Chronic AMPK activation reduces synaptic plasticity markers in Neuronal SH‐SY5Y Cells

Abstract

Neuronal synapse function and growth rely on the abundance proteins at the synapse. This abundance is regulated by key metabolic markers: AMP-Kinase (AMPK) and the Mechanistic Target of Rapamycin Complex 1 (mTORC1). AMPK is a serine/threonine kinase highly expressed in brain. When activated, AMPK stimulates catabolic processes, and inhibits anabolic processes through mTORC1 inhibition. Defects in AMPK signaling have been reported in peripheral metabolic disorders and the manipulation of AMPK activity has been an attractive therapeutic target for diseases in which altered energy metabolism contributes to etiology (Obesity type-2-diabetes). Recent evidence also suggests that impaired AMPK activity plays a role in Alzheimer's disease. However, there is ambiguity surrounding the role of AMPK activation in neuronal metabolic regulation and its affects on neuron growth and health. The objective of this study was to determine the direct effect of chronic AMPK modulation on markers related to synapse growth and function in a neuronal cell culture model. Retinoic acid differentiated (1g/mL) SH-SY5Y Human Neuroblastoma cells were treated with: 1) Vehicle control; 2) A-769662 (100uM; AMPK agonist); or 3) compound C (30uM; AMPK inhibitor). Cells were treated for 1, 3, and 5 days to examine chronic AMPK activation or inhibition. Cell lysates were collected for western blotting (WB) to examine AMPK activation (AMPK T172), a marker of mTORC1 formation and cellular proliferation (raptor), and markers of neurogenesis and pre/post-synaptic proteins (synaptophysin, homer-1, PSD-95, synaptophysin, BDNF). PSD-95, homer-1, synaptophysin, and BDNF are significant markers in maintaining/developing proper synapse function and strength. AMPK T172 phosphorylation following treatment with A-769662, was higher than control for all time points (24h, 3d, 5d) and resulted in higher raptor S792 phosphorylation compared to control, indicative of chronic mTORC1 inhibition. No changes in neuronal marker content was observed following 24h of AMPK activation. However, significant reductions were seen in PSD-95, homer-1, synaptophysin, and BDNF content following 3d and 5d of AMPK activation. Taken together, these findings indicate a role for AMPK activation in regulating key synaptic plasticity markers and highlights drawbacks in pursuing AMPK as a therapeutic target for metabolic diseases such as Alzheimer's disease.