Alcohol use disorder–associated gene FNDC4 alters glutamatergic and GABAergic neurogenesis in neural organoids
Xiujuan Zhu, August J. John, Sooan Kim, Li Wang, Enci Ding, Jing Zheng, Ateka Saleh, Irene Marín-Goñi, Abedalrahman Jomaa, Huanyao Gao, Meijie Wang, Ching Man Wai, Irene Moon, Cindy Chen, Alireza Agahi, Brandon J. Coombes, Tony M. Kerr, Nobuyoshi Suto, Liewei Wang, Mark A. Frye, Joanna M. Biernacka, Victor M. Karpyak, Hu Li, Richard M. Weinshilboum, Duan Liu
Xiujuan Zhu, August J. John, Sooan Kim, Li Wang, Enci Ding, Jing Zheng, Ateka Saleh, Irene Marín-Goñi, Abedalrahman Jomaa, Huanyao Gao, Meijie Wang, Ching Man Wai, Irene Moon, Cindy Chen, Alireza Agahi, Brandon J. Coombes, Tony M. Kerr, Nobuyoshi Suto, Liewei Wang, Mark A. Frye, Joanna M. Biernacka, Victor M. Karpyak, Hu Li, Richard M. Weinshilboum, Duan Liu
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Research Article Genetics Neuroscience

Alcohol use disorder–associated gene FNDC4 alters glutamatergic and GABAergic neurogenesis in neural organoids

Abstract

Large-cohort GWAS for alcohol use disorder (AUD) drug treatment outcomes and AUD risk have repeatedly identified genetic loci that are splicing quantitative trait loci for the fibronectin III domain containing 4 (FNDC4) gene in the brain. However, FNDC4 function in the brain and how it might contribute to AUD pathophysiology remain unclear. In the present study, we characterized GWAS loci–associated FNDC4 splice isoforms and demonstrated that FNDC4 alternative splicing results in loss of function for FNDC4. We also investigated FNDC4 function using CRISPR/Cas9 editing and the creation of human induced pluripotent stem cell–derived (iPSC-derived) neural organoids joined with single-nucleus RNA sequencing, a series of studies that showed that FNDC4 KO resulted in a striking shift in the relative proportions of glutamatergic and GABAergic neurons in iPSC-derived forebrain organoids as well as changes in their electrical activity. We further explored a potential mechanism(s) of FNDC4-dependent neurogenesis, and the results suggested a role for FNDC4 in mediating neural cell surface interactions. In summary, this series of experiments indicates that FNDC4 plays a role in regulating cerebral cortical neurogenesis in the brain. This regulation may contribute to the response to AUD pharmacotherapy as well as the effects of alcohol on the brain.

Authors

Xiujuan Zhu, August J. John, Sooan Kim, Li Wang, Enci Ding, Jing Zheng, Ateka Saleh, Irene Marín-Goñi, Abedalrahman Jomaa, Huanyao Gao, Meijie Wang, Ching Man Wai, Irene Moon, Cindy Chen, Alireza Agahi, Brandon J. Coombes, Tony M. Kerr, Nobuyoshi Suto, Liewei Wang, Mark A. Frye, Joanna M. Biernacka, Victor M. Karpyak, Hu Li, Richard M. Weinshilboum, Duan Liu

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Figure 1

AUD drug treatment response and FNDC4 alternative splicing in the brain.

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AUD drug treatment response and FNDC4 alternative splicing in the brain....
(A) Kaplan-Meier plot showing the percentage of patients with AUD who did not relapse to heavy alcohol drinking during 90 days of pharmacotherapy. A smaller percentage of patients who carried the rs56951679 SNP variant allele C remained abstinent from heavy alcohol drinking, suggesting that worse drug treatment response is seen in patients carrying the rs56951679 SNP C allele. The P value was calculated using the log-rank (Mantel-Cox) test. The Kaplan-Meier plot was generated based on our published GWAS data (9). (B) The rs56951679 SNP is an sQTL for the FNDC4 gene in many human brain regions. The SNP genotype was associated with FNDC4 RNA splicing in brain cortex (left) and nucleus accumbens (right) based on the RNA-seq data generated by GTEx (v10) (10). The variant allele C was associated with an increased level of intron excision on chromosome 2 (chr.2): 27,492,478-27,493,389 (hg38). (C) Depiction of the rs56951679 SNP-associated alternative splice sites and FNDC4 RNA splice isoforms. The physical position of the FNDC4 gene on chr.2 based on human genome assembly hg38 with sQTL splice sites highlighted in purple (top); the reference FNDC4 mRNA with 7 exons (middle) and FNDC4 splice variant with exon 6 excision (bottom) are numbered below. FNDC4 transcripts were annotated by RefSeq. (D) Depiction of the FNDC4 protein encoded by the reference (canonical) and spliced (truncated) FNDC4 RNA isoforms. The canonical FNDC4 protein includes a signal peptide (SP), a FN3 domain, a TM domain, and a cytosolic C-terminus. The truncated FNDC4 protein displays a frameshift after amino acid 182 that disrupts the TM domain.