(M1030-04-22) Amyloid Beta Peptides Inhibit Insulin Trafficking at the Blood-Brain-Barrier in Alzheimer's disease Brain

Purpose: Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting elderly above 65 years of age¹, and is characterized by cognitive decline. Pathologically, AD is characterized by the presence of amyloid beta (Aβ) plaques and hyperphosphorylated tau protein in the brain parenchyma². The main risk factors for AD include aging, genetic predisposition, and metabolic syndrome, which manifest insulin resistance³. The brain insulin resistance is evident by diminished insulin levels within the brain and disrupted insulin signaling³ that are associated with perturbations in energy metabolism and neuronal function. These pathological changes were previously shown to contribute to neurodegeneration and cognitive decline in AD⁴. Therefore, researchers have attempted to restore brain insulin levels through the intranasal administration of the insulin⁵. While this method has improved cognition to some extent, higher insulin concentrations have triggered the off target side effects⁶. So, it is critical to investigate the molecular mechanisms underlying the insulin depletion in AD brains to develop target therapies to address brain insulin resistance. Insulin is predominantly produced in the periphery and enters the brain through the blood brain barrier (BBB) via a saturable transport mechanisms^7,8. Specifically, the transport process involves receptor mediated binding, endocytosis, intracellular vesicular trafficking, and transcytosis⁹. Recent reports have claimed that insulin uptake into the brain is independent of the insulin receptor^10,11. Even if the insulin receptor is not directly involved in insulin transcytosis at the BBB, the role of insulin signaling in regulating insulin trafficking cannot be ignored. Our lab has previously investigated the effect of Aβ peptides on the insulin uptake at the BBB endothelium and found that Aβ40 and Aβ42 decrease the brain insulin uptake at the BBB in AD transgenic mouse models (APP/PS1). Further, Reverse Phase Protein Array (RPPA) analysis has identified that Aβ peptides differentially disrupt metabolic (PI3K/AKT) and mitogenic (MEK/ERK) arms of insulin signaling pathways. We hypothesize that Aβ peptides accumulating in the AD brain inhibit insulin trafficking at the BBB. However, there is limited evidence to link insulin signaling and insulin uptake. Therefore, in the study, we aim to investigate the role of insulin signaling in the insulin uptake in BBB endothelial monolayers in vitro.

Methods: Western blots were performed to investigate the optimal time and concentration for pharmacological inhibition of the insulin signaling inhibitors. The hCMEC/D3 cells were treated with an AKT phosphorylation inhibitor MK2206 (65, 650, and 6.5 μM) and MEK phosphorylation inhibitor trametinib (1.8 nM, 18 nM, and 180 nM) at various time points (1, 2, 3, 6, and 24 hours) and co-incubated with insulin (50 nM) for 30 minutes. Density of proteins in the blots were analyzed using LICOR imaging. Flow cytometry studies were conducted to investigate the effect of insulin signaling inhibitors on AF647-insulin uptake. The hCMEC/D3 cells were pretreated with MK2206 (66 nM or  650 nM) and trametinib (1.8nM or 18 nM) for 1.5 hours followed by 30 minute co-incubation with 50 nM AF647-insulin. Intracellular fluorescence was measured using flow cytometry and data was analyzed using FlowJo software.

Results: Treatment with MK2206 (65 nM) for 1, 2, or 3 hours inhibited AKT phosphorylation (**p-value ≤0.01) compared to the insulin treated group as shown in Fig 1 A and B. Further, treatment with higher concentrations of MK2206 (65 nM, 650 nM or 6.5 μM) for 2 hours inhibited AKT phosphorylation to a greater extent (**p-value≤0.01, ***p-value≤0.001, ****p-value≤0.0001). Trametinib treatment at 1.8 nM concentration for 2, 6,  or 24 hours inhibited the ERK phosphorylation (***p-value ≤0.001) at all the time points compared to the insulin treated group (Fig. 2 A and B).  Further, treatment with higher concentrations of trametinib (1.8 nM, 18 nM, or 180 nM) for 2 hours has a greater effect on ERK phosphorylation (Fig. 2 C and D)(**p-value ≤0.01, ***p-value ≤0.001). The accumulation of AF647-insulin accumulation decreased in the MK2206 (650 nM) pretreated group compared to the AF647-insulin treated group (Fig. 3 A and B). This data indicates the role of AKT activation in insulin uptake, whereas, trametinib treatment has no impact on AF647-insulin uptake (Fig. 3C and D). This data suggests that the insulin uptake is independent of the ERK pathway.

Conclusion: In the current study, we have identified the possible mechanisms of how amyloid beta peptides inhibit the insulin uptake at the BBB endothelium. Flow cytometry studies have shown that the AKT pathway is involved in the insulin uptake. Also, RPPA data generated in our lab previously suggests that Aβ peptides downregulate the PI3K/AKT pathway. Taken together,  Aβ peptides most likely disrupt insulin uptake by inhibiting PI3K/AKT pathway rather than the MEK/ERK pathway. Our future studies will focus on investigating the impact of insulin signaling on the endocytosis of Aβ peptides.

References: (1) Arnold, S. E.; Arvanitakis, Z.; Macauley-Rambach, S. L.; Koenig, A. M.; Wang, H.-Y.; Ahima, R. S.; Craft, S.; Gandy, S.; Buettner, C.; Stoeckel, L. E.; Holtzman, D. M.; Nathan, D. M. Brain Insulin Resistance in Type 2 Diabetes and Alzheimer Disease: Concepts and Conundrums. Nat. Rev. Neurol. 2018, 14 (3), 168–181. https://doi.org/10.1038/nrneurol.2017.185.
(2) Farkas, E.; Luiten, P. G. M. Cerebral Microvascular Pathology in Aging and Alzheimer’s Disease. Prog. Neurobiol. 2001, 64 (6), 575–611. https://doi.org/10.1016/S0301-0082(00)00068-X.
(3) Abou-Raya, S.; Abdou, A.; Kamel, M. A.; Ramadan, A. M. A. Insulin Resistance and Alzheimer’s Disease: The Role of Defective Insulin Signaling and Inflammation. 2018, 4 (2), 38. https://doi.org/10.4103/ejode.ejode_4_18.
(4) Biessels, G. J.; Kappelle, L. J. Increased Risk of Alzheimer’s Disease in Type II Diabetes: Insulin Resistance of the Brain or Insulin-Induced Amyloid Pathology? Biochem. Soc. Trans. 2005, 33 (5), 1041–1044. https://doi.org/10.1042/bst0331041.
(5) Benedict, C.; Hallschmid, M.; Schultes, B.; Born, J.; Kern, W. Intranasal Insulin to Improve Memory Function in Humans. Neuroendocrinology 2007, 86 (2), 136–142. https://doi.org/10.1159/000106378.
(6) Hanson, L. R.; Frey, W. H. Intranasal Delivery Bypasses the Blood-Brain Barrier to Target Therapeutic Agents to the Central Nervous System and Treat Neurodegenerative Disease. BMC Neurosci. 2008, 9 (S3), S5. https://doi.org/10.1186/1471-2202-9-S3-S5.
(7) Greco, A. V.; Ghirlanda, G.; Fedeli, G.; Gambassi, G. Insulin in the Cerebro Spinal Fluid of Man. Eur. Neurol. 1970, 3 (5), 303–307. https://doi.org/10.1159/000113983.
(8) Margolis, R. U.; Altszuler, N. Insulin in the Cerebrospinal Fluid. Nature 1967, 215 (5108), 1375–1376. https://doi.org/10.1038/2151375a0.
(9) Pulgar, V. M. Transcytosis to Cross the Blood Brain Barrier, New Advancements and Challenges. Front. Neurosci. 2019, 12, 1019. https://doi.org/10.3389/fnins.2018.01019.
(10) Rhea, E. M.; Rask‐Madsen, C.; Banks, W. A. Insulin Transport across the Blood–Brain Barrier Can Occur Independently of the Insulin Receptor. J. Physiol. 2018, 596 (19), 4753–4765. https://doi.org/10.1113/JP276149.
(11) Hersom, M.; Helms, H. C.; Schmalz, C.; Pedersen, T. Å.; Buckley, S. T.; Brodin, B. The Insulin Receptor Is Expressed and Functional in Cultured Blood-Brain Barrier Endothelial Cells but Does Not Mediate Insulin Entry from Blood to Brain. Am. J. Physiol.-Endocrinol. Metab. 2018, 315 (4), E531–E542. https://doi.org/10.1152/ajpendo.00350.2016.

Acknowledgements: This work was supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke R01NS125437


Figure 1: Pharmacological activity of AKT inhibitor MK2206 was determined by pretreating the hCMEC/D3 cells MK2206 and co-incubation with insulin, 50 nM for 30 minutes. MK2206 has decreased AKT phosphorylation with 65 nM concentration with 1, 2 or 3 hours treatment compared to the insulin alone treated group with blots shown in A and quantification in B. The MK2206 has decreased AKT phosphorylation with 2 hours treatment at 65 nM, 650 nM, and 6.5 μM concentration compared to the insulin alone treated group with blots shown in C and quantification in D. **p-value ≤0.01,***p-value ≤0.001, ****p-value ≤0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test.



Figure 2: Pharmacological activity of MEK inhibitor trametinib was determined by pretreating the hCMEC/D3 cells trametinib and co-incubation with insulin, 50 nM for 30 minutes. Trametinib has decreased ERK phosphorylation with 1.8 nM concentration with 2, 6 or 24 hours treatment compared to the insulin alone treated group with blots shown in A and quantification in B. Trametinib has decreased ERK phosphorylation with 2 hours treatment at 1.8 nM, 18 nM or 180 nM concentration compared to the insulin alone treated group with blots shown in C and quantification in D. **p-value ≤0.01,***p-value ≤0.001, ****p-value ≤0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test.


Figure 3: Effect of insulin signaling inhibitors is investigated by pretreating the hCMEC/D3 cells with MK2206 (65 nM or 650 nM) or trametinib (1.8 nM or 18 nM) for 1.5 hours followed by co-incubation with AF647-insulin (50 nM) for 30 minutes. Treatment with MK2206 (650 nM) has decreased AF647-Insulin compared to the AF647-insulin alone group with histogram shown in A and bar graph comparison in B. Treatment with trametinib has no effect on the AF647-insulin uptake with histogram shown in C and bar graph comparison in D. *p-value ≤0.05, one-way ANOVA followed by Dunnett’s multiple comparison test.