Embryonic alcohol exposure disrupts the ubiquitin-proteasome system
Olivia Weeks, Bess M. Miller, Brian J. Pepe-Mooney, Isaac M. Oderberg, Scott H. Freeburg, Colton J. Smith, Trista E. North, Wolfram Goessling
Olivia Weeks, Bess M. Miller, Brian J. Pepe-Mooney, Isaac M. Oderberg, Scott H. Freeburg, Colton J. Smith, Trista E. North, Wolfram Goessling
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Embryonic alcohol exposure disrupts the ubiquitin-proteasome system

Abstract

Ethanol (EtOH) is a commonly encountered teratogen that can disrupt organ development and lead to fetal alcohol spectrum disorders (FASDs); many mechanisms of developmental toxicity are unknown. Here, we used transcriptomic analysis in an established zebrafish model of embryonic alcohol exposure (EAE) to identify the ubiquitin-proteasome system (UPS) as a critical target of EtOH during development. Surprisingly, EAE alters 20S, 19S, and 11S proteasome gene expression and increases ubiquitylated protein load. EtOH and its metabolite acetaldehyde decrease proteasomal peptidase activity in a cell type–specific manner. Proteasome 20S subunit β 1 (psmb1hi2939Tg) and proteasome 26S subunit, ATPase 6 (psmc6hi3593Tg), genetic KOs define the developmental impact of decreased proteasome function. Importantly, loss of psmb1 or psmc6 results in widespread developmental abnormalities resembling EAE phenotypes, including growth restriction, abnormal craniofacial structure, neurodevelopmental defects, and failed hepatopancreas maturation. Furthermore, pharmacologic inhibition of chymotrypsin-like proteasome activity potentiates the teratogenic effects of EAE on craniofacial structure, the nervous system, and the endoderm. Our studies identify the proteasome as a target of EtOH exposure and signify that UPS disruptions contribute to craniofacial, neurological, and endodermal phenotypes in FASDs.

Authors

Olivia Weeks, Bess M. Miller, Brian J. Pepe-Mooney, Isaac M. Oderberg, Scott H. Freeburg, Colton J. Smith, Trista E. North, Wolfram Goessling

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Figure 2

EtOH modulates the ubiquitin proteasome system and chymotrypsin-like proteasome peptidase activity in a cell type–specific manner.

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EtOH modulates the ubiquitin proteasome system and chymotrypsin-like pro...
(A) Western blot analysis of ubiquitylated protein and the 20S proteasome after exposure to 0% or 1% EtOH. See Supplemental Figure 2A for loading controls. (B) ImageJ quantification of normalized ubiquitylated protein levels in embryos treated with 0% and 1% EtOH (12–120 hpf; 1-sided unpaired t test, *P ≤ 0.05; n = 3). (C and D) Proteasome activity assay in protein extracts of from whole homogenized 5 dpf larvae. Chymotrypsin-like proteasome activity is impaired by 1% EtOH exposure (12 hpf–5 dpf) and 0.01% MeCHO exposure (104–120 hpf; *P < 0.05, ****P ≤ 0.0001, 2-sided t test per time point; n = 5). (E) Time course ISH for EtOH and MeCHO metabolism genes. For most genes, expression after 72 hpf is noted in the liver and intestine. (F) Confocal imaging of 2D-plated hepatic and biliary epithelial cell (BEC) organoids after 24 hours of treatment with 0 or 100mM EtOH. (G and H) Exposure to 100 mM EtOH impairs chymotrypsin-like proteasome activity in 2D hepatic organoids but not BECs (****P < 0.0001, 2-sided t test; n = 5). Scale bars: 100 μm. For B, G, and H, data represent mean ± SEM. For C and D, data represent mean ± SD.