Cerebral endothelial cell derived small extracellular vesicles improve cognitive function in aged diabetic rats

Abstract

Small extracellular vesicles (sEVs) mediate cell-cell communication by transferring their cargo biological materials into recipient cells. Diabetes mellitus (DM) induces cerebral vascular dysfunction and neurogenesis impairment, which are associated with cognitive decline and an increased risk of developing dementia. Whether the sEVs are involved in DM-induced cerebral vascular disease, is unknown. Therefore, we studied sEVs derived from cerebral endothelial cells (CEC-sEVs) of aged DM rats (DM-CEC-sEVs) and found that DM-CEC-sEVs robustly inhibited neural stem cell (NSC) generation of new neuroblasts and damaged cerebral endothelial function. Treatment of aged DM-rats with CEC-sEVs derived from adult healthy normal rats (N-CEC-sEVs) ameliorated cognitive deficits and improved cerebral vascular function and enhanced neurogenesis. Intravenously administered N-CEC-sEVs crossed the blood brain barrier and were internalized by neural stem cells in the neurogenic region, which were associated with augmentation of miR-1 and -146a and reduction of myeloid differentiation primary response gene 88 and thrombospondin 1 proteins. In addition, uptake of N-CEC-sEVs by the recipient cells was mediated by clathrin and caveolin dependent endocytosis signaling pathways. The present study provides ex vivo and in vivo evidence that DM-CEC-sEVs induce cerebral vascular dysfunction and neurogenesis impairment and that N-CEC-sEVs have a therapeutic effect on improvement of cognitive function by ameliorating dysfunction of cerebral vessels and increasing neurogenesis in aged DM rats, respectively.

Keywords: cerebral endothelial cell; cognitive function; diabete mellitus; exosomes; neural stem cells; rat; small extracellular vesicles.

FIGURE 1

Characterization of CEC-sEVs. NTA (A) shows size and distribution of N-CEC-sEVs and DM-CEC-sEVs. TEM image (B) shows the cup-shaped structures of N-CEC-sEVs and DM-CEC-sEVs. Western blot images (C) show sEV membrane protein markers, but not intracellular protein calnexin.

FIGURE 2

The effects of DM-CEC-sEVs and N-CEC-sEVs on NSCs. The schematic outlines the experimental protocol for the effect of DM-CEC-sEVs on healthy non-DM-NSCs and the representative images of non-DM-NSCs culture (A). Quantitative data show DM-CEC-sEVs reduced the number and size of neurospheres, BrdU (green) positive cells, and Tuj1 (green) positive cells (B). The schematic outlines the experimental protocol for the effect of N-CEC-sEVs on DM-NSCs and the representative images of DM-NSCs culture (C). Quantitative data show N-CEC-sEVs increased number and size of neurospheres, BrdU positive cells, and Tuj1 positive cells (D). *p < 0.05 versus indicated groups.

FIGURE 3

N-CEC-sEV treatment promotes neurogenesis. Representative microscopic images (A,B) and quantitative data (C) of the BrdU positive cells (green, arrow) in the SVZ (A) and the DG (B) of age-matched non-DM rats and aged-DM rats treated with saline and N-CEC-sEVs. Representative microscopic images (D,E) and quantitative data (F) show DCX⁺ neuroblasts (red) in the SVZ (D) and the DG (E). Representative microscopic images of BrdU⁺ cells double labeled with DCX (G, arrow) and Nestin cells (H, arrow) in the SVZ of aged-DM rats treated with N-CEC-sEVs. Bar graphs in (G,H) are the quantitative data of BrdU⁺/DCX⁺ and BrdU⁺/Nestin⁺ double labeled cells in the SVZ of aged-DM treated with saline and N-CEC-sEVs. Representative confocal microscopic images of DCX⁺ neuroblasts (red) in the DG (I) of aged-DM rats. Inserts in I show a DCX⁺ neuroblast (arrow in Saline) with short processes, whereas a DCX⁺ neuroblast (arrow in N-CEC-sEVs) with long and branched processes. Bar graphs in (J) show N-CEC-sEVs increased branch number and length of DCX⁺ neuroblast. LV: lateral ventricle. *p < 0.05 versus indicated groups.

FIGURE 4

N-CEC-sEV treatment reduces cognitive deficits in aged DM rats. Odor recognition test (A), social interaction test (B), Morris water maze (C), blood glucose level (D), and body weight (E) in aged-DM rats treated with saline and N-CEC-sEVs. *P < 0.05 vs. the saline group. N = 10/group.

FIGURE 5

N-CEC-sEV treatment reduces cerebral vascular damage. Representative confocal microscopic images (A) and quantitative data (B) of fibrin/fibrinogen immunoreactivity (green) in the hippocampus of aged-DM rats treated with saline or N-CEC-sEVs. *p < 0.05 versus indicated groups.

FIGURE 6

DM-CEC-sEVs increase healthy CEC permeability, whereas N-CEC-sEVs reduce DM-augmented permeability. Blockage of clathrin- and caveolin-pathways abolishes the effects of sEVs on CEC permeability. Quantitative data (A) of endothelial monolayer barrier permeability assay in N-CECs with and without DM-CEC-sEVs, and in DM-CECs with and without N-CEC-sEVs in the presence and absence of endocytosis inhibitors. Representative images and quantitative data (B) of sEVs internalization by N-CECs in the presence and absence of endocytosis inhibitors. *p < 0.05 versus indicated groups.

FIGURE 7

N-CEC-sEVs intravenously administered cross the BBB and are internalized by NSCs. Representative confocal microscopic images show GFP signals (green, arrows) in cells lining cerebral vessels (A) and cells within the SVZ (B) and DG of hippocampus (C) after IV injection of N-CEC-sEVs-GFP. Representative TEM images (D–F) show GFP positive immunogold particles (red arrows) within endothelial cells that lining the cerebral blood vessel luminal surface (D), where tight junction was intact (black arrowhead in D). GFP positive immunogold particles (10 nm, E,F) were within sEVs (red arrows) localized to cytoplasm and nucleus of SVZ cells. LV, lateral ventricle. Bars = 25 μm in (A–C). Bars = 200 nm in (D,F).

FIGURE 8

N-CEC-sEV treatment elevates miR-1 and –146a, and reduces MYD88 and TSP1 expression in DG and SVZ tissues isolated from aged-DM rats. Quantitative RT-PCR data show miR-1 and –146a levels in N-CEC-sEVs and DM-CEC-sEVs (A) and in DG and SVZ tissues (B) isolated from age-matched non-DM rats, and aged-DM rats treated with saline and N-CEC-sEVs. Representative Western blots (C) and quantitative data (D) of MYD88 and TSP1 levels in the DG and the SVZ. Data are presented as fold change from the non-DM group. *p < 0.05 versus indicated groups.