Loss of ciliary gene Bbs8 results in physiological defects in the retinal pigment epithelium

Primary cilia are sensory organelles vital for developmental and physiological processes. Their dysfunction causes a range of phenotypes including retinopathies. Although primary cilia have been described in the retinal pigment epithelium (RPE), little is known about their contribution to biological processes within this tissue. Ciliary proteins are increasingly being identified in non-ciliary locations and might carry out additional functions, disruption of which possibly contributes to pathology. The RPE is essential for maintaining photoreceptor cells and visual function. We demonstrate that upon loss of Bbs8, predominantly thought to be a ciliary gene, the RPE shows changes in gene and protein expression initially involved in signaling pathways and developmental processes, and at a later time point RPE homeostasis and function. Differentially regulated molecules affecting the cytoskeleton and cellular adhesion, led to defective cellular polarization and morphology associated with a possible epithelial-to-mesenchymal transition (EMT)-like phenotype. Our data highlights the benefit of combinatorial “omics” approaches with in vivo data for investigating the function of ciliopathy proteins. It also emphasizes the importance of ciliary proteins in the RPE and their contribution to visual disorders, which must be considered when designing treatment strategies for retinal degeneration

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins

A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes

BACKGROUND: Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21. RESULTS: To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis. CONCLUSIONS: We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders

Transcription Factor EB Controls Metabolic Flexibility during Exercise

The transcription factor EB (TFEB) is an essential component of lysosomal biogenesis and autophagy for the adaptive response to food deprivation. To address the physiological function of TFEB in skeletal muscle, we have used muscle-specific gain- and loss-of-function approaches. Here, we show that TFEB controls metabolic flexibility in muscle during exercise and that this action is independent of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Indeed, TFEB translocates into the myonuclei during physical activity and regulates glucose uptake and glycogen content by controlling expression of glucose transporters, glycolytic enzymes, and pathways related to glucose homeostasis. In addition, TFEB induces the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation. This coordinated action optimizes mitochondrial substrate utilization, thus enhancing ATP production and exercise capacity. These findings identify TFEB as a critical mediator of the beneficial effects of exercise on metabolism

Role of uL3 in the Crosstalk between Nucleolar Stress and Autophagy in Colon Cancer Cells

The nucleolus is the site of ribosome biogenesis and has been recently described as important sensor for a variety of cellular stressors. In the last two decades, it has been largely demonstrated that many chemotherapeutics act by inhibiting early or late rRNA processing steps with consequent alteration of ribosome biogenesis and activation of nucleolar stress response. The overall result is cell cycle arrest and/or apoptotic cell death of cancer cells. Our previously data demonstrated that ribosomal protein uL3 is a key sensor of nucleolar stress activated by common chemotherapeutic agents in cancer cells lacking p53. We have also demonstrated that uL3 status is associated to chemoresistance; down-regulation of uL3 makes some chemotherapeutic drugs ineffective. Here, we demonstrate that in colon cancer cells, the uL3 status affects rRNA synthesis and processing with consequent activation of uL3-mediated nucleolar stress pathway. Transcriptome analysis of HCT 116p53−/− cells expressing uL3 and of a cell sub line stably depleted of uL3 treated with Actinomycin D suggests a new extra-ribosomal role of uL3 in the regulation of autophagic process. By using confocal microscopy and Western blotting experiments, we demonstrated that uL3 acts as inhibitory factor of autophagic process; the absence of uL3 is associated to increase of autophagic flux and to chemoresistance. Furthermore, experiments conducted in presence of chloroquine, a known inhibitor of autophagy, indicate a role of uL3 in chloroquine-mediated inhibition of autophagy. On the basis of these results and our previous findings, we hypothesize that the absence of uL3 in cancer cells might inhibit cancer cell response to drug treatment through the activation of cytoprotective autophagy. The restoration of uL3 could enhance the activity of many drugs thanks to its pro-apoptotic and anti-autophagic activity

A gene toolbox for monitoring autophagy transcription

Autophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the "induction list" by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps

Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure

BACKGROUND & AIMS: Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. METHODS: Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. RESULTS: Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. CONCLUSION: PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. LAY SUMMARY: Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes