Mechanisms of Oestrogenic Modulation of Neural Circuitry

In the UK it is estimated that 1 in 4 adults experience mental health problems in any one year, and 1 in 6 experiences this at any given time (The Office for National Statistics Psychiatric Morbidity report, 2001). One example of this are people who with schizophrenia. In the UK alone, there are over 250,000 people diagnosed with schizophrenia, and this is estimated to cost £6.7 billion to treat these patients. Although schizophrenia is widespread, therapies for patients are often ineffective or fail to treat all aspects of the disorder. Multiple hypotheses have been proposed that implicate a number of brain systems and their dysfunction in schizophrenia. Some examples of these theories include abnormal brain chemistry and reduced connections between brain cells, also known as neurons. Multiple studies suggest that connections between neurons, called synapses, are reduced in the brains of suffers of schizophrenia. This may be in part caused by genetic modifications in molecules that shape synapses during brain development. Indeed, it has been proposed that by developing drugs that control how neurons are shaped or control the number of connections in the brain, neurons can re-connect with each other, offering a novel and effective treatment for schizophrenia. Currently, no effective treatment has been developed that specifically increase the number of connections between neurons. This is due in part to our poor understanding of how wiring in the brain is achieved. Therefore, it is vital to understand whether, and how, such potential agents can allow neurons to re-connect with each other, such that new and more effective treatments can be generated for this pervasive disorder. It has been known for some time that oestrogens can have a positive effect on brain function. More recently, it has also been suggested that these oestrogens, in combination with antipsychotic medications, can improve treatment of schizophrenia in men and women. But how oestrogens are beneficial in schizophrenia is not known. Moreover, long-term treatment with oestrogens is associated with increased risk of developing heart conditions and even cancer. One way to avoid such adverse side-effects is to understand how oestrogens are beneficial for schizophrenic patients, which would allow for the development of more effective and specific drugs. Our previous studies have demonstrated that oestrogens control the number of synapses between neurons, and thus control the amount of information that passes between them. We believe that this process is part of how oestrogens can be beneficial in schizophrenia. Our research uses neurons grown in dishes, a technique which provides a starting point to investigating the role of oestrogens in controlling synapses, and allows us to ask specific questions about their role in models of diseased cells. In this proposal, we will determine whether oestrogens can shape how neurons are formed, and how they can increase the number of synapses on neurons. We will use sophisticated and cutting-edge cellular imaging technique to ask these questions. Excitingly, these studies will allow us to understand how effective oestrogens are in controlling the number of connections between neurons with mutant proteins that are considered to be important for disorders such as schizophrenia. The results of these experiments will provide important information about how potential oestrogen-based therapies can control the way that neurons connect with each other in cellular models of schizophrenia. Answering these questions is essential for us to start to developing potential new therapeutic strategy in treating the debilitating symptoms that schizophrenic patients experience.

Oestrogen is a powerful regulator of cognition and neural circuitry. Recent data indicate that oestrogens are an effective adjunct treatment for schizophrenia. Oestrogens can influence the development of cortical neurons and I have shown that oestrogens increase dendritic spine number and regulate synaptic expression of AMPA and NMDA glutamate receptors. However, the role of brain-synthesised oestrogens in the development of cortical neurons is not known. Furthermore, we do not have a detailed understanding of the molecular mechanisms underlying oestrogenic modulation of dendritic spines and glutamate receptors are not known.
Using a multidisciplinary approach integrating molecular and cellular studies with advanced imaging techniques, I will pursue these Specific Aims:
1) I will use in utero electroporation to express shRNA against aromatase, the key enzyme needed for the production of oestrogens in the brain. I will then use 2-photon laser scanning microscopy to image brain sections to determine the role of brain-synthesised oestrogens on cortical neuron migration, dendrite formation and synaptogenesis.
2) I will use a combination of pharmacological agents, shRNA, FRET-biosensors and mutant constructs with confocal imaging of fixed or live cultured cortical neurons to dissect the molecular pathways required for oestrogen-modulation of neuronal signalling and dendritic spines.
3) To assess the ability of oestrogens to modulate glutamate receptors synaptic expression, I will use combination of pharmacological agents, shRNA, mutant constructs, labelling of surface receptors and ph-sensitive GFP-tagged constructs with confocal imaging of fixed or live cultured cortical neurons dissect the molecular pathways required for oestrogen-regulation of glutamate receptor trafficking.

The proposed project focuses on understanding the relevance, and underlying mechanisms, of estrogenic-signalling as a potential therapeutic agent in the treatment of schizophrenia. Clinical studies have shown estrogens to have limited success in ameliorating schizophrenic symptoms in male and female patients, highlighting their therapeutic potential. However, long-term treatment with estrogens is associated the increased risk for the development of cardiovascular problems and cancers. Therefore, it is essential to elucidate the molecular mechanisms underlying the beneficial effects of estrogens in order to develop more effective and safer estrogen-based therapies. This work outline in the current project will directly and indirectly benefit a number of groups. In addition to the academic groups outlined in the Academic Beneficiaries section, these include:
1) Clinical researchers. Whist this work is a basic, pre-clinical study, results from the project will help to guide clinicians in the potential therapeutic value of estrogenic-based therapies. Upon completion of these studies we will have a greater understanding if the mechanisms by which estrogens may exert its beneficial effects in schizophrenia. Moreover, as these studies will also utilize neurons derived directly from human patients with schizophrenia and thus accurately model the genetic complexities of this pervasive disorder, these studies will provide a solid basis from which translational studies can be proposed.
2) Pharmaceutical companies. As highlighted by the PI's previous interactions with pharmaceutical companies, and the current support offered by researchers at the same company, it is clear that pharmaceutical companies will also benefit greatly from the current project. Research into the molecular mechanisms underlying psychiatric disorders is being conducted in a number of institutions, in an effort to identify novel therapeutic targets. However, the current project will directly test the effectiveness of estrogens in modulating synaptic connectivity in a disease context, highly relevant for schizophrenia. Upon completion of this project, we will be able to provide a more comprehensive insight into the molecular mechanisms essential for the beneficial effects of estrogens. Based on these data, pharmaceutical companies will be able to identify novel drug targets for the development of novel and safer estrogen-based therapies.
3) Public. Schizophrenia is a devastating disorder affecting 1% of the public; in the UK alone there are over 250,000 diagnosed patients. Because of the disease's complexity, therapies for schizophrenic patients are often ineffective or fail to treat all aspects of the disorder, highlighting the urgent need to develop more effective and safer treatments for this disorder. Through public dissemination plans, this work will provide the public with a greater understanding of the underlying mechanisms important for this disorder, and the potential relevance of estrogenic-based therapies for schizophrenia. Moreover, upon completion of this work, researchers and pharmaceutical companies will be able to identify novel drug targets and develop treatment for this pervasive disorder, which will lead to the novel treatment and directly impact patients and their families, by improving their quality of life.
4) Government agencies. It is estimated that treatment of schizophrenia costs in excess of £6.7 billion. In order to reduce this burden of cost for care, it is essential to develop novel treatment for schizophrenia for the more effective treatment of patients. Results from this study will guide future studies in the development of novel therapeutic strategies aim at treating schizophrenia, which will reduce the cost of treatment, and allow patients, previously unable to work, to return to employment, fostering economic performance.