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. 2023 Jan 3;6(1):2.
doi: 10.1038/s42003-022-04398-2.

Aβ efflux impairment and inflammation linked to cerebrovascular accumulation of amyloid-forming amylin secreted from pancreas

Affiliations

Aβ efflux impairment and inflammation linked to cerebrovascular accumulation of amyloid-forming amylin secreted from pancreas

Nirmal Verma et al. Commun Biol. .

Abstract

Impairment of vascular pathways of cerebral β-amyloid (Aβ) elimination contributes to Alzheimer disease (AD). Vascular damage is commonly associated with diabetes. Here we show in human tissues and AD-model rats that bloodborne islet amyloid polypeptide (amylin) secreted from the pancreas perturbs cerebral Aβ clearance. Blood amylin concentrations are higher in AD than in cognitively unaffected persons. Amyloid-forming amylin accumulates in circulating monocytes and co-deposits with Aβ within the brain microvasculature, possibly involving inflammation. In rats, pancreatic expression of amyloid-forming human amylin indeed induces cerebrovascular inflammation and amylin-Aβ co-deposits. LRP1-mediated Aβ transport across the blood-brain barrier and Aβ clearance through interstitial fluid drainage along vascular walls are impaired, as indicated by Aβ deposition in perivascular spaces. At the molecular level, cerebrovascular amylin deposits alter immune and hypoxia-related brain gene expression. These converging data from humans and laboratory animals suggest that altering bloodborne amylin could potentially reduce cerebrovascular amylin deposits and Aβ pathology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The hypothesis of amylin-induced impairment of brain Aβ clearance.
Putative amylin function (green panel) and pathology (red panels) (a) along with work flow and methods (b). c Blood amylin concentrations in dementia (DEM; n = 19), mild cognitive impairment (MCI; n = 19), and cognitively unimpaired (CU; n = 42) individuals. d Pairwise correlation coefficient (r) between amylin and insulin concentrations in same blood samples as in (c). e Flow cytometry sorting of amylin positive (Q2) or negative (Q1) circulating CD14+ monocytes in blood with lower quartile vs. upper quartile amylin concentrations. f Confocal microscopic images showing amylin engulfed in CD14+ monocytes (n = 3). Pairwise correlation coefficient (r) between amylin and Aβ42 concentrations in human brains, including persons with sAD (n = 42) and without AD (n = 18) (g), and between matched antemortem plasma amylin concentration and amylin concentration in autopsied brain tissue, including persons with sAD (n = 12) and without AD (n = 8) individuals (h) (potential outliers were removed from the analysis; shown in Supplemental Fig. S1e). i IHC analysis using anti-amylin (brown) and anti-Aβ (green) antibodies on serial sections from a sAD brain (n = 18). j Confocal microscopic analysis and amylin-Aβ proximity ligation assay (PLA) showing vascular amylin-Aβ deposits in a sAD brain. IHC analysis of fAD brains showing Aβ deposits in perivascular spaces and vessel walls with amylin accumulation within the lumen (k, l), and amylin deposits in the vessel wall (m) or vessel wall (n), and Aβ deposits in perivascular spaces (n = 32). Data are presented as box and whiskers or correlation analyses; Kruskal–Wallis one-way of variance, Data are means ± SEM.
Fig. 2
Fig. 2. Systemic and cerebrovascular inflammation in rats with pancreatic expression of amyloid-forming human amylin (HIP rats).
a Cross-sectional blood amylin and glucose concentrations in HIP rats age 6–8 months (n = 6), age 10–12 months (n = 13), and age 15–16 months (n = 12). b Blood amylin concentrations in humans with dementia (DEM) vs. HIP rats; same rats as in (n = 16) (a). c Flow cytometry sorting of circulating CD14+ monocytes positive for amylin in blood from same rats as in (b) (n = 10 males/group). d Confocal microscopic images of circulating monocytes stained for CD14+ (red) and amylin (green) in blood from the same rats as in (b) (n = 5 blood samples/group). e Interleukin (IL)-1β ELISA in plasma from HIP vs WT rats similar to groups in (b) (n = 10 males/group). f Confocal microscopic images showing IL-1β and amylin deposits in brain blood vessels in rats studied in (b) (n = 3 males/group). g IHC analysis of brain sections from HIP and WT rats from the same groups as in (c) showing vascular deposits of amylin (brown) and astroglial reactions (green stains for glial fibrillar acidic protein; GFAP) (n = 5 males/group). IHC analysis of phagocytic microglia (CD68) (h) and vascular monocyte recruitment (CD11b) (i) in brain sections from HIP vs WT rats from the same groups as in (b) (n = 10 males/group). Data are means ± SEM; unpaired t-test for all panels.
Fig. 3
Fig. 3. Cerebrovascular amylin-Aβ deposition induced by amyloid-forming human amylin secreted from the pancreas.
a Thioflavin T (Th T) fluorescence signal intensities in blood lysates from HIP rats and WT littermates (age 15-16-months; n = 10 males/group). b Amylin concentrations in brain microvessel lysates in HIP and WT rats similar to those in (a) (n = 10 males/group). c IHC analysis of HIP rat brains showing Aβ deposits (green) in perivascular spaces and amylin accumulation (brown) within the lumen. (n = 5 males/group; age 15–16-months). d Average Thioflavin T (Th T) fluorescence signal intensities in blood lysates from APP/PS1/HIP and APP/PS1 littermates age 15–16-months (n = 10 rat males/group). e Amylin concentrations in brain microvessel lysates from same rats as above. f Representative IHC micrographs of brain sections from APP/PS1/HIP and APP/PS1 rats co-stained with anti-amylin (brown) and anti-Aβ (green) antibodies (n = 5 males/group; age 15–16-months) (3 slides/brain). Representative IHC images and analysis of phagocytic microglia (CD68) (g) and vascular monocyte recruitment (CD11b) (h) in brain sections from APP/PS1/HIP vs APP/PS1 rat males (n = 10 males/group; age 16-months). Data are means ± SEM; unpaired t-test for all panels.
Fig. 4
Fig. 4. Altered relaxation of vascular smooth muscle cells by increased blood amylin concentrations.
a T2-weighted MRI and longitudinal ventricular hyperintensity volumes in HIP vs. WT rats (n = 4–5 males/group). b CBF maps and global CBF in HIP and WT rats, age 15-16 months (9 males/group). c Cross-sectional concentrations of plasma nitrite and nitrate in HIP and WT rats (n = 6 males/group/age). Diameter traces in pial arteries from HIP and WT rat males at different intravascular pressures (d), and arterial tone of pial arteries (e) measured at the indicated intravascular pressure (2–3 arteries/ rat, n = 6–7 males/group, age 15–16 months). f Same as in (e) in posterior cerebral arteries from WT and amylin knockout (AKO) rats, and in AKO rats intravenously injected with human amylin (n = 3 males/group, age 9–10 months). g, h Lipid peroxidation in pial artery SMCs from WT and diabetic HIP rat males measured with Liperfluo (g; N = 62 SMC from 4 WT rats and 70 cells from 4 HIP rats) and C11-BODIPY581/591 (h; N = 87 SMC from 7 WT rats and 68 cells from 4 HIP rats). i Arginase activity and arginase-1 and arginase-2 concentrations in HIP vs. WT brain microvessel lysates (n = 7 males/group; age 15–16 months). j Proposed mechanism: chronically increased concentrations of pancreatic amyloid-forming amylin in blood cause oxidative stress within the vascular wall leading to NO-arginase dysregulation and impaired SMC function and myogenic tone. Data are means ± SEM. Mann–Whitney non-parametric test for panels (g, h), unpaired t-test for the other panels.
Fig. 5
Fig. 5. Impaired Aβ efflux from the brain induced by amyloid-forming human amylin secreted from the pancreas.
a Western blot analyses of brain tissue Aβ in WT and HIP rats (n = 4–5 males/group; age 15–16-months) with rat Aβ40 and APP/PS1 rat brain homogenate used as positive controls for Aβ immunoreactivity signal. b Estimated brain Aβ efflux through immunoprecipitation and Western blot analyses of Aβ in plasma and brain homogenates from WT and HIP rats (similar to those in a), with APP/PS1 rat brain homogenate used as positive control for Aβ immunoreactivity signal. c Proposed amylin action on the Aβ transport across the BBB. d Confocal microscopy and STORM analysis of amylin in brain microvessels isolated from HIP (n = 47 microvessels) and WT (n = 21 microvessels) rats (n = 3 males/group; age 15–16 months). Confocal microscopy analysis of serial sections from HIP rat brains stained with Thioflavin S (Thio S) or amylin (e) and with the lipid peroxidation marker 4-HNE or amylin (f) (n = 3 males similar to those in d). Western blot analysis of P-gp (g) and LRP1 (h) in brain microvessel lysates from HIP and WT rats (n = 5–7 males/group similar to those in a), and in brain microvascular ECs incubated with human amylin. Data are means ± SEM.
Fig. 6
Fig. 6. In vitro test of amylin-induced impairment of Aβ efflux across the BBB.
a Cartoon representation of the in vitro BBB model (ECs monolayer - luminal chamber; astrocytes - abluminal chamber) used in Aβ transcytosis experiments. b Transendothelial electrical resistance (TEER) in EC monolayers (n = 20 preparations) as a function of days in culture. c Representative Western blot and densitometry quantification of LRP1 in lysates from primary rat brain microvascular vascular ECs treated with vehicle or various concentrations of human amylin (500 nM, 1 µM, 5 µM, and 10 µM) for 24 h (n = 3 preparations/test). d Percent cell viability from the MTS assay in ECs treated with amyloid-forming human amylin (500 nM, 1 µM, 5 µM, and 10 µM) or vehicle, for 24 h. e The Aβ42 transcytosis quotient (TQ) across the in vitro BBB, in vehicle- and human amylin-treated EC monolayers. f LRP1 mRNA levels (fold difference using 2−ΔΔCt method) measured with qRT-PCR in lysates from ECs treated with vehicle, human amylin or rat amylin. g LRP1 mRNA levels measured by qRT-PCR in brain capillary lysates from same rats as in Fig. 5h. h, i miRNA (miR)-103 and miR-107 expression levels measured by qRT-PCR in lysates from ECs treated with vehicle or human amylin (same as in Fig. 5h), and in brain capillary lysates from same rats as in Fig. 5h. j TargetScan schematic showing consensus regions for miR-205, miR200bc-3p/429, and miR-103 and miR-107. Western blot analyses of LRP1 from miRNA (miR) 103 and miR-107 treated ECs compared to miR-control (n = 3 preparations/group) (k), as well as of LRP1 (l) and P-gp (m) from antagomir (amiR) 103 and amiR-107 treated ECs compared to amiR-control treated cells (n = 3 preparations/group). Data are mean ± SEM. one-way ANOVA with Dunnett’s post hoc (F). unpaired t test for the other panels.
Fig. 7
Fig. 7. The genes predicted to influence the impact of cerebrovascular amylin deposition on the central nervous system.
a The Log10 (p-value) versus Log10 (fold change) of the DE genes in HIP vs. WT rat brains. Each dot represents one gene, with upregulated in red color (HIP vs. WT, ≥ 1.5-fold change), downregulated in blue color (HIP vs. WT, ≥ 1.5-fold change), and < 1.5-fold change in black color. b Top 5 canonical pathways identified by Ingenuity Pathways Analysis of differentially expressed (DE) genes detected by RNA-seq analysis of hippocampal tissue from the HIP vs. WT rat groups (P < 0.05) (n = 10 males/group). c Hierarchical clustering of 25 DE genes identified the enrichment of top 5 canonical pathways in (b). d Top 5 Gene Ontology (GO) biological processes enriched in HIP rat group compared to WT rat group.

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