Epilepsy-associated digenic variants affecting an actin/mitochondria/glutamate pathway promote seizure susceptibility
Shenzhao Lu, Mengqi Ma, Shabab B. Hannan, Mingxi Deng, Hu Chen, Zhijian Yu, Lindsey D. Goodman, Haein Kim, Yun Zhao, Sandeep Kumar Dubey, Wen-Wen Lin, Xueyang Pan, Debdeep Dutta, Vishnu Anand Cuddapah, Jill A. Rosenfeld, Xi Luo, Zhandong Liu, Joshua M. Shulman, Hugo J. Bellen
Shenzhao Lu, Mengqi Ma, Shabab B. Hannan, Mingxi Deng, Hu Chen, Zhijian Yu, Lindsey D. Goodman, Haein Kim, Yun Zhao, Sandeep Kumar Dubey, Wen-Wen Lin, Xueyang Pan, Debdeep Dutta, Vishnu Anand Cuddapah, Jill A. Rosenfeld, Xi Luo, Zhandong Liu, Joshua M. Shulman, Hugo J. Bellen
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Research Article Development Genetics Neuroscience

Epilepsy-associated digenic variants affecting an actin/mitochondria/glutamate pathway promote seizure susceptibility

Abstract

Epilepsy affects approximately 50 million people worldwide, yet more than half of individuals with a presumed genetic cause still lack a molecular diagnosis despite the identification of over 1,000 monogenic epilepsy genes. This diagnostic gap is unlikely to be resolved by improved variant detection alone, suggesting that variants affecting the same biological pathway may combine to cause disease. By studying epilepsy-associated actin regulatory genes, we identified a conserved actin/mitochondria/glutamate (AMG) pathway. We demonstrate that reduced actin polymerization promoted DRP1-mediated mitochondrial fission, increased ROS levels, and enhanced glutamatergic transmission, leading to seizures. The glial innate immune pathway, a recently recognized contributor to epilepsy, is activated when the AMG pathway is affected. Reducing mitochondrial fission with the mitochondria division inhibitor (Mdivi-1), or suppressing ROS with N-acetyl-l-cysteine amide (NACA), significantly alleviated seizures. Importantly, digenic heterozygous loss-of-function variants in AMG pathway genes combined to cause seizures, and individuals with epilepsy of unknown etiology showed an increased burden of such variants when compared with the controls. Modeling patient-specific digenic combinations in Drosophila confirmed that many combinations promote seizure susceptibility. Together, these findings establish the AMG pathway as a mechanistic framework for identifying digenic etiologies in epilepsy and highlight potential therapeutic targets.

Authors

Shenzhao Lu, Mengqi Ma, Shabab B. Hannan, Mingxi Deng, Hu Chen, Zhijian Yu, Lindsey D. Goodman, Haein Kim, Yun Zhao, Sandeep Kumar Dubey, Wen-Wen Lin, Xueyang Pan, Debdeep Dutta, Vishnu Anand Cuddapah, Jill A. Rosenfeld, Xi Luo, Zhandong Liu, Joshua M. Shulman, Hugo J. Bellen

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Figure 5

Inhibition of mitochondrial fission significantly rescues sif LoF phenotypes.

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Inhibition of mitochondrial fission significantly rescues sif LoF phenot...
(A) Graphical illustration of actin filaments mediated mitochondrial fission via recruitment of DRP1 (created with BioRender). (B) Protein levels in sif rescue and sif-mutant animals were determined by Western blotting. Third instar larvae were collected for protein extraction and analysis. (C) Quantification of protein levels. Data points represent the relative protein levels per independent repeat (n = 5). (D) Survival rate of sif-mutant animals with the expression of UAS-Drp1 RNAi. The survival rate was measured using the expected Mendelian ratio by counting flies that eclosed and were viable after 24 hours. (n = 3–9, with over 300 progenies counted for each genotype). (E) Survival rate of sif-mutant animals fed different doses of Mdivi-1 (0, 20, 50, and 100 μM in food) (n = 4 for 0 μM and 100 μM groups, n = 1 for 20 μM and 50 μM groups). (F and G) Quantification of heat sensitivity for control (w1118), sif heterozygote (sifT2A-GAL4/+), sif-mutant (w UAS-lacZ/+ sifT2A-GAL4/Df), and sif-mutant flies fed different doses of Mdivi-1. The flies were tested at 3–5 dpe. Data points in F represent replicates (n = 5–8), and each replicate contains 5–10 animals; data points in G represent the recovery time for each animal (n = 24–54). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired 2 tailed t test with Welch’s correction for unequal variances (C–G).