Poster BB18 - Glitches in the meningeal lymphatics system: a contributing factor in the progression of Alzheimer’s Disease

Introduction: Alzheimer’s Disease (AD) is a neurodegenerative brain disorder characterized by the abnormal accumulation of protein aggregates in the brain called amyloid-beta (Aβ) plaques. Amyloid beta (Aß) is part of the brain excretory waste and is cleared out by the lymphatics system of the brain (called glymphatics) or phagocytized by specialized glial cells called the microglia. In a healthy brain, Aβ is cleared from the brain via a vascular network called the meningeal lymphatic system. However, in AD there is a disruption in the waste clearance mechanism. Glymphatics abnormalities lead to a disruption in this drainage and subsequent accumulation of Aβ and the formation of plaques. These plaque formations also activate microglia to turn on the synaptic elements of the brain, phagocytizing them and also destroy microtubules which are the supporting framework of neurons1. Although the role of Aß has recently caught the attention of the neuroscience community, the correlation with the lymphatics system remains largely unexplored. Perhaps delving into the mechanisms of clearance could provide us with possible therapeutic measures for AD.

Materials and

Methods: Using our recently developed in vitro meningeal lymphatics (ML) co-culture system at the Munson Lab, we are able to recapitulate the lumen of a meningeal lymphatic vessel2. Our model consists of a monolayer of human lymph node lymphatic endothelial cells (LECs) on the underside of tissue culture insert and human meningeal cells (hMCs) within a tissue culture insert, to resemble the lumen of a meningeal lymphatic vessel. After allowing sufficient time for the co-culture monolayers to develop, the model is dosed with different isomers of Aß at varying concentrations. While soluble fibrillar Aß is present in a healthy brain as a waste product, plaque formation is indicative of fluid impedance. Our current model examines both soluble Aß as well as aggregated Aß. Testing and analysis of Aß dosages between 1uM to 20 uM of different forms have yielded quantifiable results. The synthetic Aß peptide from Anaspec(1-42) is aggregated over 7 days using a previously existing protocol3. The ML models are fixed and processed for immunohistochemistry staining before being imaged on an AxioObserver.

Results, Conclusions, and Discussions: When taken together, our results show significant decreases in the percentage area intact of the LEC monolayers with increasing Aß concentrations (Figure 2). We also observe morphological distension in the ML with an increase in Aß dosage. It is also seen that increasing dosages of soluble amyloid beta tend to self-aggregate while successively causing disruption of the junctions. Further SEM imaging on the LEC monolayers treated with Aß show an increase in fibrillar sheering with an increase in soluble Aß dosage. We also assessed the effects of Aß on transcription factors responsible for oxidative stress. We can conclude that aggregated Aß causes significantly more damage to the LEC monolayers as opposed to soluble Aß of the same dosage. Soluble Aß elicits detrimental effects at higher dosages due to possible self-aggregation of Aß which was confirmed by SEM imaging. Oxidative stress profiling arrays reveal nuclear level damage to cells. This imminent deterioration of the lymphatic drainage system of the brain can be linked to being a definitive contributor to the progression of AD. Our ML model also avails application in the microfluidic chip designed by our collaborators in the Pompano Lab designed to study in vitro crosstalk between the brain, lymph node and meningeal lymphatics barrier.

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