Neuronal differentiation and activity drive nucleocytoplasmic shuttling of the intellectual disability kinase TLK2

Introduction: Autosomal dominant intellectual developmental disorder 57 (MRD57) is a rare neurodevelopmental disorder characterised by delayed language and psychomotor development, intellectual disability, hypotonia, gastrointestinal issues and facial dysmorphia. It is linked to genetic mutations in the serine/threonine kinase TLK2, which generally cause haploinsufficiency. TLK2 is an established cell cycle regulator that has been extensively studied in mitotic cells. It is upregulated in cancers, driving tumour growth, however, the role of TLK2 in postmitotic neurons is not understood. We therefore aimed to determine where TLK2 is expressed in the brain and its subcellular localisation during neuronal differentiation. Methods: We analysed TLK2 transcript or protein expression and localisation in public RNAseq datasets, mouse brain sections, and a rat neuroblastoma cell line model of neuronal differentiation. Results: Human and mouse brain transcriptomic data revealed splice variant diversity in the N-terminus of TLK2, which contains its nuclear localisation sequence (NLS). Using splice-specific in situ hybridisation probes, we observed expression of TLK2 transcripts that contain and lack the NLS in the mouse hippocampus and cerebellum. Surprisingly, TLK2 protein was predominantly cytoplasmic in the adult mouse brain. Similarly, in rat neuroblastoma cells, we found that neuronal differentiation enhances a cytoplasmic pool of TLK2 by two mechanisms: nuclear export of full length TLK2 and increased expression of TLK2 splice variants lacking the NLS. Finally, acute stimuli that mimic synaptic activity were sufficient to elicit nuclear export of TLK2. Discussion: Our data highlight a previously unrecognised role of cytoplasmic TLK2 in neurons and future studies should determine how the loss of TLK2 activity in MRD57 impacts cytoplasmic TLK2 substrates in the developing and mature brain.