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 7

psmb1 and psmc6 are necessary for hepatopancreatic development.

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psmb1 and psmc6 are necessary for hepatopancreatic development. (A) ISH...
(A) ISH for exocrine pancreas marker cpa5 and hepatocyte marker fabp10a. EtOH (1%) (12–72 hpf) reduced exocrine pancreas and liver size. (B and C) ImageJ quantification of pancreas and liver size (****P ≤ 0.0001, unpaired 2-tailed t test, n ≥13). (D and E) ISH for exocrine pancreas marker trypsin and hepatocyte markers prox1a and fabp10a in psmb1hi2939 and psmc6hi3593 (72 hpf) mutants. (F–I) Quantification of pancreas and liver size using ImageJ. psmb1+/–, psmb1–/–, and psmc6–/– have significantly reduced exocrine pancreas size. psmb1–/– and psmc6–/– have significantly reduced liver size (n > 4, ****P ≤ 0.0001, **P < 0.01, ordinary 1-way ANOVA with Dunnett’s multiple-comparison test). (J–L) ISH for liver marker fabp10a and exocrine pancreas marker trypsin, followed by quantification of organ size using ImageJ area calculation at 96 hpf. EtOH (1%) (12–96 hpf) significantly reduced liver and pancreas size. Treatment with BTZ significantly exacerbated the effects of EAE on liver and pancreas area (*P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA with Sidak’s multiple comparisons). BTZ concentration = 2.5 μM (12–96 hpf). Scale bars: 100 μm. From left, column sample n = 7, 16, 13 (F); n = 6, 20, 7 (G); n = 6, 20, 7 (H); n = 5, 12, 12 (I); n = 19, 21, 25, 19 (K); and n = 19, 19, 15, 25 (L). Data represent mean ± SD.