Loss-of-function mutation of c-Ret causes cerebellar hypoplasia in mice with Hirschsprung disease and Down's syndrome

Nobutaka Ohgami,Akira Iizuka,Hirokazu Hirai,Ichiro Yajima,Machiko Iida,Atsuyoshi Shimada,Toyonori Tsuzuki,Mayumi Jijiwa,Naoya Asai,Masahide Takahashi,Masashi Kato

doi:10.1016/j.jbc.2021.100389

Abstract

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.

Highlights

The c-RET proto-oncogene encodes a receptor-tyrosine kinase
Increased phosphorylation of c-Ret Y1062 in Purkinje cells during postnatal development and motor impairments in cRet-KIYF/YF-mice c-Ret protein was constantly detectable in PCs until postnatal day (P) 15, while phosphorylated Y1062 in c-Ret was detectable in PCs after P8 and gradually increased until P15 in wild-type (WT) mice, several days before WT mice had complete cerebellar development (Fig. 1, A and B)
Eighteen-day-old c-Ret-KIYF/YF-mice showed a higher density of granule cell progenitors (GCPs) in the external germinal layer (EGL) than that in WT mice, while the density of GCPs in the EGL in WT mice and that in c-Ret-KIYF/YF-mice were comparable on P2.5 (Fig. 2, C and D). c-Ret-KIYF/YF-mice showed a decreased number of GABA A receptor alpha 6 (GABRA6)positive matured granule cells (GCs) compared with that in WT mice on P13 (Fig. 2, G and H)

Summary

Introduction

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down’s syndrome (HSCR-DS) in humans. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. C-Ret mutant mice showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS. In this study, impaired coordinate performance was shown in Ednrbknockout (−/−) mice (Fig. S1), suggesting that cerebellar hypoplasia develops in Ednrb-knock-out (−/−) mice with HSCR including deafness [18].

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Results

Conclusion