Developmental defects and impaired network excitability in a cerebral organoid\xa0model of\xa0KCNJ11 p.V59M-related neonatal diabetes

Gokhan Dalgin,Alfredo J Garcia,Louis H Philipson,Andrew K Tryba,Siri Atma W Greeley,Ashley P Cohen,Soo-Young Park

doi:10.1038/s41598-021-00939-7

Abstract

The gene KCNJ11 encodes Kir6.2 a major subunit of the ATP-sensitive potassium channel (KATP) expressed in both the pancreas and brain. Heterozygous gain of function mutations in KCNJ11 can cause neonatal diabetes mellitus (NDM). In addition, many patients exhibit neurological defects ranging from modest learning disorders to severe cognitive dysfunction and seizures. However, it remains unclear to what extent these neurological deficits are due to direct brain-specific activity of mutant KATP. We have generated cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) possessing the KCNJ11 mutation p.Val59Met (V59M) and from non-pathogenic/normal hiPSCs (i.e., control/WT). Control cerebral organoids developed neural networks that could generate stable synchronized bursting neuronal activity whereas those derived from V59M cerebral organoids showed reduced synchronization. Histocytochemical studies revealed a marked reduction in neurons localized to upper cortical layer-like structures in V59M cerebral organoids suggesting dysfunction in the development of cortical neuronal network. Examination of temporal transcriptional profiles of neural stem cell markers revealed an extended window of SOX2 expression in V59M cerebral organoids. Continuous treatment of V59M cerebral organoids with the KATP blocker tolbutamide partially rescued the neurodevelopmental differences. Our study demonstrates the utility of human cerebral organoids as an investigative platform for studying the effects of KCNJ11 mutations on neurophysiological outcome.

Highlights

The gene KCNJ11 encodes Kir6.2 a major subunit of the ATP-sensitive potassium channel (KATP) expressed in both the pancreas and brain
These mutations commonly cause sustained activation of the K ATP channel that in turn, produces a sustained hyperpolarization within cells. These gain-of-function mutations in the K ATP channel cause hyperglycemia due to failed insulin secretion from pancreatic beta cells2. KATP channels are expressed in the pancreas and in other organs such as brain and muscle, where the activating mutation may have a direct contribution to tissue specific dysfunction observed in patients with neonatal diabetes
Sanger sequencing confirmed that the presence of the mutant channel allele was sustained after reprograming (Fig. S1, panels E, F). Both induced pluripotent stem cells (iPSCs) lines expressed pluripotent markers OCT4 and TRA-1–60 (Fig. S1, panels G, H) and OCT4 and SOX2 (Fig. S1, panels I, J) and pluripotency was demonstrated by direct differentiation into three embryonic germ layers (Fig. S1, panels K-P)

Summary

Introduction

The gene KCNJ11 encodes Kir6.2 a major subunit of the ATP-sensitive potassium channel (KATP) expressed in both the pancreas and brain. We have generated cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) possessing the KCNJ11 mutation p.Val59Met (V59M) and from non-pathogenic/normal hiPSCs (i.e., control/WT). Neonatal diabetes mellitus is a monogenic disorder of which about 30% are caused by dominant heterozygous mutations in KCNJ111 These mutations commonly cause sustained activation of the K ATP channel that in turn, produces a sustained hyperpolarization within cells. KATP channels are expressed in the pancreas and in other organs such as brain and muscle, where the activating mutation may have a direct contribution to tissue specific dysfunction observed in patients with neonatal diabetes. We generated cerebral organoids from human induced pluripotent stem cells (hiPSCs) carrying either a non-pathogenic/control or KCNJ11 V59M mutant allele, to compare the effects of this gain of function K ATP mutation on maturation of the cortical network activity. This study provides the first direct evidence that a mutant KCNJ11 channel causes neurological deficits in patient hiPSC derived brain tissue

Methods

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Conclusion