Reversible stabilisation and translocation strategies for the study of neuronal PTEN in vitro and in vivo
Lead Research Organisation:
King's College London
Department Name: MRC Ctr for Developmental Neurobiology
Abstract
A fundamental question in neuroscience concerns the mechanisms that control the assembly and the maintenance of appropriate connectivity in the brain. Neuronal signalling mediated by specific enzymes, PI3K, and the principle antagonist of PI3K, the lipid phosphatase PTEN, has been identified as a major regulator in this process. Changes in the function of PI3K or PTEN in humans are associated with high incidences of neurological symptoms, including epilepsy, autism and mental retardation, which may be a direct consequence of underlying changes in the connections of neurons. Therefore, it is critical to gain understanding of the molecular mechanisms of how PI3K/PTEN signalling regulates neuronal connections. These mechanisms may also underlie some types of adult neural plasticity, since alterations in PI3K signalling at a time when neurons are already functionally integrated in neuronal circuits are associated with changes in neuronal connections.
We will investigate how PTEN regulates the morphology of neurons, which directly influences the wiring of the nervous system. We will also investigate the possibility of alleviating the progressive changes that occur in neuronal connections in the brain when PTEN is lost. We will perform experiments in which neurons will lack PTEN for different periods of time, before re-installing PTEN expression. The information we find out from this will be essential for building a better understanding the degree of plasticity within the specific neuronal connections that are important for normal learning and social behaviour.
This research will help in the development of novel treatment strategies that disrupt the formation of abnormal brain circuits associated with several neurological conditions, including autism.
We will investigate how PTEN regulates the morphology of neurons, which directly influences the wiring of the nervous system. We will also investigate the possibility of alleviating the progressive changes that occur in neuronal connections in the brain when PTEN is lost. We will perform experiments in which neurons will lack PTEN for different periods of time, before re-installing PTEN expression. The information we find out from this will be essential for building a better understanding the degree of plasticity within the specific neuronal connections that are important for normal learning and social behaviour.
This research will help in the development of novel treatment strategies that disrupt the formation of abnormal brain circuits associated with several neurological conditions, including autism.
Technical Summary
Recent work indicates that restricted loss of PTEN in neuronal populations in the forebrain of mice results in progressive macrocephaly and exuberant sprouting of neuronal processes. The appearance of enlarged neuronal soma and macrocephaly (both of which are indicative of specific forms of ASD in humans), together with the development of neurological symptoms that include deficits in social interaction, seizures and decreased learning, suggests that these mice may provide a direct causal link between PTEN deficiency and ASD.
The project will consolidate and extend the use of novel tools that complement currently exploited gene targeting strategies by adding the potential to regulate PTEN in a specific, fast, reversible and tuneable manner. With these new tools, we propose: 1) to determine the function of nuclear PTEN in neurons. We will use a chemical-induced dimerization system in order to restrict PTEN to the nucleus or to exclude PTEN from the nucleus. We will monitor the activation status of PI3K signalling, but also PTEN-regulated, potentially PI3K independent signalling pathways. The results will be correlated with experiments that are aimed at investigating role of nuclear PTEN on the establishment and maintenance of proper neuronal morphology. We also propose: 2) to generate tuneable PTEN mice. These will permit conditional control over protein levels of PTEN in dependence of a synthetic ligand. We will combine the tuneable PTEN mouse with a specific CRE driver in order to evaluate the role of inactivating PI3K signalling on neuronal morphology of forebrain neurons in vivo. We will also create mice in which conditional PTEN loss will be directly coupled with the expression of our tuneable PTEN. This will allow us to address if progressive changes in the cortex and hippocampus of mice (as a consequence of loss of PTEN) can be antagonised by restoring PTEN. We will allow different time windows for neurons to be PTEN deficient, before re-installing PTEN and analyses by morphological and behavioural tests.
This research is essential for the attempt to gain fundamental insights into the significance of PI3K signalling in the formation and maintenance of neuronal circuits, as well as into the degree of plasticity within specific neuronal connections that are important for normal learning and social behaviour. We believe that this research has, potentially, direct implications for the development and validation of pharmacological therapeutics for the treatment of various neurological disorders, including ASD.
The project will consolidate and extend the use of novel tools that complement currently exploited gene targeting strategies by adding the potential to regulate PTEN in a specific, fast, reversible and tuneable manner. With these new tools, we propose: 1) to determine the function of nuclear PTEN in neurons. We will use a chemical-induced dimerization system in order to restrict PTEN to the nucleus or to exclude PTEN from the nucleus. We will monitor the activation status of PI3K signalling, but also PTEN-regulated, potentially PI3K independent signalling pathways. The results will be correlated with experiments that are aimed at investigating role of nuclear PTEN on the establishment and maintenance of proper neuronal morphology. We also propose: 2) to generate tuneable PTEN mice. These will permit conditional control over protein levels of PTEN in dependence of a synthetic ligand. We will combine the tuneable PTEN mouse with a specific CRE driver in order to evaluate the role of inactivating PI3K signalling on neuronal morphology of forebrain neurons in vivo. We will also create mice in which conditional PTEN loss will be directly coupled with the expression of our tuneable PTEN. This will allow us to address if progressive changes in the cortex and hippocampus of mice (as a consequence of loss of PTEN) can be antagonised by restoring PTEN. We will allow different time windows for neurons to be PTEN deficient, before re-installing PTEN and analyses by morphological and behavioural tests.
This research is essential for the attempt to gain fundamental insights into the significance of PI3K signalling in the formation and maintenance of neuronal circuits, as well as into the degree of plasticity within specific neuronal connections that are important for normal learning and social behaviour. We believe that this research has, potentially, direct implications for the development and validation of pharmacological therapeutics for the treatment of various neurological disorders, including ASD.