Sox9 Is a Modifier of the Liver Disease Severity in a Mouse Model of Alagille Syndrome.

BACKGROUND AND AIMS: Alagille syndrome (ALGS) is a multisystem developmental disorder characterized by bile duct (BD) paucity, caused primarily by haploinsufficiency of the Notch ligand jagged1. The course of the liver disease is highly variable in ALGS. However, the genetic basis for ALGS phenotypic variability is unknown. Previous studies have reported decreased expression of the transcription factor SOX9 (sex determining region Y-box 9) in late embryonic and neonatal livers of Jag1-deficient mice. Here, we investigated the effects of altering the Sox9 gene dosage on the severity of liver disease in an ALGS mouse model. APPROACH AND RESULTS: Conditional removal of one copy of Sox9 in Jag1+/- livers impairs the biliary commitment of cholangiocytes and enhances the inflammatory reaction and liver fibrosis. Loss of both copies of Sox9 in Jag1+/- livers further worsens the phenotypes and results in partial lethality. Ink injection experiments reveal impaired biliary tree formation in the periphery of P30 Jag1+/- livers, which is improved by 5 months of age. Sox9 heterozygosity worsens the P30 biliary tree phenotype and impairs the partial recovery in 5-month-old animals. Notably, Sox9 overexpression improves BD paucity and liver phenotypes in Jag1+/- mice without ectopic hepatocyte-to-cholangiocyte transdifferentiation or long-term liver abnormalities. Notch2 expression in the liver is increased following Sox9 overexpression, and SOX9 binds the Notch2 regulatory region in the liver. Histological analysis shows a correlation between the level and pattern of SOX9 expression in the liver and outcome of the liver disease in patients with ALGS. CONCLUSIONS: Our results establish Sox9 as a dosage-sensitive modifier of Jag1+/- liver phenotypes with a permissive role in biliary development. Our data further suggest that liver-specific increase in SOX9 levels is a potential therapeutic approach for BD paucity in ALGS.

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action.

Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected under standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism, whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and ß-lactamase assays) and the inner membrane (Escherichia coli ß-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine-azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coli-derived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine-azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine-azithromycin synergy, we screened a polyamine analogue library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

NGLY1 Deficiency, a Congenital Disorder of Deglycosylation: From Disease Gene Function to Pathophysiology.

N-Glycanase 1 (NGLY1) is a cytosolic enzyme involved in removing N-linked glycans of misfolded N-glycoproteins and is considered to be a component of endoplasmic reticulum-associated degradation (ERAD). The 2012 identification of recessive NGLY1 mutations in a rare multisystem disorder has led to intense research efforts on the roles of NGLY1 in animal development and physiology, as well as the pathophysiology of NGLY1 deficiency. Here, we present a review of the NGLY1-deficient patient phenotypes, along with insights into the function of this gene from studies in rodent and invertebrate animal models, as well as cell culture and biochemical experiments. We will discuss critical processes affected by the loss of NGLY1, including proteasome bounce-back response, mitochondrial function and homeostasis, and bone morphogenetic protein (BMP) signaling. We will also cover the biologically relevant targets of NGLY1 and the genetic modifiers of NGLY1 deficiency phenotypes in animal models. Together, these discoveries and disease models have provided a number of avenues for preclinical testing of potential therapeutic approaches for this disease.

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Determining Bile Duct Density in the Mouse Liver.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

The Roles of Notch Signaling in Liver Development and Disease.

The Notch signaling pathway plays major roles in organ development across animal species. In the mammalian liver, Notch has been found critical in development, regeneration and disease. In this review, we highlight the major advances in our understanding of the role of Notch activity in proper liver development and function. Specifically, we discuss the latest discoveries on how Notch, in conjunction with other signaling pathways, aids in proper liver development, regeneration and repair. In addition, we review the latest in the role of Notch signaling in the pathogenesis of liver fibrosis and chronic liver disease. Finally, recent evidence has shed light on the emerging connection between Notch signaling and glucose and lipid metabolism. We hope that highlighting the major advances in the roles of Notch signaling in the liver will stimulate further research in this exciting field and generate additional ideas for therapeutic manipulation of the Notch pathway in liver diseases.