The exocyst regulates exocytosis of the insulin-sensitive GLUT4 channel in neurons

Abstract

Alzheimer’s Disease (AD) is the leading cause of dementia, impacting an estimated 6.7 million Americans, and the prevalence is projected to double in the next 25 years. This irreversible neurodegenerative disease manifests as severe memory loss and cognitive decline due to a progressive loss of neurons in the brain. Meta-analytic data demonstrate a substantially increased risk of AD among individuals with type 2 diabetes mellitus (T2DM), and up to 81% of people living with AD are estimated to have T2DM. Racial and ethnic groups suffering from high rates of T2DM, such as African Americans and Native Hawaiians and other Pacific Islanders, also have disproportionately higher rates of AD and show an earlier onset of cognitive decline. Despite the breadth and rigor of clinical data, we still have a poor understanding of the mechanistic relationship between insulin levels in the brain and the etiology of AD. Although the causes of AD are not yet fully understood, a classic pathological hallmark of the AD brain is the accumulation of amyloid beta (Aβ) plaques. The generation of these plaques involve the improper neuronal cleavage of the transmembrane amyloid precursor protein (APP), yielding small Aβ fragments that aggregate in the extracellular space. We have discovered that the exocyst protein complex may be an insulin-sensitive regulator of APP trafficking and processing in neurons, which would represent a new molecular mechanism directly linking insulin signaling and AD. Using both mouse hippocampal neurons and human SH-SY5Y neuroblastoma cells differentiated into neurons, we have shown that exocyst subunits are co-localized with APP on intracellular transport vesicles, however this APP-exocyst interaction is dramatically decreased within minutes of insulin treatment. In these SH-SY5Y neuronal cells expressing an APP transgene with familial AD mutations, RNAi silencing of exocyst genes led to substantial decreases in Aβ production. In this thesis work, we have engineered SH-SY5Y neurons to express combinations of fluorescently-labeled APP and exocyst proteins for live-cell total internal reflection fluorescence (TIRF) microscopy measurement of coordinated movement of APP and exocyst subunits. To further study these phenomena, we have engineered SH-SY5Y neurons to express both a pH-sensitive fluorescent glucose transporter 4 (GLUT4) with an mScarlet tag measuring total GLUT4. Through this tool, we have found evidence that the exocyst regulates GLUT4 translocation to the plasma membrane of neurons in response to insulin, which has only been previously shown in adipocytes and muscle cells. The anticipated impact of this research is the identification of a novel molecular pathway that directly links insulin signaling in neurons with early AD pathogenesis, providing a better understanding of how insulin dysregulation increases the risk of AD.