Small GTPase activity and astrocyte morphology as determinants of astrocyte Ca2+ signalling

• Applicants: Professor Dr. Christian Henneberger; Dr. Andre Zeug
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Biophysics
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 284079634

Astrocytes, an abundant type of glia in the brain, play important roles in maintaining neuronal network function through multiple mechanisms, e.g. energy supply and neurotransmitter clearance. Their unique morphology enables single astrocytes to fulfil these functions at thousands of synapses. In addition, it has become evident that astrocytes are able to directly sense and modulate signalling and plasticity at these synapses. Astrocyte Ca2+ signalling is thereby heavily implicated. The properties and propagation of the largely store-dependent Ca2+ signals depend on diffusion and therefore on astrocyte morphology, which is itself dynamic. However, which signalling cascades are critical in controlling astrocyte morphology and, in turn, Ca2+ signalling remains poorly understood. We therefore propose to uncover the mechanisms by which small GTPases of the Rho family control astrocyte morphology and astrocyte Ca2+ signalling. Our central hypothesis thereby is that activity of small GTPases of the Rho family dynamically determine astrocyte morphology and consequently the Ca2+ signals implicated in neuron-glia interactions. Our preliminary experiments confirmed that astrocytes Ca2+ dynamics follow power law. Interestingly, we found the power law exponent to depend on astrocyte morphology, in line with our main hypothesis. Transient expression of active and dominant negative form of small GTPases of the Rho family revealed their profound impact on astrocyte morphology. These suggest that, indeed, small GTPase activity modifies astrocyte Ca2+ signalling through structural changes. To complement these in vitro studies, we have developed a novel procedure to study astrocyte morphology and its changes in situ using two-photon excitation fluorescence imaging. Preliminary data obtained in situ suggest that epileptiform activity, for instance, is a potent trigger of astrocyte morphology changes requiring intact RhoA signalling. Together, these data indicate that small GTPases could be a critical determinant of astrocyte morphology and Ca2+ signalling in vitro and in situ and may be of pathophysiological relevance. Thus, the goal of this proposal is to uncover the specific mechanisms that govern this relationship. To this end, we will manipulate small GTPase by molecular, pharmacological and optogenetic tools in vitro and in situ. We will then visualize and quantify Ca2+ signalling in parallel to astrocyte morphology changes. What triggers or controls small GTPase activity is also of physiological importance. Therefore, we have already established biosensor monitoring of GTPase activity, which will allow us to test what neuronal activity recruits GTPase activity. Finally, we apply the newly gained insight to reveal how GTPase-mediated astrocyte restructuring is involved in epileptogenesis. In summary, the proposed work will provide novel insights into the fundamental mechanisms that shape astrocyte Ca2+ signaling in physiological and pathological conditions.

• DFG Programme: Research Grants
• International Connection: Russia
• Cooperation Partner: Professor Dr. Alexey Semyanov