Alzheimer’s disease-linked ACE variants increase ACE1 catalytic activity and production of angiotensin II

Figures

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  Figure 1. ACE1 stable cell lines display unaffected protein degradation dynamics and survival phenotypes.A, expression of ACE1 protein in differentiated SH-SY5Y ACE stable cell lines. Scale bar 15 μm. B, Western blot showing proper clearance of ubiquitinated proteins in stable cell line whole cell lysate. 24-h treatment of 1 μM MG132 was used as a positive control for improper ubiquitin-proteasome system function. C, Western blot showing no increase in ER-associated stress. 24-h treatment of 1 μM thapsigargin (Th) was used as a positive control to induce ER-stress. pEIF2α normalized to EIF2α quantified below (one-way ANOVA with Tukey’s multiple comparisons test, p < 0.0001 Thapsigargin vs all other columns, ACE1 mutant cell line columns compared to each other were not significant). D, representative images of DAPI- and TUNEL-positive nuclei in stable cell lines. Scale bar 50 μm. One Vector coverglass was incubated with 1000 U/ml for 30 min to induce DNA double-stranded breaks as a positive control (Pos Ctrl). E, quantification of the percentage of TUNEL-positive nuclei per cell line. Results from 4 differentiations, 1 coverglass per experiment, points plotted as the average of three images…

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  Figure 2. The R1279Q ACE1 mutant causes increased abundance of ACE1 protein on the cell-surface.A, ACE1 protein measured in WCE via ELISA, three differentiations (two differentiations for R1279Q). Brown-Forsythe ANOVA test (p < 0.0001), Dunnett’s T3 multiple comparisons test Wild-type vs each mutant (vs Vector p < 0.0001, vs T916M p < 0.0001, vs N1036K p = 0.0001, vs R1279Q p = 0.0096). B, surface ACE1 protein measured by flow cytometry. Each data point represents the mean fluorescence intensity (MFI) of single cells from 20,000 cells counted per well, n = 3 wells per cell line. One representative experiment showed two separate differentiations. One-way ANOVA (p < 0.0001), Dunnett’s multiple comparisons test Wild-type vs each mutant (vs Vector p < 0.0001, vs T916M p < 0.0001, vs N1036K p = 0.5619, vs R1279Q p = 0.0013). C, ACE1 shedding assessed via ELISA measurement of ACE1 in conditioned media and normalized to ACE1 in WCE, three differentiations (two differentiations for R1279Q). Brown-Forsythe ANOVA test p < 0.0001), Dunnett’s T3 multiple comparisons test Wild-type versus each mutant (vs T916M p < 0.0001, vs N1036K p = 0.1427, vs R1279Q p = 0.0548).

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  Figure 3. T916M and N1036K mutants have increased membrane-bound ACE1 catalytic activity.A, figure chematic of membrane-bound ACE1. Created with BioRender. B, production of fluorescent o-aminobenzoyl peptide over 3 hours, three differentiations (2 differentiations for R1279Q) (two-way ANOVA, Dunnett’s multiple comparisons test, see Data Set 1 for details). C, ACE1 activity in WCE measured by area under the curve (AUC) of o-aminobenzoyl peptide over 3 hours. Plotted as fold of Wild-type (WT). Brown-Forsythe ANOVA test (p < 0.0001), Dunnett’s T3 multiple comparisons test Wild-type vs each cell line (vs Vector p < 0.0001, vs T916M p < 0.0001, vs N1036K p = 0.0006, vs R1279Q p = 0.0004). Captopril-treated sample average: 0.0465 AUC Fold WT. D, AUC normalized to WCE ACE1 protein per reaction (2 differentiations for R1279Q). Plotted as Fold WT. One-way ANOVA (p < 0.0001), Dunnett’s multiple comparisons test Wild-type vs each mutant (vs T916M p = 0.0024, vs N1036K p < 0.0001, vs R1279Q p = 0.0693). E, pg/ml of Ang II in stable cell line conditioned media after cells were treated with 500 nM Ang I for 1 h, plotted as fold of WT. Brown-Forsythe ANOVA test (p < 0.0001), Dunnett’s T3 multiple…

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  Figure 4. Summary of primary findings and predicted effects in the central nervous system. ACE1 is a membrane-bound dipeptidyl-peptidase but can be cleaved primarily by ADAM10 to release the two catalytic domains into the extracellular space (far left panel). Under normal conditions, membrane-bound ACE1 cleaves Ang I to form Ang II (yellow peptide icon) which can bind to receptors such as AT1R and activate intracellular signaling cascades. Ang II can also be further degraded into other angiotensin peptide products by proteases like ACE2 and aminopeptidase A. sACE1 can also cleave Ang I to Ang II, albeit at a lower rate than membrane-bound ACE1 depending on the microenvironment. Previous work has shown the AD-associated R1279Q mutation in knock-in mice increases ACE1 presence at the neuronal membrane which caused increased Ang II production and AT1R activation, leading to neuronal death (far right panel). We have demonstrated in this work that R1279Q stable cell lines have increased membrane-bound ACE1. Two additional AD-associated ACE1 mutations, T916M and N1036K, increase Ang II production through a different mechanism than R1279Q. These mutations increased the catalytic activity of…