Examination of the effects of chronic AMPK modulation on synaptic proteins in neuronal SH-SY5Y cells

Abstract

Neuronal synapse function and growth are dependent on protein abundance at the synapse and are regulated by key metabolic markers: AMPK and 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. 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 the affects on neuron growth and health. This thesis aimed to determine the direct effect of chronic AMPK activation on anabolic and catabolic processes in a neuronal cell culture model. Retinoic acid differentiated (1𝜇g/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 activation following treatment with A-769662, was seen to persist for all time points (24h, 3d, 5d) and resulted in increased raptor S792 phosphorylation, indicative of chronic mTORC1 inhibition. No change in neuronal marker content was seen 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 impacting key synaptic plasticity markers and highlights drawbacks in persuing AMPK as a therapeutic target for metabolic diseases such as Alzheimer’s disease given the neuronal damage already assocatied with this disease