(T1130-05-33) High-Density Lipoprotein Mimetic Peptide 4F Reduces Amyloid Beta Peptide Accumulation in the Blood-Brain Barrier Endothelium of Alzheimer’s disease brain

Purpose: Deposition of toxic amyloid beta (Aꞵ) peptides in the brain parenchyma is one of the pathological hallmarks of Alzheimer’s disease (AD). Cerebrovascular dysfunction is observed in 90% of AD patients and Aꞵ accumulation in the blood-brain barrier (BBB) endothelium, which lines the cerebrovascular lumen, is believed to be a major contributor to cerebrovascular dysfunction. Therefore, the use of pharmacological agents to reduce Aꞵ exposure in the BBB endothelium offers a promising therapeutic strategy to improve cerebrovascular health and mitigate AD pathology. One of such molecules thus being developed is high-density lipoprotein (HDL) mimetic peptide, 4F, which possesses neuro- and vaso-protective benefits of HDL particles, while maintaining high brain permeability due to its small molecular size. Our previous studies have demonstrated that 4F could decrease blood-to-brain influx but increase brain-to-blood efflux of Aꞵ across the BBB in health mice¹, thereby potentially reducing Aꞵ accumulation in the brain parenchyma. However, the 4F effect on Aꞵ accumulation within the BBB endothelium, especially in disease condition is yet to be established. Further, the underlying molecular pathway by which 4F alters Aꞵ trafficking at the BBB remains uncharacterized.

Methods: In vivo: 4F was daily administered via intraperitoneal injection in both wide-type (WT) and APP/PS1 transgenic mice for 7 days (N=4 in each group). On the day following last 4F administration, radiolabeled ¹²⁵I-Aꞵ₄₂ was injected intravenously and the animals were immediately imaged for the following 40min by dynamic single positron emission computed tomography (SPECT)-computed tomography (CT) imaging. The dynamic heart imaging data was used to deconvolve the plasma pharmacokinetics (PK) of ¹²⁵I-Aꞵ₄₂ as described by our previous publication². Dynamic brain imaging data and deconvolved plasma PK were used to conduct graphical analysis to determine the ¹²⁵I-Aꞵ₄₂ distribution kinetics in the brain parenchyma and BBB endothelium (Figure 1A). In vitro: Impact of 4F on Aꞵ₄₂ exocytosis in the BBB endothelial cells was investigated. Primary bovine brain microvasculature endothelial (BBME) monolayers grown on 24-mm Transwell inserts were pre-treated with 4F followed by incubating with fluorescence labeled Aꞵ₄₂ (F-Aꞵ₄₂) for 1h. Afterwards, the media was replaced with fresh growth media in both luminal and abluminal chambers. Then sample aliquots were taken from both luminal and abluminal chambers at different time points for fluorescence measurement. Further, effects of Aꞵ₄₂ and 4F on p38 MAPK pathway were investigated using human cerebral microvascular endothelial cell (hCMEC/D3) monolayers by western blotting. The impact of p38 inhibition on F-Aꞵ₄₂ endocytosis in the BBB endothelial cells was determined by flow cytometry.

Results: Logan plot analysis demonstrated that 7-day treatment of 4F could significantly reduce the distributional volume of ¹²⁵I-Aꞵ₄₂ in the BBB endothelium of APP/PS1 transgenic mice (46.8 mL in 4F treated versus 81.9 mL in control mice). However, no significant difference was observed in WT mice (Figure 1B). Interestingly, Patlak plot analysis results showed that 4F could increase the irreversible influx rate of ¹²⁵I-Aꞵ₄₂ into brain parenchyma of APP/PS1 transgenic mice (1.94 mL/min in 4F treated versus 0.70 mL/min in control mice) (Figure 1C). These in vivo experiment results suggested that 4F could reduce the Aꞵ₄₂ accumulation in the BBB endothelium by facilitating Aꞵ₄₂ transport into brain parenchyma. To corroborate the in vivo findings, Aꞵ₄₂ exocytosis from the BBB endothelium to both blood and brain sides was determined. Kinetic analysis indicated that the exocytosis rate of F-Aꞵ₄₂ to the abluminal (brain) side was increased by 2 fold in 4F treated BBME monolayers. To investigate the underlying molecular mechanisms of 4F effect, the role of p38 kinase in Aꞵ₄₂ endocytosis was investigated. Reverse-phase protein array (RPPA) assay suggested that Aꞵ₄₂ increased the phosphorylation p38 kinase in BBB endothelial cells and the results were validated by Western blot. However, 4F was shown to significantly reduce the Aꞵ₄₂-induced p38 activation in hCMEC/D3 cells (Figure 2A). Further, flow cytometry analysis indicated that p38 inhibitor SB203580 significantly decreased intracellular fluorescence of F-Aꞵ₄₂ (Figure 2B), suggesting that Aꞵ₄₂ uptake by BBB endothelial cells is mediated by p38 kinase.

Conclusion: These findings demonstrated the beneficial effect of HDL mimetic peptide 4F in reducing Aꞵ₄₂ accumulation in the BBB endothelium of AD transgenic mice. This is significant because Aꞵ exposure is associated with BBB dysfunction which aggravates AD progression. We also investigated the cellular and molecular mechanisms underlying the kinetic observations. It was found that 4F could increase the exocytosis and decrease endocytosis of Aꞵ₄₂ in the BBB endothelial cell. Further, 4F decreased the Aꞵ₄₂ endocytosis potentially via inhibiting the activation of p38 MAPK pathway.

References: 1. Swaminathan SK, Zhou AL, Ahlschwede KM, Curran GL, Lowe VJ, Li L, Kandimalla KK. High-Density Lipoprotein Mimetic Peptide 4F Efficiently Crosses the Blood-Brain Barrier and Modulates Amyloid-β Distribution between Brain and Plasma. J Pharmacol Exp Ther. 2020 Nov;375(2):308-316.
2. Wang Z, Wang L, Ebbini M, Curran GL, Min PH, Siegel RA, Lowe VJ, Kandimalla KK. Deconvolution of Plasma Pharmacokinetics from Dynamic Heart Imaging Data Obtained by Single Positron Emission Computed Tomography/Computed Tomography Imaging. J Pharmacol Exp Ther. 2023 Jul;386(1):102-110.

Acknowledgements:This study was supported by the National Institutes of Health National Institute on Aging (AG058081) and National Institute of Neurological Disorders and Stroke (R01NS125437).

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