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 6

Inhibition of mitochondrial ROS using NACA significantly rescues sif LoF phenotypes.

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Inhibition of mitochondrial ROS using NACA significantly rescues sif LoF...
(A) Graphical illustration of mitochondrial fission and ROS generation (created with BioRender). (B and C) An aconitase activity assay was used to reveal high ROS in sif mutants. This high ROS was rescuable by expression of sif cDNA in these mutants (n = 9). (D and E) Survival rate of sif LoF mutants fed with different doses of NACA (0, 40, 80, and 160 μg/mL in food) (D) or Mdivi-1 and NACA (M+N) (Mdivi-1: 100 μM; NACA: 80 μg/mL) (E). n ≥3, except for NACA: 40 μg/mL group (n = 1), with over 200 progenies counted for each group. (F) Quantification of heat sensitivity for control (w1118), sif heterozygote (sifT2A-GAL4/+), sif-mutant (w UAS-lacZ/+ sifT2A-GAL4/Df), and sif-mutant flies fed Mdivi-1 (100 μM), NACA (80 μg/mL), or their combination (M+N). Flies were tested at 3–5 dpe. (G) Representative traces of spontaneous EPSPs from muscles with intact CNS of sif mutants fed normal food (top) or NACA (bottom). (H) Percentage of muscle cells with active events (events >5 mV). Fisher’s exact test. (I) Average amplitude of spontaneous EPSP events (n = 13–17). (J) Average frequency of spontaneous EPSP events (n = 13–17). 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 (B–F, I, and J).