• Neurons
• glucose
• cell death
• mitochondrial bioenergetics
• calcium homeostasis

Glucose represents the main source of energy for the mammalian brain. In fact, in the adult brain, neurons have the highest energy demand, requiring continuous delivery of glucose from blood; however, its excess strongly affects brain metabolism giving serious pathological consequences, such as metabolic and neurological disorders. It has been demonstrated that glucose metabolism provides the fuel for physiological brain function through generation of ATP, which is fundamental for neuronal and non-neuronal cellular maintenance, as well as for neurotransmitter production. Therefore, regulation of glucose metabolism is critical for brain physiology and its deregulation in the brain may result in several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, affecting not only the brain itself but also the entire organism.
In this master thesis project, we investigated the effect of increasing glucose concentrations, ranging from low (5 mM, hypoglycaemic condition) to medium (15 - 25 mM) to high (50 - 75 mM, hyperglycaemic condition), in immortalised hippocampal neurons HN9.10e, a reliable in vitro model of one of the most vulnerable regions of the central nervous system. In order to do this, we first induced neuronal cycle arrest at G0/G1 phase by exposing hippocampal neurons to serum deprivation for 48 h. Subsequently, neurons were separately treated with the above-mentioned increasing glucose concentrations for 48 h, after which serum starvation was again induced and finally, biological and chemical analyses were performed at different time points (every 48 h and over 8 days in vitro). To quantify the damage that occurs in HN9.10e hippocampal neurons following glucose-inducing cellular stress, we examined several biological parameters, including cell death, cell number and what type of stress was involved, analysing mitochondrial and calcium dysfunctions. Cell death and number were evaluated using Hoechst 33258 and Propidium Iodide of nuclear chromatin, while mitochondrial and calcium dynamics were investigated measuring mitochondrial membrane potential using TMRM, mitochondrial ROS production and intracellular calcium using fluorescent probes, including MitoTracker ROS and Fluo3-AM, respectively. We observed that glucose induces dose-dependent cell death and dose-dependent intracellular calcium and mitochondrial membrane potential dysfunctions and mitochondrial ROS production over time, suggesting that hippocampal neurons survive better in a hypoglycaemic stress environment rather than a hyperglycaemic condition. Furthermore, neuronal extracellular metabolites produced following glucose-inducing cellular stress in HN9.10e cultured hippocampal neuronal medium, i.e those of the Cori cycle, glycolysis, short-chain fatty acids and several amino acids, were analysed and quantified by HPLC-DAD and Principal Component Analysis (PCA). We specifically focused our analysis on several extracellular metabolites, including lactic, glycolic, pyruvic, fumaric, acetic, citric, propionic, butyric acids, dopamine and tryptophan. Our results showed an intense metabolic activity mostly due to glycolysis, as demonstrated by the high lactate concentration values, accompanied by an increase of oxidative stress. We also confirmed, in our paradigm, an inverse correlation of Δψm loss with ROS production and intracellular calcium, as already expected. However, the inverse correlation of Δψm depolarisation with lactic, propionic, and citric acids here observed resulted novel and will definitely need further exploration. Because of the role of hyperglycaemia in neurodegenerative diseases and the severe damage of the hippocampal region in this kind of diseases, further investigation will be needed to better evaluate metabolic alterations following high glucose exposure in hippocampal neuronal cells.