Fine-tuning of mTOR signaling by the UBE4B-KLHL22 E3 ubiquitin ligase cascade in brain development

ABSTRACT Spatiotemporal regulation of the mechanistic target of rapamycin (mTOR) pathway is pivotal for establishment of brain architecture. Dysregulation of mTOR signaling is associated with a variety of neurodevelopmental disorders. Here, we demonstrate that the UBE4B-KLHL22 E3 ubiquitin ligase cascade regulates mTOR activity in neurodevelopment. In a mouse model with UBE4B conditionally deleted in the nervous system, animals display severe growth defects, spontaneous seizures and premature death. Loss of UBE4B in the brains of mutant mice results in depletion of neural precursor cells and impairment of neurogenesis. Mechanistically, UBE4B polyubiquitylates and degrades KLHL22, an E3 ligase previously shown to degrade the GATOR1 component DEPDC5. Deletion of UBE4B causes upregulation of KLHL22 and hyperactivation of mTOR, leading to defective proliferation and differentiation of neural precursor cells. Suppression of KLHL22 expression reverses the elevated activity of mTOR caused by acute local deletion of UBE4B. Prenatal treatment with the mTOR inhibitor rapamycin rescues neurogenesis defects in Ube4b mutant mice. Taken together, these findings demonstrate that UBE4B and KLHL22 are essential for maintenance and differentiation of the precursor pool through fine-tuning of mTOR activity.

Two LoxP sites were inserted in introns 2 and 4 by CRISPR. Ube4b f/f and Ube4b f/+ ; Nestin-Cre/+ were crossed to obtain heterozygous and homozygous conditional knockout progeny.
(B) Validation of Ube4b deletion by RNAscope in sagittal brain sections from a pair of Ube4b f/f (top) and Ube4b  (E) Dendritic arborization shown by microtubule-associated protein 2 (MAP2) staining in P0 cortical slices. Disorganization of dendritic trees was noticed in the S1 cortex of CKO animals. Scale bar, 50 μm.
(F) Representative behaviors of a 1-month-old CKO mouse that was experiencing a tonic-clonic seizure. Onset of the seizure episode started with sudden body stiffness with curling tail and hunched back. Then the animal showed rhythmic twitching of its body and muscle contraction, followed by body rolling. At the end of the seizure, the animal became quiet for a few seconds and then crawled away (Recovery).    were introduced into Neuro2A cells. Following drug selection, single clones were expanded and immunoblotted for UBE4B. γ-tubulin was used as the loading control. Multiple heterozygous and homozygous knockout clones were obtained. The KO#11 clone was used for the CHX experiment in Figure 3E. (B) Temporal expression of UBE4B, KLHL22 and mTOR in mouse brains starting from E15.5 to 6 weeks.
γ-tubulin was used as the loading control.
(B-C) Quantification of average intensity of pS6 signals in the layers II-IV (B) and V-VI (C). Brain slides from 3 pairs of animals were quantified for the control and rapamycin groups, respectively. ***p < 0.001 relative to Ube4b f/f treated with vehicle control; ### p < 0.001 relative to CKO treated with vehicle control; one-way ANOVA.
(D) Effects of rapamycin treatment on the percentage of pS6 + CTIP2 + GCs in the S1 cortex. N = 3 animals per group. *p < 0.05 relative to Ube4b f/f treated with vehicle control; # p < 0.05 relative to CKO treated with vehicle control; one-way ANOVA.
(E-G) Immunohistochemistry of PAX6 in the DG of rapamycin-treated newborn Ube4b f/f and Ube4b f/f ; Nestin-Cre/+ animals. Scale bar, 100 μm. No significant difference (ns) in the total numbers of PAX6 + NPCs (F) or DAPI-marked nuclei (G) was observed between quantified littermates.
(H) A model of mTOR regulation by the UBE4B-KLHL22 E3 cascade. UBE4B restricts the level of KLHL22 and keeps the activity of mTOR at a level optimal for NPC proliferation and differentiation. In the absence of UBE4B, accumulated KLHL22 causes mTOR hyperactivation, which leads to impaired NPC proliferation and differentiation. Table S1. List of antibodies, plasmids, mouse strains and software Table S2. Lists of upregulated and downregulated proteins in P0, P7, 7 M, 9 M and 11 M brains.
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