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Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis. Mitochondria are found within virtually all of our body’s cells and are best known as their power plants. Damaged mitochondria cause many diseases in humans – from rare, inherited metabolic disorders that cause symptoms including muscle weakness and developmental problems, to age-related diseases such as diabetes and Parkinson’s disease. How does mitochondrial damage lead to such a variety of symptoms and conditions? To answer this question, researchers must understand how cells respond to and compensate for such damage. To mimic mitochondrial failure, Bao et al. reduced the amount of DNA in the mitochondria of human cells and observed that this caused the cells to accumulate more of an amino acid called serine. Further investigation showed that this accumulation comes in part from cells producing more serine, and that a protein called Activating Transcription Factor 4 is responsible for increasing the expression of the genes needed to produce serine in the cells. Bao et al. also found that damaged mitochondria are less able to consume serine to produce a compound called formate, which is a precursor for DNA building blocks. If cells cannot acquire enough extra serine to compensate for this inefficiency, they cannot produce some of the building blocks required to make DNA and other critical compounds in the cell. Supplementing the cells with formate or the DNA building blocks enabled the cells to recover, which suggests that formate supplements may help to treat some mitochondrial disorders. At a higher level, these results suggest that the mitochondrion’s role as a major chemical factory in the cell, and not just as the power plant, may also contribute to disease when the mitochondria are broken. Further work is now needed to investigate how cells know to turn on Activating Transcription Factor 4 when their mitochondria are damaged. It also remains to be discovered whether this reduces or exacerbates the symptoms of mitochondrial disease.

Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability. Genetic or small-molecule activation of the hypoxia response is protective against mitochondrial toxicity in cultured cells and zebrafish models. Chronic hypoxia leads to a marked improvement in survival, body weight, body temperature, behavior, neuropathology, and disease biomarkers in a genetic mouse model of Leigh syndrome, the most common pediatric manifestation of mitochondrial disease. Further preclinical studies are required to assess whether hypoxic exposure can be developed into a safe and effective treatment for human diseases associated with mitochondrial dysfunction.

Hepatic macrophages are key components of the liver immunity and consist of two main populations. Liver resident macrophages, known as Kupffer cells in mammals, are crucial for maintaining normal liver homeostasis. Upon injury, they become activated to release proinflammatory cytokines and chemokines and recruit a large population of inflammatory monocyte-derived macrophages to the liver. During the progression of liver diseases, macrophages are highly plastic and have opposing functions depending on the signaling cues that they receive from the microenvironment. A comprehensive understanding of liver macrophages is essential for developing therapeutic interventions that target these cells in acute and chronic liver diseases. Mouse studies have provided the bulk of our current knowledge of liver macrophages. The emergence of various liver disease models and availability of transgenic tools to visualize and manipulate macrophages have made the teleost zebrafish ( ) an attractive new vertebrate model to study liver macrophages. In this review, we summarize the origin and behaviors of macrophages in healthy and injured livers in zebrafish. We highlight the roles of macrophages in zebrafish models of alcoholic and non-alcoholic liver diseases, hepatocellular carcinoma, and liver regeneration, and how they compare with the roles that have been described in mammals. We also discuss the advantages and challenges of using zebrafish to study liver macrophages.

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. The cells of nearly all organisms need iron as this metal plays an important role in a wide range of biological processes. However, iron can also trigger the formation of harmful molecules that can damage cells. It is therefore crucial that the amount of iron in cells is tightly controlled and that any extra iron is safely stored away. Most of the iron in the body is stored within a protein called ferritin, which is then broken down to release iron as it is needed, in a process known as ferritinophagy. Cells use several systems to break down proteins, one of which, called autophagy, has been linked to ferritinophagy. During autophagy, a bubble-like structure called an autophagosome engulfs proteins that need to be removed and delivers them to a compartment in the cell where they can be broken down. In 2014, researchers showed that a protein called NCOA4 on the surface of autophagosomes targets ferritin for destruction. When iron levels are high in the cell, the amount of NCOA4 on the autophagosomes is low. This leads to fewer ferritin molecules being broken down. In contrast, low iron levels lead to an increase of NCOA4 on autophagosomes, which promotes ferritinophagy and increases the amount of iron in the cell. Now, Mancias, Vaites et al—including several of the researchers involved in the 2014 work—investigate the role of NCOA4 in ferritinophagy in more detail. Biochemical experiments revealed that a region of NCOA4 directly interacts with a particular subunit of ferritin and this interaction is necessary to deliver ferritin to autophagosomes. Mancias, Vaites et al. then used laboratory grown-cells to investigate why the amount of NCOA4 changes in response to the amount of iron in the cell. The experiments show the amount of NCOA4 varies depending on whether it interacts with another protein called HERC2, which targets proteins for destruction by a structure called the proteasome. HERC2 only binds to NCOA4 when iron levels are high, which leads to NCOA4 being broken down by the proteasome. When iron levels are low, HERC2 does not interact with NCOA4. The presence of more NCOA4 then leads to more ferritinophagy, and so increases the amount of iron in the cell. Mancias, Vaites et al. also found that red blood cells, which depend highly on iron, do not develop properly in zebrafish that have lower amounts of the NCOA4 protein. Further work is needed to see whether NCOA4 is also important for the development of other cells and tissues.

The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/β-catenin signaling. Interestingly, transcriptional compensation is most prominent in non-proliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation. The liver possesses the ability to regenerate following sudden injury. Here, the authors use single-cell RNA-sequencing and in situ transcriptional analyses to identify a new phase of liver regeneration in mice aimed at maintaining essential functions throughout the regenerative process.

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase ( glul ) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis. Cox et al.  report that Yap induces the expression of glutamine synthetase, thereby elevating glutamine and nitrogen levels for de novo nucleotide synthesis. They show that this promotes hepatomegaly and growth of liver cancer cells in zebrafish.

A high-throughput chemical screen of primary human hepatocytes in combination with machine-learning algorithms for evaluation of imaging data identifies compounds that promote expansion of primary human hepatocytes and maturation from human iPS cells toward an adult-like hepatocyte phenotype. Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo , and human hepatocyte sourcing has limited many fields of research for decades. Here we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. The first class induced functional proliferation of primary human hepatocytes in vitro . The second class enhanced hepatocyte functions and promoted the differentiation of induced pluripotent stem cell–derived hepatocytes toward a more mature phenotype than what was previously obtainable. The identification of these small molecules can help address a major challenge affecting many facets of liver research and may lead to the development of new therapeutics for liver diseases.

The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR-dependent manner.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc–Flt4 signalling. Ddx21 function is essential for Vegfc–Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc–Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation. Hogan and colleagues report that the RNA helicase Ddx21 mediates Vegfc-stimulated lymphangiogenesis during zebrafish development through controlling rDNA transcription and ribosome biogenesis in endothelial cells.

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to ‘segregate’ ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left–right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP–UBXN10 in ciliogenesis. Through proteomics, Harper and colleagues identify proteins interacting with UBXD adaptors for the multifunctional AAA-ATPase VCP and reveal a role for UBXN10 in ciliogenesis.