Intracellular Trafficking of Amyloid Precursor Protein by the Exocyst: Mechanisms in Alzheimer's Disease and Insulin Signaling

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Figure 19 - Close Up Epi Exoc4-mNeonGreen_mutAPP-mScarlet (Red Channel Only) - APP vesicles moving through a differentiated SHSY-5Y cell.avi (20.88 MB)
Figure 19 - Epi Exoc4-mNeonGreen_mutAPP-mScarlet (Red Channel Only) - APP vesicles moving through a differentiated SHSY-5Y cell.avi (2.07 MB)
Figure 20C - Close Up Epi Exoc7-mNeonGreen_mutAPP-mScarlet - APP moving through a differentiated SHSY-5Y cell and projection outlined by Exoc7 fluorescence.avi (1.29 MB)
Figure 20C - Epi Exoc7-mNeonGreen_mutAPP-mScarlet - APP moving through a differentiated SHSY-5Y cell and projection outlined by Exoc7 fluorescence.avi (6.74 MB)

Date

2021

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Fogelgren, Benjamin

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Cell and Molecular Biology

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Abstract

Alzheimer’s disease is a devastating progressive neurodegenerative disease that causes memory loss, personality changes, and impaired reasoning. It currently affects approximately 6 million people in the US, and is America’s 6th leading cause of death. Research on the disease has been extensive for decades, however there are still no effective therapies and the pathogenic mechanisms are not fully understood. Two of the pathways implicated in causing the damage seen in Alzheimer’s disease focus around amyloid beta peptide aggregates, that form plaques, and hyperphosphorylated tau, which forms tangles. Studies on the Amyloid hypothesis focus on the generation and aggregation of the peptide, which is cleaved from the amyloid precursor protein (APP). How APP is cut and whether it will lead formation of the amyloid peptide has been shown to be influenced by the trafficking of APP bringing it into contact with different secretases. Based on the intracellular trafficking patterns of APP, and hints from the literature, we hypothesized that the eight-protein exocyst complex regulates APP trafficking and amyloid beta generation in neurons. For our research, we used two cell models: the SH-SY5Y cell line, a human neuroblastoma cell line able to be differentiated into neuronal cells using retinoic acid, and mouse primary hippocampal neurons. For our experiments, we generated several transgenic SH-SY5Y lines, including a mutant APP overexpressing line that secretes high levels of amyloid beta, as well as lines that co-express fluorescent-tagged mutant APP and exocyst proteins for use in live cell imaging. We found that when several members of the exocyst family are knocked down using siRNA in our mutAPP overexpressing line, the amount of amyloid beta released in the media significantly decreases. Using the proximity ligation assay (PLA), we also discovered that Exoc5 and APP closely co-localize (within 40nm) in mouse primary hippocampal neurons. Since the exocyst regulates the insulin-induced trafficking of Glut4 glucose transporters in adipocytes and muscle cells, we tested the effect of insulin signaling in neurons on the exocyst and its relationship to APP and Glut4. When insulin was present, the exocyst holocomplex assembly increased significantly, as did its association with Glut4, however APP association with the exocyst was largely abolished. The data suggests that insulin signaling is able to switch the exocyst complex away from APP-containing vesicles and potentially reduce amyloid beta generation. These findings point to the regulation of APP trafficking by the exocyst complex and a direct connection between the exocyst’s influence on the production of the amyloid beta peptide and insulin signaling effects. Understanding the involvement of the exocyst in Alzheimer’s disease could provide further targets for drug development and help bridge the gap in our understanding of connections between insulin signaling in the brain and Alzheimer’s disease.

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Molecular biology, Cellular biology, Neurosciences, APP, Exoc3L2, exocyst, PLA, trafficking, vesicles

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http://hdl.handle.net/10125/75906

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74 pages

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M.S. - Cell and Molecular Biology
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