ALTERED ASTROCYTE-NEURON INTERACTIONS AND EPILEPTOGENESIS IN TUBEROUS SCLEROSIS COMPLEX DISORDER

Background: Astrocytes are involved in the regulation of dendritic growth, synaptogenesis, synapse number, synaptic function, and synaptic activity. Functional abnormalities in astrocytes contribute to the pathophysiology of human epilepsy and also in animal models of epilepsy. Recent evidence suggested astrogliosis as a primary pathology in tuberous sclerosis (TS) cortical tubers. Astrocyte pathology may result from mTOR cascade alterations with decreased glutamate and potassium uptake, leading to neuronal hyperexcitation with epileptogenesis.Objective/Hypothesis: A failure in pruning excessive excitatory synapses may contribute to the pathogensis of epilepsy in TS by producing neuronal over-excitability, due in part to effects mediated by TSC in astrocytes. We will determine glial mechanisms for modulating epileptogenicity, specifically focusing on effects of TSC particular to astrocytes that alter glutamatergic and GABAergic synapse formations during development.Specific Aims and Study Design: Aim 1: Determine the effect of astrocyte TSC mutation on pre- and post-synaptic function. Hypothesis: Enhanced mTOR activity blocks normal function of astrocytes in TSC2+/- and mouse GFAP cre mediated TSC1 KO astrocytes, independent of tuber formation.Design: Primary forebrain astrocyte cultures derived from TSC2 wild-type (+/+) and TSC-deficient mice (TSC2+/- or TSC1mGFAP CKO mice) will be analyzed for a panel of biomarkers for astrocyte function. We will measure astrocyte calcium oscillation and calcium waves using multi-photon imaging techniques. Astrocyte proteins involved in the regulation of neuronal morphology and plasticity will be examined. We will assess the effect of astrocyte TSC deficiency on spine development in cortical neuronal co-cultures. Cortical neurons will be co-cultured with wild-type and TSC2+/- or TSC1-CKO astrocytes. Dendrites and spine morphology will be assayed during maturation by confocal microscopy.Aim 2: Analyze whether astrocyte dysfunction involves in postnatal spine pruning defect, which may result in abnormally enhanced excitatory synaptic connectivity and epileptogenicity.Hypothesis: Astrocyte dysfunction modulates mTOR-dependent spine pruning deficit in TSC, which underlies neuronal hyperexcitability and epileptogenicity in TSC.Design: We will label pyramidal neurons in brain slices from astrocyte-specific TSC1 CKO with DiOlistic labeling. We will also immunostain and immunoblot these brain slices for pre- and post-synaptic markers. Dendritic spines in sensory cortex layer V pyramidal neurons from wild-type and TSC1 KO mice will be examined during the primary period of developmental pruning. We will examine seizure susceptibility in vitro and in vivo. Synaptic, epileptiform activity and EEG recordings will be undertaken, and the comparative threshold for seizure induction will be characterized.Aim 3: Analyze tuber, peri-tuber and non-tuber tissue from TS and control human brain tissue for mTOR, astrocyte function and synaptic formation.Hypothesis: Astrocyte dysfunction correlates with excitatory neuronal hyperactivity in brains from TSC patients.Design: Biopsied and autopsied tissue from TS patients and control cortex will be examined to biomarkers for astrocyte function. We will examine tuber and non-tuber cortical brain tissue from TSC patients and controls for neuronal morphology (dendrites and spines) using modified Golgi label and electrophysiology.Innovation: While the molecular basis of TS is established, far less is known about the pathogenetic mechanisms of epileptogenesis in TS. Previous studies mostly focused on focal epileptogenesis in cortical tubers. We propose that non-tuber cortex presents an abnormally excitable neuronal network that could underlie seizure generation. We will concentrate on a non-neuronal mechanism may exist in regulating synaptic function and thus epileptogenesis in TSC. We will use a wide variety of experimental approaches, including electrophysiology (patch-clamping, video-EEG monitoring), histological (conventional and fluorescent assays), molecular biological, and cellular imaging (confocal and two-photon microscopy). We will measure biomarkers for astrocyte and neuronal functions and provide an index of glial transmission in the cortex of TSC-deficient mouse models and peri-tuber or non-tuber tissue from TSC human brain. We will determine whether astrocyte dysfunction leads to failure in pruning excessive excitatory synapses during development, which underlies epilepsy in young TSC patients. We will examine non-tuberal cortex from autopsy and biopsy TS brain to find correlates between astrocyte pathology and markers for neuronal activity in TS.Impact: A key, unresolved issue is the cause of the neurological symptoms in TS patients. We will investigate astrocytic mechanisms in pruning excessive excitatory synapse during development, which may produce neuronal overexcitability and thus underlie epileptogenesis in astrocyte-specific TS KO mouse models.