EXPLORING THE INTERACTION BETWEEN TSC2, PTEN, AND THE NMDA RECEPTOR IN ANIMAL MODELS OF TUBEROUS SCLEROSIS

Background: Deletions in either the Tsc1 or Tsc2 gene cause tuberous sclerosis, a disease characterized by tumor susceptibility and neurological manifestations. The Tsc1 and Tsc2 proteins form a complex that suppresses the activity of mTOR, a critical regulator of protein synthesis and cell growth. Thus, loss of the TSC complex results in excessive mTOR activity. However, loss of Tsc2 also results in the upregulation of Pten, an upstream suppressor of mTOR. Pten has been recently shown to regulate the expression of the NMDA receptor, which is important for cognitive function. Thus, it is possible that in tuberous sclerosis both Pten and NMDA receptor function are disrupted.Hypothesis: I hypothesize that loss of Tsc2 function causes disruptions in NMDA receptor subunit expression that are mediated by alterations in Pten and direct protein-protein interactions, and not by changes in mTOR signaling.Specific Aims: (1) To examine the subunit composition of the NMDA receptor in PTEN and Tsc2 knockout mice. (2) To determine whether the receptor abnormalities in Tsc2 mutants are dependent on the elevated activity of PTEN or mTOR.Study Design: Aim 1: We will study the developmental profile of the NMDA receptor in conditional Pten and Tsc2 mutant and control littermates by Western blot analysis and also determine the synaptic localization of the subunits by protein fractionation. Aim 2: In one set of experiments, we will cross conditional Tsc2 and Pten mutant mice to generate double mutants and examine their NMDA receptor composition. In another set of experiments, we will treat Tsc2 mutant mice with rapamycin (or vehicle control) to inhibit mTOR activity. If our hypothesis is correct, we will see restored NMDA receptor expression in double Tsc2/Pten mutants, but not in rapamycin-treated Tsc2 mutant mice.Innovation: This proposed work is highly innovative because despite its importance for cognition, very little is known about NMDA receptor regulation in tuberous sclerosis or in animal models of the disease. Furthermore, most studies on tuberous sclerosis focus on the role of mTOR whereas this study focuses on Pten and the NMDA receptor, and may potentially reveal a novel mechanism of disease.Impact: These studies will advance our understanding of the etiology of cognitive deficits in tuberous sclerosis. If we successfully identify specific alterations in NMDA receptor composition in our mouse models and understand the molecular mechanism that mediates them, future studies can be designed to restore normal receptor activity through pharmacological manipulations of the receptor or pharmacological inhibition of Pten. Initial studies will be conducted in animal models. In addition, further studies with human tissue will be conducted using brain biopsies derived from patients with tuberous sclerosis complex. We will analyze the patient tissue to determine whether abnormalities similar to those observed in mouse models are present. If this is the case, Pten inhibitors or NMDA receptor antagonists may be evaluated as novel therapies to improve cognition defects in TSC patients. These novel strategies may lead to an improvement of the neurological symptoms of tuberous sclerosis.