The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy

Abstract

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs * 3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs * 3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs * 3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs * 3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs * 3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

The epilepsy and intellectual disability-associated protein TBC1D24 regulates the maintenance of excitatory synapses and animal behaviors

“…However, the F251L knock-in mice display severe seizure at around postnatal day 21 that leads to lethal convulsion. While this manuscript was under preparation, a very recent study reported generation of TBC1D24 knock-in mice that carry non-sense mutation leading to truncation of the protein [88]. Similar to the TBC1D24 F251L/251L mice in our study, the homozygous mice carrying non-sense TBC1D24 mutation also undergo early death at three weeks after birth due to spontaneous seizure [88].…”

“…While this manuscript was under preparation, a very recent study reported generation of TBC1D24 knock-in mice that carry non-sense mutation leading to truncation of the protein [88]. Similar to the TBC1D24 F251L/251L mice in our study, the homozygous mice carrying non-sense TBC1D24 mutation also undergo early death at three weeks after birth due to spontaneous seizure [88]. The F251L knock-in mice in our study and transgenic mice harboring non-sense mutation reported by Tona et al [88] will be valuable for future elucidation of the mechanistic link between TBC1D24 deficiency and epilepsy.…”

“…Given the strong association of TBC1D24 mutations with ID, it is important to characterize how TBC1D24 deficiency in vivo affects animal behaviors including learning and memory. Despite the emergence of TBC1D24 transgenic mouse models recently [61,88], the behavioral changes associated with TBC1D24 gene mutations have not been studied, probably because of early lethality of the mice. The TBC1D24 F251L/F251L mice in our current study also suffer early death around three weeks after birth.…”

Mouse Models of Human Pathogenic Variants of TBC1D24 Associated with Non-Syndromic Deafness DFNB86 and DFNA65 and Syndromes Involving Deafness

“…The wild type 325 bp amplicon was digested by NlaIIII and produced 115 bp and 210 bp restriction fragments, while the p.His336Glnfs*12 allele generated a 324 bp amplicon and was uncut by NlaIIII. The engineering and genotyping of mice carrying the p.Ser324Thrfs*3 allele were described in Tona et al 2019 [ 13 ].…”

“…There are several alternative transcripts of human TBC1D24 including a transcript that skips micro exon 3 [ 10 , 13 ]. The largest TBC1D24 transcript encodes a TBC domain (Tre-2-Bub2-Cdc16) and a TLDc domain (TBC, LysM, domain catalytic) ( Figure 1 A).…”