Investigation of mTOR-dependent neuronal markers, gene expression and central metabolism in dopaminergic neurons

The mechanistic target of Rapamycin (mTOR) signalling pathway is conserved in all eukaryotes and regulates growth and metabolism in response to environmental cues such as nutrients and growth stimuli. The heart of the pathway is the TOR kinase that operates within two functionally and structurally distinct complexes, TORC1 and TORC2. Analyses of transcriptomics data from previous experiments have identified transcription factors linked to development/metabolism i.e., LHX9 and GATA4, showing significant change in expression following pharmacological inhibition of mTOR via an ATP-competitive inhibitor of both TOR complexes, Torin1. Lim Homeobox 9 (LHX9), a transcription factor, is crucial for nervous system development and is implicated in glycolysis. The purpose of this research was to define the function of the mTOR pathway in controlling gene expression programmes during differentiation, growth and neurodegeneration as well as its implications into glycolysis by implementing a combination of pharmacological interventions in a time course manner using Torin1 (TORC1/2 inhibitor) and RapaLink-1 (TORC1 inhibitor), followed by an investigation into the role of LHX9 in mediating mTOR-dependent effects during growth and neurodegeneration. Western blotting was used to validate RNA-seq derived outputs by examining LHX9 expression in differentiated SH-SY5Y cells. The results show an upregulation of LHX9 in differentiated SH-SY5Y cells after Rapalink-1 and Torin1 treatments, with an increased expression with TORC1 and TORC2 inhibition. All the glycolytic enzymes tested did not have significant changes in differentiated SH-SY5Y cells with Torin1 and RapaLink-1 treatment showing that glycolysis runs normally. Due to these results, we can assume that LHX9 does not have an effect on glycolysis.