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 our 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 the postsynaptic scaffold protein 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. Another major health care issue is intrinsic to the fact that a large number of patients develop pharmacoresistance, that is, they become non-responsive to medication. Therefore, new therapeutic strategies that tackle pathogenic mechanisms of the disease are clearly required to satisfy the variable and essential demands of patients.
Prior to the development of effective individualized therapies, it must be determined whether a disease-associated mechanism represents either an adaptive form of plasticity that is able to compensate for the disease-causing insult, or such maladaptive neuronal plasticity that sustains disease progression. In a recent study for example, 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 that were resected 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). To investigate this further, we generated an animal model that allows targeted and neuron type-specific expression of this RNA-edited GlyR variant to study cognitive function, learning and memory, and emotional behavior (J Clin Invest 124:696, 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. In more detail, targeted expression of the RNA-edited GlyR in excitatory glutamatergic neurons provoked seizure-like activity and cognitive dysfunction, and its 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” research approach. It identified a maladaptive and disease-causing mechanism of neuronal plasticity that can serve as a good starting point for the development of individualized treatments. We are expanding these experiments to target the expression of the RNA-edited GlyR variant to other neuronal types and neurotransmitter systems. However, as we still need to find out which neuron types actually increase the GlyR RNA editing in temporal lobe epilepsy we are furthermore applying advanced imaging techniques with molecular RNA editing sensor constructs as well as forced intercalation (FIT) probes to gain insights into neuron type-specific regulation of RNA editing and perform high throughput screens for identification of small molecule inhibitors.
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 our of hippocampi that were resected from 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 GABAergic transmission at inhibitory synapses (Brain 133:3778, 2010). We discovered that neuronal activity or 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. This system permits induction of protein expression in response to cellular stress that accompanies epileptic seizures. We are asking which candidate proteins (or combinations) can suppress seizure activity and eventually epileptogenesis by inference with maladaptive forms of neuronal plasticity. Using optogenetics, we are also characterizing network activities with regard to their potential to cause cellular stress and induce candidate gene expression. Hopefully, our efforts will allow identification of other disease causing mechanisms of plasticity than those described above, for instance changes in the regulation of neuronal pH and chloride, energy metabolism, calcium-dependent proteolysis, and inhibitory actions of GABA, in order to identify good starting points for the development of effective epilepsy therapies.
Finally, we recently identified a key mechanism of brain tumor formation. GlyRs carry a nucleus import signal. In glioma cells, this signal is active and prevents GlyRs from functioning as neurotransmitter receptors. Rather, GlyRs invade the nucleus and regulate stem cell gene expression and self-renewal capacity, and thereby tumorigenesis in vivo (J Cell Sci 127:3687, 2014). This study also identified the relevant intracellular GlyR signaling domain, which shall provide the basis for a promising therapeutic concept to block glioma formation in vivo.
Notably, 425 gene products undergo amino acid recoding RNA editing (www.rnaedit.com), but we currently do not know whether RNA editing of these candidate sites changes in epilepsy, and whether or how amino acid recoding editing affects protein function, thereby eventually contributing to maladaptive forms of neuronal plasticity. Using a combination of state of the art research approaches and advanced technologies including microfluidics-based multiplex PCR combined with deep sequencing, we want to identify and target more gene products with disease-specific regulation. This approach shall provide us with pivotal knowledge about still other maladaptive forms of neuronal plasticity, which is mandatory to the development of personalized medical care.