Excitatory and inhibitory transmission between neurons in the brain needs to be thoroughly regulated and coordinated. Deregulation of this coordination ultimately results in nervous system disorders. A core aspect of my work concerns the study of the brain at the molecular level, by investigating RNA editing and alternative RNA splicing. In particular, we analyze key components of the molecular machine responsible for inhibition of electrical impulses, i.e. glycine receptors (GlyR) and GABA type A receptors (GABA(A)R) as well as gephyrin. We elucidate the function of these molecules in physiological and pathophysiological processes on a molecular, cellular and systemic level.
Synaptic, neuronal and network mechanisms of disease
Epilepsy is a devastating neurodegenerative disease that severely deteriorates life quality due to unpredictable occurrence of seizures and associated cognitive dysfunction. Moreover, epilepsy patients suffer from severe psychiatric comorbidities including anxiety and depression. Most epilepsy syndromes have no discernable genetic component. This indicates that epileptogenesis is governed by disease-promoting molecular and cellular mechanisms of neuronal plasticity, which may vary from patient to patient, resulting in diverse clinical pictures of cryptogenic/idiopathic epilepsies. Therefore, new therapeutic strategies are needed to satisfy the variable demands of patients.
Prior to the development of effective individualized therapies, it must be elucidated whether a disease-associated mechanism represents either an adaptive form of plasticity that is able to compensate for the disease-causing insult, or a maladaptive form of plasticity that sustains disease progression. In a recent study, we identified a maladaptive form of neuronal plasticity by studying RNA editing of the neurotransmitter receptor for glycine (GlyR). The initial observation was that the expression of an RNA-edited GlyR variant is increased in hippocampi from patients with pharmacoresistant temporal lobe epilepsy (Nature Neurosci 8:736, 2005; J Cell Mol Med 12:2848, 2008; Eur J Neurosci 30:1077, 2009). We therefore generated a new animal model that allows the targeted and neuron type-specific expression of the RNA-edited GlyR variant. This model was then used to study cognitive function, learning and memory, and emotional behavior (J Clin Invest doi:10.1172/JCI71472, 2014). We found that the RNA-edited GlyR is specifically expressed at presynaptic terminals and that its presence increases the functional weight of such synapses in the hippocampal network. Thereby, homeostatic control of synaptic transmission and neural network excitability is changed, and the mice display symptoms that are reminiscent of the pathology of epilepsy. Notably, targeted expression of the RNA-edited GlyR in excitatory glutamatergic neurons provoked seizure-like activity and cognitive dysfunction, and expression in inhibitory synapses of fast-spiking parvalbumin-positive interneurons resulted in anxiety. Thus, the same molecule triggered distinct psychopathological symptoms of epilepsy. This study is exemplary for a successful “bedside-to-bench” translational research strategy. It identified a maladaptive and disease-causing mechanism of neuronal plasticity that will serve as a good starting point for the development of an individualized treatment.
We are expanding these experiments to target the expression of the RNA-edited GlyR variant to other neuronal types and neurotransmitter systems. Furthermore, we are applying advanced imaging techniques using molecular beacons to gain insights into the neuron type-specific regulation of RNA editing.
Gephyrin is a postsynaptic scaffold protein required for stabilization of GlyR and GABA(A)R at glycinergic/GABAergic synapses (Nature Neurosci 4:253, 2001; Eur J Neurosci 37:544, 2013). We found that considerable functional heterogeneity of gephyrin arises from alternative mRNA splicing (Mol Cell Neurosci 16:566, 2000; J Neurosci 24:1398, 2004; J Cell Sci 120:1371, 2007). We furthermore identified gephyrin splice variants that are specifically expressed in glial or neuronal cells, indicating that gephyrin RNA splicing is regulated in a cell type-specific way (J Biol Chem 283:17370, 2008). Recently, we isolated from hippocampi of patients with temporal lobe epilepsy irregularly spliced gephyrin RNA variants. These variants lack several exons and encode neuronal gephyrins with dominant-negative activities that de-stabilize postsynaptic receptors and weaken transmission at inhibitory synapses (Brain 133:3778, 2010). We discovered that neuronal activity or calcium-dependent cellular stress induces the skipping of exons in the gephyrin-coding mRNA, leading to frameshift and premature termination of protein synthesis (Brain 133:3778, 2010). Thus, changes in RNA processing of GlyR-coding mRNA and impaired splicing of the gephyrin-coding mRNA may cooperate in the vicious circle of pathogenic mechanisms that impair neural network homeostasis and sustain the disease progression.
Several possibilities exist that can interrupt the vicious circle of impaired plasticity and disease progression. First, we are performing high-throughput drug screening to identify specific antagonists of the pathogenic GlyR variant produced by RNA editing. The identified compounds will be validated in the mouse model described above. Second, based on the finding that exon skipping in gephyrin-coding mRNA induces frameshift in the protein coding sequence, we developed a new molecular tool for neuronal self-defense (cf. ’CIPRESS’ collaborative research project funded by the BMBF; www.neuron-eranet.eu/en/317.php). This system permits induction of protein expression in response to cellular stress that accompanies epileptogenesis. We are asking which candidate proteins (or combinations) can suppress seizure activity in vivo by using in utero electroporation of different expression constructs, or by lentiviral expression in slice culture of the human epileptic hippocampus. We will also determine the characteristics of the network activity that causes cellular stress and candidate gene expression using optogenetics. I expect that this work will allow the identification of disease causing mechanisms of plasticity, for instance changes in the regulation of neuronal pH and chloride, energy metabolism, calcium-dependent proteolysis, and inhibitory actions of GABA. My work will provide insights into mechanisms of epileptogenesis and hopefully lead to the development of new therapies.