KETOGENIC DIET: An in vitro single-cell imaging and molecular analysis approach to determine and therapeutically target the control principles of neuronal bioenergetics for the treatment of epilepsy with ketogenic diet

Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS)CNS. Glutamate receptor activation imposes a significant work load and energy demand on neurons which requires neurons to increase ATP production. However, excessive activation of glutamate receptors induces neuronal dysfunction and nerve cell death, a process termed ‘excitotoxicity’. Interestingly, neuronal excitation and excitotoxic injury is substantially modulated by alterations in energy substrates. Protective effects of ketone bodies and fatty acids on neuronal excitotoxic injury and in the setting of epilepsy treatment in patients have been described. Their mechanism of action however is poorly understood.

The main aim of this PhD project is to analyse the effects of the metabolic switch imposed by a ketogenic diet on mitochondrial function and neuronal bioenergetics, by using a combined single cell imaging and molecular deep phenotyping approach. First, the candidate will analyse the influence of acetoacetate, betahydroxybutyrate and decanoic acid (core components of a ketogenic diet) on basal bioenergetics and neuronal excitability at the single cell level by employing time-lapse confocal microscopy studies. He/she will avail of key a set of established, fluorescent reporters (FRET probes) that were previously characterized by the host laboratory (Connolly et al., J Neurosci, 2014; D’Orsi et al., J Neurosci, 2015). Studies will be done in primary hippocampal neurons under baseline conditions, under conditions of hyperexcitation, and after a prolonged glutamate challenge which is normally toxic to neurons. Next the candidate will perform a systems approach to fully understand and optimise the effects of ketogenic diet on neuronal bioenergetics and excitability. The candidate will perform RNA sequencing studies to analyse the transciptome of neurons exposed to ketogenic diet and to provide insights into the mechanism of action of this novel treatment. Alterations in gene expression in response to ketogenic diet will be validated by PCR and Western blotting, and further interrogated using genetic manipulation of differentially expressed genes that are of relevance for neuronal energy metabolism, excitation and excitotoxic cell death.

Unravelling the mechanisms by which ketogenic diet modulates neuronal bioenergetics, excitability and survival is pivotal for the use and optimization of dietary approaches for the treatment of epilepsy.