Dysfunction of GABA and glycine transporters in human neurological disease

The central nervous system is a complex, intricate network of nerve cells (neurones) whose primary function is to transmit and receive messages. This communication occurs at specialised sites of contact known as synapses. At these sites, an arriving nerve impulse causes the release of a chemical (neurotransmitter) from the presynaptic cell which then interacts with receptor molecules embedded in the cell membrane of a neighbouring postsynaptic neurone. Some types of these receptors (e.g. glycine and GABA-A receptors) possess specific ion-permeable channels. The opening of these channels in response to neurotransmitter alters the electrical state of the cell either transmitting or subtly altering the incoming nerve impulse. Neurotransmitters are then recovered from the synapse by transporters located in neighbouring glial cells or the corresponding presynaptic cell. The mechanisms that regulate synaptic transmission and nerve impulse activity are important in understanding normal and diseased states of the brain. Indeed, many drugs in use or under development act primarily via GABA or glycine receptors and their transporters. The therapeutic nature of these agents provides a compelling reason for further understanding the molecular details of the structure and function of these proteins. This proposal will benefit research in this area by enhancing our knowledge concerning transporters for GABA and glycine. In a recent study we were able to show that genetic defects in the glycine transporter GlyT2 were responsible for causing a rare illness called hyperekplexia. This affects newborn children and is characterised by noise or touch-induced seizures which result in breath-holding episodes. In some instances hyperekplexia can lead to brain damage or sudden infant death. Our major aims are: i) to study the consequences of GlyT2 mutations to reveal how these defects disable the transporter, and to investigate whether defects in a glycine transporter found on synaptic vesicles (VIAAT) or proteins that associate with GlyT2 can also cause hyperekplexia; ii) to determine whether genetic mutations in a second glycine transporter, GlyT1, is responsible for cases of a different childhood illness, glycine encephalopathy, which can lead to severe brain damage or death; iii) since defects in GABA receptors are found in some types of epilepsy, we will investigate whether mutations in GABA transporter genes also cause epilepsy. It is our hope that a detailed understanding of the genetic defects responsible for these illnesses will enable better diagnosis and treatment of affected individuals.

We have recently proven that a human neurological disorder, hyperekplexia, can be caused by mutations in the gene encoding the presynaptic glycine transporter GlyT2 (Rees et al 2006, Nature Genetics 38:801-806). Prior to this, hyperekplexia was typically associated with mutations in the genes for postsynaptic inhibitory glycine receptor subunits and associated clustering proteins. Having conclusively demonstrated that hyperekplexia can be triggered by presynaptic deficits, this raises the intriguing possibility that presynaptic causes of disease may also exist in related disorders, such as idiopathic generalised epilepsies, where mutations in postsynaptic inhibitory GABA-A receptor subunit genes have already been identified. This programme aims to examine the role of GABA and glycine transporters in hyperekplexia, glycine encephalopathy and idiopathic generalised epilepsies. For glycinergic synapses, we will examine the functional consequences of newly identified GlyT2 hyperekplexia mutations and variants of GlyT2 generated by alternative splicing. We will also perform genetic screening of genes required for presynaptic vesicular packaging of glycine (e.g. the vesicular inhibitory amino acid transporter VIAAT) and two GlyT2 interacting proteins (ULIP-6 and syntenin-1) thought to be important for localisation and turnover of GlyT2. Since knockout mice for a second glycine transporter (GlyT1) show phenotypic similarities to glycine encephalopathy, we will establish whether defects in the human GlyT1 gene underlie this severe disorder. For GABAergic synapses, we will examine genes crucial for GABA vesicular packaging and uptake (i.e. VIAAT and neuronal/glial GABA transporters) in a cohort of ~1000 patients with idiopathic generalised epilepsies. Structural and functional deficits in these proteins will be studied using appropriate cellular models and functional assays (e.g. neurotransmitter uptake assays and electrophysiology). This study will bring together genetic and molecular expertise to define the causes of these neurological disorders, while providing functional insights into inhibitory neurotransmitter transporters.