INSULIN TRAFFICKING PERTURBATIONS AT THE BLOOD-BRAIN BARRIER IN ALZHEIMER’S DISEASE MODELS

Abstract

Alzheimer’s disease (AD) is recognized as a multifactorial disease and a major cause of dementia in the elderly. Pathological hallmarks of AD include brain amyloid beta (Aβ) deposits and intraneuronal tangles of hyperphosphorylated tau protein. Reduction of brain Aβ burden is widely considered as a viable therapeutic strategy for AD, and developing methods to promote brain Aβ clearance has been at the forefront of AD research. Recent clinical trials conducted in a small group of AD patients demonstrated the efficacy of intranasally-administered insulin in improving memory and reducing brain amyloid burden (Craft et al., 2012). Although the mechanism of insulin action is not clear, epidemiological studies have linked type 2 diabetes (T2DM), characterized by hyperinsulinemia and peripheral insulin resistance, with cognitive decline and amyloid burden in AD (Matsuzaki et al., 2010). One possibility is that hyperinsulinemia damages the cerebrovascular endothelium, referred to as the blood brain barrier (BBB). The BBB is a major signaling and material trafficking pathway between plasma and brain that not only delivers glucose and insulin to brain but also serves as the primary clearance portal for brain Aβ (Storck et al., 2016; Yuede et al., 2016). Hence, T2DM could augment AD pathology by inhibiting insulin delivery and disrupting brain Aβ clearance. Although, intranasal insulin may remedy the situation, this approach may limited, due to variable absorption of insulin into CNS. Moreover, insulin is a growth factor with multiple physiological targets; hence, chronic insulin therapy will have off-target effects. As a fundamental approach, critical insulin-responsive cellular and molecular pathways that facilitate Aβ trafficking should be identified. Only through elucidating these dysfunctions in AD/T2DM can novel therapeutic targets be rationally sought. In this work, the key questions concern the dynamics of insulin transport from systemic circulation into brain via the BBB and the disruption in insulin trafficking caused by Aβ peptides in AD brain. Importantly, molecular components in the insulin trafficking/signaling pathway that are disturbed by Aβ exposure were identified as well as the perturbed insulin delivery and brain insulin resistance. This new knowledge will facilitate the search for novel therapeutic targets for AD. I have been investigating mechanisms triggering insulin scarcity in AD brains by conducting kinetic biodistribution and SPECT/CT imaging assays in Aß peptide over-producing transgenic mice (APP/PS1), insulin resistant db/db (leptin receptor deficient) and aged WT mice. I analyzed plasma pharmacokinetic and dynamic brain uptake data by 3-compartmental analysis on SAAM© and discovered differences in distribution rate constants between WT and the insulin-resistant mouse groups. Upon simulating tissue distribution of a given dose of insulin in a 3-compartmental Stella© model with the estimated rate constants, I discovered that APP/PS1, db/db aged, and Aβ40/Aβ42 pre-treated WT mouse display higher plasma AUC and lower brain AUC levels of insulin, as compared to healthy, young WT mice. This led us to a hypothesis that Aß40 and Aß42 interfere with the transport of insulin into the brain parenchyma. Now, the appearance of systemic insulin in the brain parenchyma is contingent upon insulin receptors (IR) expressed by BBB endothelial cells. Therefore, I employed human cerebrovascular microendothelial cells (hCMEC) and over-expressed hCMEC monolayers with IR to investigate mechanistic interactions between Aß peptides and insulin transcytosis across the BBB. The processes involved in insulin transcytosis across BBB endothelial cells are postulated to operate in tandem with downstream signaling cascades. I studied variables in IR-mediated transport processes and correlated the results with shifts in phosphorylation of proteins expressed in downstream insulin signaling pathways, as a consequence of Aß peptide exposure. Through flow cytometry and radioactive Transwell© transport assays, I discovered that the IR-mediated uptake, permeability and exocytosis of insulin across hCMEC/D3 cells were significantly impaired by pre-exposure to Aβ40 and Aβ42. With help of FRAP/FLIP imaging analyses, I found that the half-life of lateral diffusion and exocytic recycling of IR in response to insulin was increased in presence of Aβ40 and Aβ42. With respect to hCMEC/D3 signal transduction downstream of IR, Western Blot analyses confirmed that Aβ40 and Aβ42 excessively activated the phosphorylation of the IR-β subunit at Tyr1162/1163 and decreased the phosphorylation of Akt at Ser 473. In addition, reverse-phase protein array (RPPA) assays revealed that phosphorylation of MAPK3 was increased whereas that of caveolin-1 was decreased due to Aβ40 and Aβ42 exposure. My results lead to conclude that Aβ peptides interfere with insulin signaling/trafficking at the BBB and reduce insulin availability in the AD brain. It is therefore imperative to rectify motifs which result in impaired insulin signaling at the BBB to improve its transport into the brain parenchyma.