Chronic mTORC1 inhibition rescues behavioral and biochemical deficits resulting from neuronal Depdc5 loss in mice

Abstract

DEPDC5 is now recognized as one of the genes most often implicated in familial/inherited focal epilepsy and brain malformations. Individuals with pathogenic variants in DEPDC5 are at risk for epilepsy, associated neuropsychiatric comorbidities and sudden unexplained death in epilepsy. Depdc5flox/flox-Syn1Cre (Depdc5cc+) neuronal-specific Depdc5 knockout mice exhibit seizures and neuronal mTORC1 hyperactivation. It is not known if Depdc5cc+ mice have a hyperactivity/anxiety phenotype, die early from terminal seizures or whether mTOR inhibitors rescue DEPDC5-related seizures and associated comorbidities. Herein, we report that Depdc5cc+ mice were hyperactive in open-field testing but did not display anxiety-like behaviors on the elevated-plus maze. Unlike many other mTOR-related models, Depdc5cc+ mice had minimal epileptiform activity and rare seizures prior to seizure-induced death, as confirmed by video-EEG monitoring. Treatment with the mTORC1 inhibitor rapamycin starting after 3 weeks of age significantly prolonged the survival of Depdc5cc+ mice and partially rescued the behavioral hyperactivity. Rapamycin decreased the enlarged brain size of Depdc5cc+ mice with corresponding decrease in neuronal soma size. Loss of Depdc5 led to a decrease in the other GATOR1 protein levels (NPRL2 and NPRL3). Rapamycin failed to rescue GATOR1 protein levels but rather rescued downstream mTORC1 hyperactivity as measured by phosphorylation of S6. Collectively, our data provide the first evidence of behavioral alterations in mice with Depdc5 loss and support mTOR inhibition as a rational therapeutic strategy for DEPDC5-related epilepsy in humans.

NPRL3: Direct Effects on Human Phenotypic Variability, mTOR Signaling, Subcellular mTOR Localization, Cortical Lamination, and Seizure Susceptibility

“…When treated with rapamycin (50 nM) or torin1 (50 nM) for 24 hours, these N2aC showed a reduction in soma diameter (Fig. 2C,D) equivalent to WT cell diameter (p<0.001) demonstrating that Nprl3 loss resulted in mTOR-dependent soma enlargement 14,15 .…”

“…In our study, CRISPR/Cas9 KO of Nprl3 resulted in persistent and inappropriate localization of mTOR to the lysosomal membrane during amino acid starvation. Indeed, loss of Depdc5 in mouse renders GATOR1 non-functional 14 and previous studies have shown that shRNA mediated knockdown of Depdc5 or Nprl3 resulted in increased localization of mTOR to the lysosomal surface even during amino acid starvation 15 .…”

“…However, when intracellular amino acids are low, GATOR1 alters the nucleotide bound state of the Rag proteins and prevents translocation of mTORC1 to the lysosomal surface (remaining cytoplasmic) thereby inhibiting mTOR activity 12 . GATOR1 subunits are highly expressed in brain 13,14 and the effects of for example, DEPDC5 variants on cell size, motility, and firing as well as cortical lamination have been shown in several experimental models [15][16][17][18][19][20] and in human brain specimens 4,7 . While human tissue studies show a link between NPRL3 variants and mTOR activation 9,[21][22][23] , surprisingly few studies have investigated the role of NPRL3 in neuronal morphology, cortical lamination, and cortical circuitry.…”

Protective effects of P2X7R antagonist in sepsis‐induced acute lung injury in mice via regulation of circ_0001679 and circ_0001212 and downstream Pln, Cdh2, and Nprl3 expression

“…The protein encoded by Cdh2, N-cadherin, is involved in ALI and pulmonary fibrosis. 38 Some studies have shown that Nprl3 (GATOR1 complex subunit) participates in the pathogenesis of sepsis and mediates the expression of many inflammatory factors via the mammalian target of rapamycin (mTOR) pathway, [39][40][41] suggesting that Nprl3 contributes to sepsis-induced ALI, perhaps through a regulatory interaction with mmu_circ_0001679 or mmu_circ_0001212.…”

FKBP39 controls nutrient dependent Nprl3 expression and TORC1 activity in Drosophila

“…Upon amino acid starvation, KLHL22 is bound and trapped by 14-3-3 proteins in the nucleus, resulting in a dramatic increase in DEPDC5 protein stability 26 . Interestingly, depletion of DEPDC5 results in the decreased expression of the other two GATOR1 components NPRL2 and NPRL3 23 , 27 . We reasoned that under amino acid starvation the expression levels of Nprl2 and Nprl3 might also be regulated, especially in the invertebrates that have no orthologs of KICSTOR and CASTOR1.…”