Cell-autonomous function of Dyrk1A kinase hyperactivity in neuronal maturation

• Applicant: Professor Dr. Reinhard W. Köster
• Subject Area: Developmental Neurobiology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 241961032

The highly conserved vertebrate cerebellum regulates locomotor, socio-emotional behavior and higher cognitive functions. Within the cerebellar circuitry Purkinje cells (PCs) represent central neurons as they process input signals from numerous synapses into a cerebellar output signal. These PCs are organized as a single cell layer across the cerebellar cortex. In nearly transparent zebrafish larvae this PC layer is located just underneath the skin of the hindbrain. Therefore, these PCs are easily accessible for bioimaging to monitor their developmental maturation based on dendrite outgrowth and branching, spine formation and axonal projection. Yet sparse labeling techniques are necessary to separately characterize individual PCs. Recent studies have revealed that during PC maturation these neurons spatially organize themselves into clusters dedicated to control different behaviors suggesting that axonal projections of PCs are also regionally clustered. Furthermore, PC maturation by increasing spine density depends on neuronal activity of PCs themselves. Synaptogenesis and dendrite branch formation is reduced upon overexpression of endogenously expressed Dyrk1A kinase in PCs as it occurs in Down Syndrome (DS), since Dyrk1A is located in the Down Syndrome cluster on human chromosome 21. Leucettinib is a recently developed high-affinity Dyrk1A kinase inhibitor active in a nanomolar range that has not been evaluated yet in vivo for its potential to rescue neuronal deficits caused by Dyrk1A hyperactivity. We aim to fine-tune recently established compound- and light-inducible Cre-recombinase self-excising cassettes to achieve a sparse but efficient genetic labeling technique for monitoring the morphology of individual PCs in zebrafish larvae by confocal microscopy. Furthermore, specific dendritic and axonal fluorescent reporters expressed in individual PCs of transgenic zebrafish with fluorescent protein expression throughout the PC population will allow for mapping individual PC morphologies onto a standardized zebrafish brain atlas. This will reveal a regionalized organization of zebrafish PCs within the grossly homogenous PC population to define PC subcompartments. Furthermore, coexpression of Dyrk1A together with axonal and dendritic fluorescent reporters in individual PCs will not only elucidate the influence of Dyrk1A hyperactivity on PC maturation, but reveal the ability of the Dyrk1A-inhibitor Leucettinib to revert these neurological phenotypes. These studies will provide a novel tunable genetic sparse labeling technique for monitoring and expressing transgenes in individual cells in zebrafish. With its application, a regionalized organization of cerebellar PCs based on dendritic and axonal projection will be revealed and the influence of DS-associated Dyrk1A kinase in mediating PC maturation elucidated. Therefore, these studies will crucially impact on the understanding of cellular and molecular mechanisms of neuronal maturation processes.

• DFG Programme: Research Grants