Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

Abstract

Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. We solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, we showed that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P concentrations through synaptojanin mutations. Hence, we discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function.

A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay

“…This pocket is necessary for binding to the lipid membrane via phosphoinositides phosphorylated at the 4 and 5 positions. Abrogation of the cationic pocket by introduction of two human TBC1D24 pathogenic variants found in DOORS syndrome led to impaired synaptic vesicle trafficking and seizures in drosophila [16]. TBC1D24 is the only gene in the MOR that is associated with autosomal dominant and recessive human disease phenotypes.…”

Phosphatidylinositol 4,5‐bisphosphate controls Rab7 and PLEKHM 1 membrane cycling during autophagosome–lysosome fusion

“…We also tested the localizations of the GFP-tagged versions of these proteins and their impact on Rab7 distribution and found that only TBC1D9A and TBC1D5 showed localization to endosomes and several of them (TBC1D2, TBC1D5, TBC1D9B and TBC1D15) caused Rab7 endosomes to localize at the cell periphery ( Fig EV5). The Drosophila orthologue of TBC1D24 (Skywalker) has been shown to bind PI(4,5) P 2 (Fischer et al, 2016), and indeed, it showed prominent plasma membrane and some endosomal localization ( Fig EV5). Moreover, generation of PI(4,5)P 2 on Rab7 vesicles by the recruitable PIP5K caused localization of TBC1D24 to Rab7 endosomes (Appendix Fig S1A).…”

Case Report: Novel mutations in TBC1D24 are associated with autosomal dominant tonic-clonic and myoclonic epilepsy and recessive Parkinsonism, psychosis, and intellectual disability

“…This variant occurs at a highly conserved position in the Rab-GAP N-terminal Tre2–Bub2–Cdc16 (TBC) domain of the protein. It was recently discovered that this domain directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function 4 . A fly model with 3 clinically pathogenic mutations (3Glu mutant) in the phosphoinositide-binding pocket causes severe neurological defects, including impaired synaptic-vesicle trafficking and seizures 4 .…”

“…Drosophila with mutations in the sky gene ( TBC1D24 orthologue) have a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release 3 . Overall, evidence demonstrates that TBC1D24 is a critical player in synaptic vesicle endocytosis, neurotransmitter release and presynaptic function 4 .…”