Analysis of the Mechanism and Function of the Ste20-like Kinase in the Outgrowth of Dendrites and the Stabilization of Inhibitory Synapses

Anne Quatraccioni1, Barbara Robens1, Delara Kamalizade1, Julika Pitsch1, Sheeva Ahmadi2, Dominic Winter2, Ashley Waardenberg3, Mark Graham4, Dirk Dietrich5, Albert Becker1, Susanne Schoch1

1 Institute of Neuropathology, University of Bonn Medical Center, Bonn, Germany
2 Institute of Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany
3 Australian Institute of Tropical Health & Medicine, James Cook University, Douglas, Australia
4 Synapse Proteomics, Children’s Medical Research Institute, The University of Sydney, Westmead, Australia
5 Institute of Neurosurgery, University of Bonn Medical Center, Bonn, Germany

The Ste20-like kinase (SLK) fulfills important and diverse functions in non-neuronal cells by influencing cellular processes such as apoptosis, cell cycle progression, cytoskeletal dynamics, and cell migration. Surprisingly, almost nothing is known about the functional relevance of SLK in neurons, even though the kinase is expressed in these cells throughout development and adulthood. A possible role of SLK in neurons was further supported through the finding that SLK expression was reduced in biopsy specimen from patients with cortical malformations and gangliogliomas.

To study the function of SLK in neuronal cells, we investigated the effect of a focal SLK knockdown (KD) in cortical neurons by in utero electroporation of a short hairpin RNA (shRNA) in mice. Reducing the expression of SLK decreased the arborization of the dendritic tree and resulted in a selective loss of inhibitory synapses after P15. The aberrant morphology of SLK knockdown (KD) neurons was also reflected on a functional level as mice with focal cortical SLK KD showed abnormal patterns and seizure-like events in EEG recordings.

To resolve the molecular mechanisms underlying these morphological and functional alterations and thereby identify SLK-dependent cellular pathways, different experimental strategies were applied. Immunocytochemical analysis of several known SLK downstream targets pointed to a reduced amount of F-actin in dendrites of SLK KD compared to control neurons, implying that SLK could be involved in actin cytoskeleton dynamics. A phosphoproteomic screen of cultured cortical neurons with reduced SLK levels revealed that cytoskeleton-organizing proteins and proteins described to be involved in neuron projections and outgrowth were less phosphorylated compared to control neurons. In a complementary approach using a proximity-dependent labeling strategy (BioID), we also identified proteins known to impact neurite outgrowth as probable cellular interaction partners of SLK.

In summary, SLK along with its potential up- and downstream proteins emerges as a novel regulator of dendritic growth and inhibitory synapse stabilization, thereby also influencing functional properties of neurons.