ42 and Aβ40

Caitlin N Suire et al. Alzheimers Res Ther. .

Abstract

Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid β-protein (Aβ) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aβ proteostasis in vivo and to more completely characterize the degradation of Aβ42 and Aβ40 by CatD.

Methods: We quantified Aβ degradation rates and levels of endogenous Aβ42 and Aβ40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aβ42 vs. Aβ40, and the degradation of aggregated Aβ42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aβ42 and Aβ40, as well as corresponding p3 fragments.

Results: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aβ42 and Aβ40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aβ42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aβ42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aβ42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aβ42 vis-à-vis Aβ40. Specifically, Aβ42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aβ42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aβ42 vs. Aβ40 also extend to p3 fragments ending at positions 42 vs. 40.

Conclusions: Our findings identify CatD as the principal intracellular Aβ-degrading protease identified to date, one that regulates Aβ42/40 ratios via differential degradation of Aβ42 vs. Aβ40. The finding that Aβ42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aβ42 and downstream pathological sequelae in AD.

Keywords: Alzheimer disease; Amyloid-β protein; Cathepsin D; Lysosomes; Proteostasis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CatD activity and protein levels in brain extracts. a Aβ degradation in soluble brain extracts from 15-day-old WT and CatD-KO mice as a function of pH. Note that the abundant Aβ-degrading activity occurring at acidic pH is essentially absent in CatD-KO extracts. Note also that the smaller peak at neutral pH is inhibited by insulin (Ins), reflecting IDE activity [28]. Data are mean ± SEM for 5 replicates. †p < 0.01. b Aβ-degrading activity in extracts from 15-day-old CatD-KO, CatD-HET, and CatD-WT mice at pH 4.0. Note that the activity in WT and CatD-HET extracts is largely inhibited by the CatD inhibitor, pepstatin A (PepA). Data are mean ± SEM for 4 replicates. †p < 0.01; ‡p < 0.001; #p < 0.0001. Inset: CatD activity in brain extracts from WT, CatD-HET, and CatD-KO mice measured directly using a selective substrate. Data are mean ± SEM for 4 replicates.; #p < 0.0001. Note also that Aβ-degrading activity in the CatD-HET extracts is not reduced by 50% as expected from deletion of one of two CTSD alleles. c, d Representative western blot (c) and quantification of multiple samples (d) showing relative CatD levels in CatD-KO, CatD-HET, and CatD-WT mice. Note that, consistent with the activity data in b, CatD levels in CatD-HET brains are not 50% of those in WT brains. Data in d are mean ± SEM for 6 samples per genotype. *p < 0.05; #p < 0.0001
Fig. 2
Fig. 2
Insoluble Aβ42 and Aβ40 levels in CatD-KO, CatD-HET, and CatD-WT brains. a, b Levels of insoluble, endogenous brain Aβ42 (a) and Aβ40 (b) in CatD-KO, CatD-HET, and CatD-WT mice as a function of age. Note that levels of both Aβ species are markedly increased in CatD-KO, but not in CatD-HET, mice relative to WT controls at all ages examined. Data are mean ± SEM for 4–6 replicates per group. †p < 0.01; ‡p < 0.001; #p < 0.0001. c Insoluble Aβ42/40 ratios are significantly increased in CatD-KO mice, but not NEP-KO, IDE-KO, or NEP/IDE-DKO mice, relative to their respective WT controls. Data are mean ± SEM for 28–30 replicates per group for CatD-KO and CatD-WT mice and 6–11 replicates per group for the other genotypes. #p < 0.0001. d, e Percent increases in insoluble, endogenous brain Aβ42 (d) and Aβ40 (e) in 15-day-old and 26-day-old CatD-KO mice as compared to 26-day-old NEP-KO, IDE-KO, and NEP/IDE double-knockout (DKO) mice, all normalized to respective WT controls. Note that 26-day-old CatD-KO mice exhibit significantly higher increases in insoluble Aβ42 and Aβ40 above their WT controls than age-matched mice lacking NEP, IDE, or both NEP and IDE. Data are mean ± SEM for 4–6 replicates per group. *p < 0.05; †p < 0.01; ‡p < 0.001; #p < 0.0001. f Intracellular endogenous Aβ42 accumulation occurs in CatD-KO mice by 3 weeks of age. Shown is immunohistochemical staining of a 26-day-old CatD-KO mouse and age-matched WT control with an anti-Aβ42 end-specific antibody [31]. Additional immunohistochemical characterization is provided in Supp. Fig. S4
Fig. 3
Fig. 3
CatD degrades Aβ42 and Aβ40 with markedly different kinetics. a, b Plots of initial velocity (vo) vs. substrate concentration for Aβ42 (a, red) and Aβ40 (b, blue). The dashed lines (gray) show the relative position of the data in a when superimposed on the same scale as is used for the data in b. Quantitative kinetic parameters are provided in Supp. Table S2. c, d Surface plasmon resonance confirms that Aβ42 exhibits markedly higher affinity for CatD than Aβ40, independent of degradation. Traces obtained for 3-fold dilutions of Aβ42 (c) and Aβ40 (d) beginning at 333 nM. Analysis of the fitted curves in (c) yielded a KM of 47.7 + 0.041 nM and a kd value of 0.266 + 7.2 × 105 min−1 for Aβ42. Consistent with the kinetics of Aβ40 binding obtained by other methods (Supp. Table S2), no significant binding of Aβ40 was observed within the conditions used. Note that, for technical reasons, these experiments were conducted at pH 4.5, precluding direct quantitative comparisons to kinetic parameters determined by other methods at pH 4.0
Fig. 4
Fig. 4
Aβ42 is a potent competitive inhibitor of CatD. a, b Competitive inhibition by Aβ42 (a) and Aβ40 (b) of CatD activity quantified by a fluorogenic substrate. Note that just 3 nM Aβ42 inhibits CatD (nominal concentration, ~ 1 nM) by more than 50%. c Comparable data for CatD activity quantified using an Aβ degradation assay, with 200 nM fluorescent Aβ alone (no Aβ) or in combination with 200 nM Aβ42 (red) or Aβ40 (blue). d Quantification of Aβ42 (red) and Aβ40 (blue) degradation either alone (100 nM) or in combination (100 nM each). Note that Aβ42 significantly inhibits Aβ40 degradation, but the converse is not true. Data are mean ± SEM for 4–8 replicates per group. †p < 0.01; ‡p < 0.001; #p < 0.0001. e Aβ40 degradation is significantly inhibited by 1/10 the concentration of Aβ42, a ratio representative of that present in vivo. f CatD is strongly inhibited by multiple Aβ peptides and fragments ending at position 42, including full-length murine Aβ (mAβ(1–42)) and the p3 fragment (Aβ(17–42)), more strongly than the corresponding peptides ending in Aβ40. The C-terminal fragment of Aβ42, Aβ(33–42), failed to inhibit significantly, while the corresponding fragment, Aβ(33–40), showed a modest but statistically significant inhibition under the conditions tested. Data are mean ± SEM for 4–8 replicates per group. #p < 0.0001. For data on Aβ peptides ending at position 40, the 2 symbols reflect the statistical significance of comparisons to buffer-only control (CTL) and to the corresponding fragments ending at position 42, respectively
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