期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：9
DOI：10.1073/pnas.2115524119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Both genetic and environmental factors contribute to epilepsy. Understanding their contributions and interactions helps disease management. However, it is often challenging to study gene–environment interaction in humans due to their heterogeneous genetic background and less controllable environmental factors. The fruit fly,
Drosophila melanogaster, has been proven to be a powerful model to study human diseases, including epilepsy. We generated knock-in flies carrying different epilepsy-associated pyridox(am)ine 5
′
-phosphate oxidase (
PNPO) alleles and studied the developmental, behavioral, electrophysiological, and fitness effects of each mutant allele under different dietary conditions. We showed that phenotypes in knock-in flies are allele and diet dependent, providing clues for timely and specific diet interventions. Our results offer biological insights into mechanisms underlying phenotypic variations and specific therapeutic strategies.
Pyridox(am)ine 5
′
-phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5
′
-phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in
PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the
PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific
PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in
Drosophila alleles,
h
WT
,
h
R
116
Q
,
h
D
33
V
, and
h
R
95
H
, in which the endogenous
Drosophila PNPO was replaced by wild-type human
PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that h
R95Hhad a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of
PNPO, specific molecular and/or genetic properties of each
PNPO variant, and differential allele–diet interactions.
