Journal: bioRxiv

Article Title: Tripeptidyl peptidase II is essential for maintaining cerebrovascular homeostasis of female mice and represents a novel therapeutic target for vascular dementia

doi: 10.64898/2026.05.05.722100

Figure Lengend Snippet: (A) Two-photon imaging of hippocampal slices loaded with Fluo-4 AM revealed reduced cytosolic Ca²⁺ levels in vascular cells of the CA1 and DG regions at absence of TPP2. (A1) Representative images of Ca 2+ in CA1 (upper) and DG (lower) regions. (A2) Statistical graphs of CTCF of the images. n = 4, ***p ≤ 0.001. Scale bar, 10 µm . (B) GSEA analysis performed on scRNA-seq data in hippocampal ECs at absence of TPP2 revealed enriched gene sets for Ca²⁺ signaling pathway, indicating altered activity in this pathway. (C) Genetically encoded Ca²⁺ indicators (GECIs)-based measurement revealed that TPP2 depletion in primary cerebrovascular ECs significantly decreases cytosolic Ca²⁺ while increasing endoplasmic reticulum Ca²⁺, and this phenotype can be rescued by E2 treatment. (C1) Representative images of cytosolic Ca 2+ detected by GCaMP2 (upper) and ER Ca 2+ detected by G-CEPIA1er (lower) . (C2) Statistical graphs of CTCF of the images. n = 6, ***p ≤ 0.001. Scale bar, 10 µm . (D) In bEnd.3 cells, TPP2 interacts with IP3R1, SERCA1, SIGMAR1, and VDAC2, and its depletion leads to the downregulation of IP3R1 and CYP19A1. (D1) Representative images of PLA and their statistical graphs of CTCF of the images. n = 6, ***p ≤ 0.001. Scale bar, 10 µm . (D2 ) Immunoblot analysis of the protein levels of IP3R1, SERCA1, SIGMAR1, VDAC2, and other related proteins. The results revealed a significant downregulation of IP3R1 and CYP19A1 upon TPP2 depletion. Data from one of three independent experiments are shown. (E) Immunofluorescence staining revealed significantly reduced levels of both CYP19A1 and CD31 in hippocampal vascular ECs of 12-month-old T2KO mice compared to WT controls. In contrast, no significant change was observed in 3-month-old mice. Shown is a representative result from three independent experiments. (F) Targeted metabolomic profiling of steroid hormones in primary vascular cells revealed that E2 was completely depleted in the absence of TPP2. Representative images of base peak (F1) and extracted ion chromatograms (F2) from four independent samples . (F3) Statistical graph of identified E2 showing that in WT vascular cells it is up to 10 pg per million cells, whereas it is undetectable in T2KO cells. n = 4, ***p ≤ 0.001. (G) PLA results indicated a severe disruption of the anticipatory UPR in TPP2-depleted primary ECs, which was restored by E2 treatment. This restoration was completely blocked by either Src inhibitor PP2 or PLCγ inhibitor CCT129957. Representative images of PLA (G1) and their statistical graphs of CTCF of the images (G2) are shown. n = 10, ***p ≤ 0.001. Scale bar, 20 µm. (H) Co-IP combined with immunoblot showed the interaction of the components of anticipatory UPR signaling pathway in both bEnd.3 cells and primary ECs is disrupted under TPP2 depletion. (H1) Immunoblot analysis of precipitates showed the decoupling among Src, ERα, and PLCγ under TPP2 depletion under TPP2 depletion. (H2) Immunoblot analysis of homogenates revealed no significant change in the protein levels of Src, ERα, and PLCγ of T2KO samples. Data from one of three independent experiments are shown. (I) In cerebrovascular ECs, TPP2 depletion leads to CYP19A1 deficiency via Ca²⁺ imbalance-mediated dysregulation of autophagic flux. This deficiency is exacerbated by CND1163, a SERCA activator that worsens cytosolic Ca²⁺ deficit. Conversely, it is rescued by treatment that elevates cytosolic Ca²⁺ (e.g., D-myo-inositol-1,2,4,5-tetrakisphosphate tetrasodium: IP4 tetrasodium, an IP3R1 activator) or treatment that inhibits autophagic flux (e.g., 3-methyladenine: 3-MA). Representive images from 12 independent experiments are shown. Scale bar, 20 µm.

Article Snippet: Chemical reagents including E2, PP2, CCT129957, 3-MA, CND1163, and D-myo-inositol-1,2,4,5-tetrakisphosphate tetrasodium salt (IP4 tetrasodium), C18:0p LPC, C18:0p/18:1 PC, 18:0p/20:4 PC, 18:0p/20:4 PE, 16:0/18:0 PC, 18:0/20:4 PC, 16:0p/16:0 PC, 16:0o/16:0o PC were obtained from MCE (Shanghai, China).

Techniques: Imaging, Activity Assay, Western Blot, Immunofluorescence, Staining, Metabolomic, Disruption, Co-Immunoprecipitation Assay