Astroglial Dysfunction in Models of CDKL5 Deficiency Disorder

Abstract

CDKL5 Deficiency Disorder (CDD) is a rare developmental epileptic encephalopathy typically caused by loss of function variants in the gene encoding the X-linked serine-threonine kinase CDKL5. CDKL5 is highly expressed in the brain during development, and key neuronal functions of the kinase include cytoskeletal organisation and synaptic stability. However, at present, little is known about the function of astroglia in CDD. Given the importance of these cells in synaptic development and homeostasis, as well as dysfunction in other epileptic diseases, it was hypothesised that astrocytes may contribute to CDD pathology. Induced pluripotent stem cells harbouring a CDKL5 loss-of-function mutation (and isogenic controls) were derived from CDD patient fibroblasts and differentiated into astrocytes (iAstros). Analysis of iAstros revealed transcriptomic, proteomic and functional dysregulation in CDKL5-mutant iAstros relating to water transport and immunological function, including a diminished response to TNF stimulation. Moreover, iAstros showed increased branching and reduced phosphorylation of the known CDKL5 target end-binding protein 2 (EB2) - indicative of disrupted cytoskeletal regulation in a manner similar to CDKL5-null neurons. Finally, we report the generation of novel in vitro models of CDD. CDKL5 was knocked down in adult and foetal human organotypic brain slices through transduction with an AAV encoding a novel CDKL5 shRNA. Slices transduced with the CDKL5 shRNA displayed increased spontaneous network activity, demonstrating the functionality of this model. Importantly, interrogation of these models revealed dysregulation of key astrocytic proteins congruous with the human glial stem cell model. Consequently, this study describes the generation of novel human models of CDD and their associated astrocytic dysfunction - paving the way for novel discovery and therapeutic intervention.