Forschungsschwerpunkte des Instituts
The neuronal microRNA laboratory
The correct wiring of millions of nerve cells within the mammalian brain is critical for higher cognitive functions, such as learning and memory. Neural circuits are remarkably plastic, undergoing extensive remodeling in response to experiences from the outside world. A substantial fraction of this experience-dependent plasticity occurs at the level of synapses, the highly specialized structures where information is communicated between individual nerve cells (Kandel, 2001). Accordingly, defects in synaptic plasticity are a hallmark of common neurological diseases, such as mental retardation, autism-spectrum and mood disorders.
At the molecular level, it is becoming increasingly clear that local protein synthesis in dendrites plays a crucial role in providing substrates for the morphological and functional reorganization of synapses (Sutton and Schuman, 2006). miRNAs were emerging as important regulators of protein synthesis in many biological systems, but their role in post-mitotic neurons remained elusive.
Using gain-and loss-of-function approaches in cultured rat hippocampal neurons, we found that the brain-specific miRNA, miR-134, negatively regulates the growth of dendritic spines, the major postsynaptic sites of excitatory synapses (Schratt et al., 2006). One critical target of miR-134 in spines appears to be Lim-domain containing protein kinase 1 (Limk1), a central regulator of the spine actin cytoskeleton. Since changes in spine size are a hallmark of synaptic plasticity, our results for the first time implicated miR-134, and the miRNA pathway in general, in higher cognitive functions.
More recently, we discovered an entire miRNA network dedicated to the fine-tuning of protein synthesis within dendritic spines (Siegel et al., 2009). One of the new miRNAs identified, miR-138, controls spine size by blocking the synthesis of acyl-protein-thioesterase 1 (APT1), an enzyme that removes palmitate moieties from signaling molecules and thereby regulates their localization and/or activity. We found that the Rho signaling component Ga13 is an important APT1 substrate in spines, providing further evidence that the actin cytoskeleton is a convergence point for miRNA-dependent translational control (Figure 1).
Finally, we could demonstrate that miRNA function itself is tightly regulated by neural activity. In the case of miR-134, this regulation occurs at two levels. First, miR-134 transcription is rapidly and transiently induced by extracellular stimulation (Fiore et al., 2009). Second, stimulation of individual synapses relieves the inhibitory effect of miR-134 on translation, a mechanism that is well suited to explain the bi-directional, synapse-specific changes in structure and function (i.e. those occurring during LTP/ LTD) (Fiore et al., 2009).
In summary, our work elucidated an entirely novel layer of gene regulation in synapse development and plasticity. Our results serve as a staring point for studies that will investigate the significance of miRNAs for higher cognitive function and behavior. Many prevalent neurological disorders are characterized by defects in synaptic plasticity and homeostasis (Ramocki and Zoghbi, 2008). It is our hope that the miRNA pathway will become a novel promising target for therapeutic intervention in diseases of complex genetic origin, i.e. mental retardation, autism and mood disorders.
Fiore, R., Khudayberdiev, S., Christensen, M., Siegel, G., Flavell, S.W., Kim, T.K., Greenberg, M.E., and Schratt, G. (2009). Mef2-mediated transcription of the miR379-410 cluster regulates activity-dependent dendritogenesis by fine-tuning Pumilio2 protein levels. Embo J.
Kandel, E.R. (2001). The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 1030-1038.
Ramocki, M.B., and Zoghbi, H.Y. (2008). Failure of neuronal homeostasis results in common neuropsychiatric phenotypes. Nature 455, 912-918.
Schratt, G.M., Tuebing, F., Nigh, E.A., Kane, C.G., Sabatini, M.E., Kiebler, M., and Greenberg, M.E. (2006). A brain-specific microRNA regulates dendritic spine development. Nature 439, 283-289.
Siegel, G., Obernosterer, G., Fiore, R., Oehmen, M., Bicker, S., Christensen, M., Khudayberdiev, S., Leuschner, P.J., Busch, C.L., Kane, C.G., et al. (2009). A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nature cell biology in press.
Sutton, M.A., and Schuman, E.M. (2006). Dendritic protein synthesis, synaptic plasticity, and memory. Cell 127, 49-58.
Future research plan
Based on the work from our and a number of other laboratories, miRNAs are emerging as important regulatory molecules in the control of synapse development and plasticity in vertebrates. These recent findings implicate miRNAs in higher cognitive functions, e.g. learning and memory, behavior and a number of neurological disorders, e.g. mental retardation, autism-spectrum disorders and schizophrenia. Despite this progress, little is still known about the physiological roles of miRNAs in the brain and the regulation of miRNA function by neuronal activity. Therefore, our future research will center around two major questions:
- What are the molecular mechanisms that couple microRNA-dependent control of local mRNA translation to the activity status of the neural network?
- What are the physiological consequences of miRNA perturbation for the development of neural circuits, cognition, and neurological diseases in the intact animal?
1. Molecular studies
Our previous work led to the identification of two miRNA-mRNA target interactions that play critical roles in the development of dendritic spines. We are currently using these interactions as paradigms to study the influence of neural activity on crucial steps of the miRNA pathway. For example, preliminary results indicate that activity regulates subcellular localization and processing of microRNA precursors. This would constitute a completely novel layer of miRNA regulation. In the future, we plan to characterize the cellular machinery that is responsible for miRNA transport to dendrites and synaptic miRNA processing. Moreover, the repressive function of synaptic miRNAs appears to be regulated by interacting RNA-binding proteins. We recently embarked on a large-scale RNAi screen to identify such proteins, which will be complemented by the biochemical characterization of miRNA-associated protein complexes at synapses. Once the components of these complexes are identified, we will be able to delineate the signaling pathways that modulate miRNA function in response to neural activity.
2. Physiological studies
Animal models provide increasing evidence for the importance of miRNAs in many biological systems, e.g. cardiovascular development and cancer, but the knowledge about miRNA function in post-mitotic neurons in the vertebrate CNS is still scarce. We plan to interrogate the in vivo function of candidate miRNAs that we had previously characterized in our primary hippocampal neuron cultures using two complementary approaches.
First, we have established a viral delivery system that allows the acute overexpression/inactivation of miRNAs in the mouse CNS by intracranial injection. This will allow us to monitor the effects of candidate miRNA on neural morphology (confocal microscopy) and synaptic function (electrophysiological recordings).
Second, we are generating genetically modified mice by deleting candidate miRNA genes using the Cre/lox system. Thereby, we will be able to investigate the systemic function of miRNAs in the brain, for example in behavioral assays related to learning and memory (e.g. Morris water maze) and neurological disease models. Ultimately, neural miRNAs could become novel targets for therapeutic intervention in neurological disease. As a first step towards this aim, we are establishing CNS delivery strategies for LNA-based miRNA inhibitory oligonucleotides (in collaboration with Santaris Pharma, Denmark), focusing on our previously identified miRNA candidates.
In conclusion, we are confident that our interdisciplinary approach consisting of biochemistry, high-resolution imaging, genetics, electrophysiology and behavior will help to increase our understanding of miRNAs in brain function and disease.