Background and aimsThe recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.MethodsThe histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.ResultsThe criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively) and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively). The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively) and very high for the analysis of inflammation (ICC = 0.931, p<0.001).ConclusionsWe established a simple NAFLD scoring system with high reproducibility that is applicable for different rodent models and for all stages of NAFLD etiology.

The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

Autosomal recessively inherited glucocerebrosidase 1 (GBA1) mutations cause the lysosomal storage disorder Gaucher's disease (GD). Heterozygous GBA1 mutations (GBA1+/−) are the most common risk factor for Parkinson's disease (PD). Previous studies typically focused on the interaction between the reduction of glucocerebrosidase (enzymatic) activity in GBA1+/− carriers and alpha-synuclein-mediated neurotoxicity. However, it is unclear whether other mechanisms also contribute to the increased risk of PD in GBA1+/− carriers. The zebrafish genome does not contain alpha-synuclein (SNCA), thus providing a unique opportunity to study pathogenic mechanisms unrelated to alpha-synuclein toxicity. Here we describe a mutant zebrafish line created by TALEN genome editing carrying a 23 bp deletion in gba1 (gba1c.1276_1298del), the zebrafish orthologue of human GBA1. Marked sphingolipid accumulation was already detected at 5 days post-fertilization with accompanying microglial activation and early, sustained up-regulation of miR-155, a master regulator of inflammation. gba1c.1276_1298del mutant zebrafish developed a rapidly worsening phenotype from 8 weeks onwards with striking reduction in motor activity by 12 weeks. Histopathologically, we observed marked Gaucher cell invasion of the brain and other organs. Dopaminergic neuronal cell count was normal through development but reduced by >30% at 12 weeks in the presence of ubiquitin-positive, intra-neuronal inclusions. This gba1c.1276_1298del zebrafish line is the first viable vertebrate model sharing key pathological features of GD in both neuronal and non-neuronal tissue. Our study also provides evidence for early microglial activation prior to alpha-synuclein-independent neuronal cell death in GBA1 deficiency and suggests upregulation of miR-155 as a common denominator across different neurodegenerative disorders.

Concerns have been raised about whether preclinical models sufficiently mimic molecular disease processes observed in nonalcoholic steatohepatitis (NASH) patients, bringing into question their translational value in studies of therapeutic interventions in the process of NASH/fibrosis. We investigated the representation of molecular disease patterns characteristic for human NASH in high‐fat diet (HFD)‐fed Ldlr‐/‐.Leiden mice and studied the effects of obeticholic acid (OCA) on these disease profiles. Multiplatform serum metabolomic profiles and genome‐wide liver transcriptome from HFD‐fed Ldlr‐/‐.Leiden mice were compared with those of NASH patients. Mice were profiled at the stage of mild (24 weeks HFD) and severe (34 weeks HFD) fibrosis, and after OCA intervention (24‐34 weeks; 10 mg/kg/day). Effects of OCA were analyzed histologically, biochemically, by immunohistochemistry, using deuterated water technology (de novo collagen formation), and by its effect on the human‐based transcriptomics and metabolomics signatures. The transcriptomics and metabolomics profile of Ldlr‐/‐.Leiden mice largely reflected the molecular signature of NASH patients. OCA modulated the expression of these molecular profiles and quenched specific proinflammatory‐profibrotic pathways. OCA attenuated specific facets of cellular inflammation in liver (F4/80‐positive cells) and reduced crown‐like structures in adipose tissue. OCA reduced de novo collagen formation and attenuated further progression of liver fibrosis, but did not reduce fibrosis below the level before intervention. Conclusion: HFD‐fed Ldlr‐/‐.Leiden mice recapitulate molecular transcriptomic and metabolomic profiles of NASH patients, and these signatures are modulated by OCA. Intervention with OCA in developing fibrosis reduces collagen deposition and de novo synthesis but does not resolve already manifest fibrosis in the period studied. These data show that human molecular signatures can be used to evaluate the translational character of preclinical models for NASH.

IntroductionGranulocytes and monocytes develop from common myeloid progenitor cells through a complex network of cell growth, differentiation, and apoptosis-regulating factors. Alterations in these processes may cause acute myeloid leukemia, which is characterized by uncontrolled proliferation of immature myeloid cells that fail to differentiate toward mature functional cells. 1,2 In leukemia, mutations occur in genes encoding tyrosine kinases such as Fms-like tyrosine kinase 3 (Flt3), c-kit, neuroblastoma ras (N-ras) and transcription factors such as AML1 (acute myeloid leukemia 1), CBFB (core binding factor ␤), GATA1 (GATA binding protein 1), PU.1, C/EBP␣ (CCAAT/enhancer binding protein ␣), and RAR␣ (retinoic acid receptor ␣). 1,2 Although the transcription factors predominantly play a role in lineage commitment, activation of tyrosine kinases is thought to result in proliferative and/or survival signals. 1,2 Recent studies have shown that several proteins involved in myelopoiesis (including proteins mutated in leukemia) are inactivated through ubiquitin (Ub)-proteasomal degradation pathways. [3][4][5][6][7] This form of targeted protein degradation is accomplished by the covalent conjugation of Ub to substrate proteins, usually in the form of a multi-Ub chain, which marks these proteins for progressive degradation by the 26S proteasome. 8,9 Protein ubiquitination is catalyzed through a cascade of reactions. Ub is first activated by the adenosine triphosphate (ATP)-dependent Ub-activating E1 enzyme and subsequently transferred to one of a set of E2 Ub-conjugating enzymes. The E2 enzymes act in conjunction with accessory E3 Ub protein ligases. In the E2-E3 complex, the E3 component binds to protein substrates, allowing the E2 to form a multi-Ub chain linked to a lysine of the substrate protein. 8,9 Thus, the E3 Ub ligases play a crucial role in this process because these proteins recognize the cellular proteins destined for ubiquitination. 10 To date, several types of E3 Ub ligases have been described. These include HECT (homologous to E6-AP carboxyl terminus), RING (really interesting new gene) finger, U-box, SOCS (suppressor of cytokine signaling)-box, F-box, and cullin ligases. [11][12][13][14][15][16][17][18][19][20] Although the first 3 types of proteins are actively involved in substrate ubiquitination, the last 3 do not ubiquitinate substrate proteins themselves but are part of larger protein complexes that exhibit Ub ligase activity. [15][16][17][18][19] From the Central Hematology Laboratory, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; the Department of Hematology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; the Institute of Hematology, Erasmus University Medical Center Rotterdam, The Netherlands. The online version of the article contains a data supplement.Reprints: Bert A. van der Reijden, Central Hematology Laboratory, University Medical Center Nijmegen, PO BOX 9101, 6500 HB Nijmegen, The Netherlands; e-mail: b.vanderreijden@chl.umcn.nl.The publi...