(T1130-10-55) The Mechanisms Governing the Positive Feedback Loop between Cerebrovascular Inflammation and Amyloid Beta Accumulation

Purpose: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that causes neuronal loss and cognitive decline. Deposition of amyloid beta (A𝛽) peptides and the accumulation of neurofibrillary tangles, formed by hyperphosphorylated tau protein, in the brain are two of the key pathological hallmarks of AD1. Around 80% of AD patients have vascular dysfunction2 and manifest cerebrovascular inflammation3, which is emerging as a third core feature of AD pathology that can potentially be detected before onset of AD symptoms. Exposure of the BBB endothelium to toxic levels of A𝛽, triggers the inflammatory signaling cascade and engenders vascular dysfunction. Since a major portion of A𝛽42 from the brain is cleared via the blood brain barrier (BBB) endothelium that lines the brain blood vessels⁴, vascular dysfunction could exacerbate amyloid deposition in AD patients. Thus, BBB inflammation and the brain amyloid deposition are connected in a feedback loop that aggravate AD progression. In this study we aim to understand the underlying effects of A𝛽 on stimulating the inflammatory cascade using vascular cell adhesion molecule-1 (VCAM-1) as a BBB endothelial inflammatory marker. Alternatively, the perturbations in A𝛽 trafficking across the BBB under an inflammatory stimulus are investigated.

Methods: We investigated the effect of A𝛽42 on the expression of an inflammatory marker, VCAM-1 in the mouse brain using 125I-VCAM1-IgG. AD transgenic mice have several underlying pathologies, which makes it difficult to identify the contributions of A𝛽 alone in increasing the VCAM-1 expression. We thus infused A𝛽42 in wild type (WT) mice via the internal carotid artery followed by femoral vein injection of 125I-VCAM1-IgG to determine the brain distribution of 125I-VCAM1-IgG (Fig 1B). Moreover, the effect of A𝛽42 on the membrane VCAM-1 expression in cerebral microvascular endothelial (hCMEC/D3) cell monolayers, was investigated using western blot analysis and confocal microscopy. While cerebrovascular inflammation is a consequence of A𝛽 exposure, inflammation may in turn exacerbate A𝛽 accumulation. It was previously identified that TNF alpha is an inflammatory cytokine whose levels have been shown to increase in the plasma of AD patients compared to age-matched controls⁵. We hypothesize that cerebrovascular inflammation triggered by TNF alpha alters A𝛽42 accumulation in the BBB. To investigate this hypothesis, we infused TNF alpha or PBS (control) in WT mice via the internal carotid artery followed by injection of 125I-A𝛽42 via the femoral vein (Fig. 2A). We next conducted fluorescent A𝛽42 (FITC-A𝛽42) cell uptake studies in a primary porcine brain endothelial cells and immortalized BBB cell line (hCMEC/D3). It is known that the mechanism for A𝛽 endocytosis in BBB endothelial cells is mediated by caveolae-dynamin apparatus. However, changes in this trafficking pathway under an inflammatory stimulus remain unknown. To investigate the mechanism of the A𝛽 trafficking pathway under an inflammatory stimulus, hCMEC/D3 cells were transfected with a dynamin negative mutant (K44A) to measure A𝛽42 BBB endothelial accumulation following TNF alpha stimulation using confocal microscopy and flow cytometry.

Results: VCAM-1 gene expression is increased in the cerebrovascular endothelium of AD transgenic mice compared to the controls (Fig. 1A). The in vivo studies demonstrated an overall increase in brain accumulation (% injected dose/brain weight) of 125I-VCAM-1 IgG in A𝛽42 infused mice compared to PBS infused mice (Fig 1C). The in vitro results also demonstrated an increase in membrane VCAM-1 expression upon A𝛽42 treatment compared to untreated controls (Fig 1D). These observations show that A𝛽42 exposure triggers cerebrovascular inflammation by increasing VCAM-1 expression at the BBB. We next analyzed the effects of TNF alpha on A𝛽42 accumulation. The accumulation (% injected dose/brain weight) of 125I-A𝛽42 was higher in TNF alpha infused mice compared to PBS infused mice (Fig. 2B). Thus, TNF alpha alters the trafficking of A𝛽42 at the BBB. The in vitro findings agreed with the in vivo results demonstrating an increase in F-A𝛽42 uptake by endothelial cells upon stimulation with TNF alpha as assessed by flow cytometry and confocal microscopy (Fig. 2C). Moreover, the dynamin negative mutant transfected cells demonstrated changes in A𝛽42 uptake in endothelial cells following TNF alpha stimulation.

Conclusion: In summary, our research demonstrates that A𝛽42 increases the expression of a vascular inflammatory marker, VCAM-1, on the BBB endothelium, which could be used as a novel early detection marker for vascular inflammation prevalent in AD. Moreover, inflammatory cytokines such as TNF alpha alter A𝛽42 trafficking into the brain and BBB endothelial cells via a dynamin mediated trafficking process. Understanding the underlying mechanisms of the feedback loop between cerebrovascular inflammation and amyloid beta deposition would aid in developing therapies targeted towards breaking this vicious cycle.

References: 1. Rainulf a. Stelzmann HNS, and F. Reed Murtagh. An English Translation of Alzheimer’s 1907 Paper, “Uber eine eigenartige Erkankung der Hirnrinde”. Accessed March 2021, https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ca.980080612
2. Scheibel AB. Alterations of the cerebral capillary bed in the senile dementia of Alzheimer. Ital J Neurol Sci. Oct 1987;8(5):457-63. doi:10.1007/BF02334602
3. Yu D, Corbett B, Yan Y, et al. Early cerebrovascular inflammation in a transgenic mouse model of Alzheimer's disease. Neurobiol Aging. Dec 2012;33(12):2942-7. doi:10.1016/j.neurobiolaging.2012.02.023
4. Deane R, Wu Z, Zlokovic BV. RAGE (yin) versus LRP (yang) balance regulates alzheimer amyloid beta-peptide clearance through transport across the blood-brain barrier. Stroke. Nov 2004;35(11 Suppl 1):2628-31. doi:10.1161/01.STR.0000143452.85382.d1
5. Zuliani G, Ranzini M, Guerra G, et al. Plasma cytokines profile in older subjects with late onset Alzheimer's disease or vascular dementia. J Psychiatr Res. Oct 2007;41(8):686-93. doi:10.1016/j.jpsychires.2006.02.008

Acknowledgements: This project is funded by Minnesota partnership for biotechnology and medical genomics (Grant 00056030) and National Institute of Health (Grant AG058081)


Effects of AD pathology and amyloid beta on cerebrovascular inflammation as evidenced by increased VCAM-1 expression. (A) Higher VCAM-1 mRNA expression in APP/PS1 mice compared to wild type (WT control mice. Increased VCAM-1expression in WT mice infused with Abeta42 (B)Experimental scheme. Mice were infused with PBS or Abeta42 via the internal carotid artery. 125I-VCAM-1 IgG (50u uCi) was injected via the femoral vein. The brain accumulation of 125 I-VCAM-1 IgG was assessed 40 min after femoral vein injection in mice infused with PBS or A􀁅42. (C) Higher % of the injected dose (I.D.) measured in the whole brain per gram of tissue in A􀁅42 infused mice compared to PBS infused mice. (Data: mean ± S.D (n=3). **p < 0.001; Student t-test). Higher VCAM-1 expression in Abeta42 treated human cerebral microvascular endothelial (hCMEC/D3) cells as shown by confocal microscopy. (D) Confocal micrographs depict PE-VCAM-1-IgG binding to hCMEC/D3 monolayers on Abeta42 (3.125 μg/mL, 3hrs.) stimulation . PE-VCAM- 1 IgG is stained red, and the nuclei is stained blue (DAPI). Scale bar (20 um)


Effects of an inflammatory cytokine, TNF alpha, on amyloid beta trafficking. Higher 125I-Abeta42 uptake in TNF alpha infused mice in comparison to PBS control mice. (A) Experimental scheme. Mice were infused with PBS or TNF alpha via the internal carotid artery. 125I- Abeta42(100 uCi) was injected via the femoral vein. Following a 30-minute interval the mice was perfused and 125I activity was assessed in a gamma counter. (B) Greater % of the injected dose (I.D.) measured in the whole brain per gram of tissue in TNF alpha infused mice compared to PBS infused mice. (Data: mean ± S.D (n=3). *p < 0.05; Student t-test). (C) Increased F- A􀁅42 uptake in hCMEC/D3 cells on stimulation with TNF alpha (20 ng/mL, 24hrs.) as demonstrated by and confocal microscopy. FITC- Abeta42 is stained green, and the nuclei is stained blue (DAPI). Scale bar (20 􀁐m)