The role of the neuronal environment in neurodevelopment and circuit formation in Tuberous Sclerosis Complex

Background: Tuberous Sclerosis Complex (TSC) results from TSC1 or TSC2 gene mutations with hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). TSC is characterised by benign hamartomas in multiple organs including the brain and neurological symptoms are often amongst the most severe and disabling manifestations. Cortical tubers, histologically characterised by disturbed cortical lamination, dysmorphic neurons, and extensive gliosis, are typical brain tumours in TSC that have been implicated in the generation of seizures.
MTORC1 is a main regulator of cellular metabolism in response to energy and nutrients supply. Malfunctioning of the TSC1:TSC2-mTORC1 pathway can disturb cellular metabolic homeostasis with severe consequences to cellular survival, proliferation, and differentiation. However, the exact impact of mTORC1 hyperactivation on the development of cortical tubers in TSC remains unclear and previous studies demonstrated evidence that tuberigenesis is likely caused by non-genetic factors in addition to disturbances of the TSC1:TSC2-mTORC1 axis.
Aims: In my DPhil project, I examined the impact of extragenetic factors on the development of cortical tubers in TSC. I chose one non-cellular and one cellular factor, namely the availability of extracellular glucose and co-culturing with TSC2-deficient astrocytes, due to two reasons: (i) mTORC1 contributes to cellular energy and glucose metabolism and (ii) elevated levels of potentially malfunctioning astrocytes are a main feature of cortical tubers in vivo.
Methods: I used in vitro stem cell-derived glial and neuronal cultures to model neurode-velopment and neuronal circuit formation in TSC. Further, I applied a Bayesian computational modelling tool, dynamic causal modeling (DCM), to multi-electrode array (MEA) recordings from neuronal cultures to extract information on the synaptic foundation of neuronal connectivity in TSC.
Results: In vitro, TSC2 deficiency causes developmental disturbances in neural stem cells (NSC) such as prolonged and increased stem cell renewal as well as transcriptional changes in neurons implicating reduced maturity and a tendency towards gliogenesis. However, these changes did not resemble a full cortical tuber-like picture as the late-stage transcriptional abnormalities did not manifest on the protein level.
Limiting cellular glucose supply drives TSC2-deficient neuronal cultures to develop a cortical tuber-like picture including an increased glia-to-neuron ratio and impaired neurogenesis.
Glucose deprivation reduces the ability to produce energy through glycolysis and mitochondrial respiration in NSCs with different compensatory coping strategies in TSC2 +/+ and TSC2 -/- cells. TSC2-deficient NSCs upregulate important glycolytic genes such as hexokinase II and lactate dehydrogenase B but fail to activate mitochondrial metabolism. These changes might induce a tendency for proliferation and abnormal survival limiting their neurogenic potential as their transcriptional state in glucose deprivation resembles a cancerous metabolism.
Lastly, I use DCM on MEA data from in vitro TSC glio-neuronal co-cultures to examine the impact of astrocytes on neuronal connectivity. First, I demonstrate the suitability of DCM analysis for in vitro neuronal recordings in a proof-of-concept study. Second, DCM analysis of recordings from glio-neuronal co-cultures provides preliminary evidence that TSC2-deficient astrocytes increase the parameterised excitation-inhibition ratio in TSC2-deficient neurons.
Conclusion: My study provides evidence that non-genetic factors such as glucose supply and glial co-culture play an important role in the development of cortical tubers. Disturbances to the environment in which TSC2 -/- neurons develop impact their capacity to create healthy neuronal cultures opening up therapeutic strategies aimed at extracellular targets outside of the classic TSC1:TSC2-mTORC1 pathway.

Keywords: Tuberous Sclerosis Complex
stem cells
epilepsy
MEA
DCM
Advisor: Cader, Z
Sen, A
Moran, R