NPRL2 Inhibition of mTORC1 Controls Sodium Channel Expression and Brain Amino Acid Homeostasis

Abstract

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the Gap Activity TOward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian Target Of Rapamycin Complex 1 (mTORC1) kinase when intracellular amino acids are low.Here we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures prior to death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials in neurons. The increased action potential strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.Significance Statement: NPRL2 is a requisite subunit of the epilepsy-linked GATOR1 complex that functions as a negative regulator of mTORC1 kinase when intracellular amino acids are limited. Here we report the generation and characterization of a new neurological model of GATOR1-dependent mTORopathy, caused by the loss of NPRL2 function in glutamatergic neurons. Loss of NPRL2 increases mTORC1 signal transduction, significantly alters amino acid homeostasis in the brain, and causes SUDEP.In addition, loss of NPRL2 increases the strength of electrically stimulated action potentials and the expression of epilepsy-linked sodium channels. These data reveal an unanticipated link between 3 intracellular amino acid signaling by NPRL2 and a novel mTORC1-dependent regulation of sodiumchannel expression in epilepsy.

Increased glycine contributes to synaptic dysfunction and early mortality in Nprl2 seizure model

“…Hoechst staining of brain slices revealed that mutant mice had significantly thicker cortices ( Figure 1 G), a consistent finding noted with mTORC1 upregulation ( Carson et al., 2012 ; Chen et al., 2019 ; Ishida et al., 2021 ; Yuskaitis et al., 2018 ). In addition to changes in size, mutant mice also demonstrated decreased survival, with no mutants surviving beyond 21 days of age which is similar to other studies ( Hui et al., 2022 ; Ishida et al., 2021 ) ( Figure 1 H), while heterozygotes displayed comparable survival to control littermates (data not included).…”

“…This increase in GFAP intensity is consistent with reactive astrogliosis as has been shown in multiple models of epilepsy ( Carson et al., 2012 ; Crowell et al., 2015 ) ( Figure S1 J). This astrogliosis occurs secondarily to neuronal Nprl2 loss, as pS6 staining was largely restricted to neurons and did not show any prominent overlap in expression with GFAP similar to other Nprl2 mouse models ( Hui et al., 2022 ; Ishida et al., 2021 ) ( Figures S1 J and S1K).…”

“…To better understand underlying mechanisms, we sought to evaluate whether synaptic dysfunction might contribute to seizure phenotypes. A previous study ( Hui et al., 2022 ) had looked at intrinsic phenotypes in the lateral habenula, but synaptic changes and intrinsic properties in the cortex have not been previously examined. We observed increased eEPSCs, decreased eIPSCs, and alterations in release probability in layer 2/3 cortical neurons, which point to a presynaptic contribution to these alterations.…”

“…Several studies have found that Nprl2 regulates amino acid and TCA cycle metabolism in yeast, Drosophila , mouse muscle, and mouse embryonic fibroblasts ( Laxman et al., 2014 ; Moloney et al., 2021 ). Although two recent studies with conditional knockout mouse models of Nprl2 have shown these mice develop seizures ( Hui et al., 2022 ; Ishida et al., 2021 ), very little is known about how loss of Nprl2 results in seizures and/or the metabolic and molecular contributions to these mechanisms.…”

“…Previous studies of NPRL2 are limited given that constitutive mouse mutants are lethal due to deficient hematopoiesis and methionine:folate homeostasis ( Dutchak et al., 2015 ). Recent studies with conditional Nprl2 knockout mice describe that homozygous mutants have mTORC1-dependent seizures, enlarged neurons, and changes in certain metabolites ( Hui et al., 2022 ; Ishida et al., 2021 ). However, how these alterations might impact synaptic function and how they might contribute to seizure activity has yet to be explored.…”