Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration.

Wnt signalling induces a gradient of stem/progenitor cell proliferation along the crypt-villus axis of the intestine, which becomes expanded during intestinal regeneration or tumour formation. The YAP transcriptional co-activator is known to be required for intestinal regeneration, but its mode of regulation remains controversial. Here we show that the YAP-TEAD transcription factor is a key downstream effector of Wnt signalling in the intestine. Loss of YAP activity by Yap/Taz conditional knockout results in sensitivity of crypt stem cells to apoptosis and reduced cell proliferation during regeneration. Gain of YAP activity by Lats1/2 conditional knockout is sufficient to drive a crypt hyperproliferation response. In particular, Wnt signalling acts transcriptionally to induce YAP and TEAD1/2/4 expression. YAP normally localises to the nucleus only in crypt base stem cells, but becomes nuclear in most intestinal epithelial cells during intestinal regeneration after irradiation, or during organoid growth, in a Src family kinase-dependent manner. YAP-driven crypt expansion during regeneration involves an elongation and flattening of the Wnt signalling gradient. Thus, Wnt and Src-YAP signals cooperate to drive intestinal regeneration.

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor.

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/ß-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.

Hallmarks of intestinal stem cells.

Intestinal stem cells (ISCs) are highly proliferative cells that fuel the continuous renewal of the intestinal epithelium. Understanding their regulatory mechanisms during tissue homeostasis is key to delineating their roles in development and regeneration, as well as diseases such as bowel cancer and inflammatory bowel disease. Previous studies of ISCs focused mainly on the position of these cells along the intestinal crypt and their capacity for multipotency. However, evidence increasingly suggests that ISCs also exist in distinct cellular states, which can be an acquired rather than a hardwired intrinsic property. In this Review, we summarise the recent findings into how ISC identity can be defined by proliferation state, signalling crosstalk, epigenetics and metabolism, and propose an update on the hallmarks of ISCs. We further discuss how these properties contribute to intestinal development and the dynamics of injury-induced regeneration.

STARD1 promotes NASH-driven HCC by sustaining the generation of bile acids through the alternative mitochondrial pathway.

BACKGROUND & AIMS: Besides their physiological role in bile formation and fat digestion, bile acids (BAs) synthesised from cholesterol in hepatocytes act as signalling molecules that modulate hepatocellular carcinoma (HCC). Trafficking of cholesterol to mitochondria through steroidogenic acute regulatory protein 1 (STARD1) is the rate-limiting step in the alternative pathway of BA generation, the physiological relevance of which is not well understood. Moreover, the specific contribution of the STARD1-dependent BA synthesis pathway to HCC has not been previously explored. METHODS: STARD1 expression was analyzed in a cohort of human non-alcoholic steatohepatitis (NASH)-derived HCC specimens. Experimental NASH-driven HCC models included MUP-uPA mice fed a high-fat high-cholesterol (HFHC) diet and diethylnitrosamine (DEN) treatment in wild-type (WT) mice fed a HFHC diet. Molecular species of BAs and oxysterols were analyzed by mass spectrometry. Effects of NASH-derived BA profiles were investigated in tumour-initiated stem-like cells (TICs) and primary mouse hepatocytes (PMHs). RESULTS: Patients with NASH-associated HCC exhibited increased hepatic expression of STARD1 and an enhanced BA pool. Using NASH-driven HCC models, STARD1 overexpression in WT mice increased liver tumour multiplicity, whereas hepatocyte-specific STARD1 deletion (Stard1ΔHep) in WT or MUP-uPA mice reduced tumour burden. These findings mirrored the levels of unconjugated primary BAs, ß-muricholic acid and cholic acid, and their tauroconjugates in STARD1-overexpressing and Stard1ΔHep mice. Incubation of TICs or PMHs with a mix of BAs mimicking this profile stimulated expression of genes involved in pluripotency, stemness and inflammation. CONCLUSIONS: The study reveals a previously unrecognised role of STARD1 in HCC pathogenesis, wherein it promotes the synthesis of primary BAs through the mitochondrial pathway, the products of which act in TICs to stimulate self-renewal, stemness and inflammation. LAY SUMMARY: Effective therapy for hepatocellular carcinoma (HCC) is limited because of our incomplete understanding of its pathogenesis. The contribution of the alternative pathway of bile acid (BA) synthesis to HCC development is unknown. We uncover a key role for steroidogenic acute regulatory protein 1 (STARD1) in non-alcoholic steatohepatitis-driven HCC, wherein it stimulates the generation of BAs in the mitochondrial acidic pathway, the products of which stimulate hepatocyte pluripotency and self-renewal, as well as inflammation.

The Transcription Co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation Into Enterocyte vs Secretory Lineages.

BACKGROUND & AIMS: Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established. METHODS: We sorted cells from Lgr5-GFP knockin intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription polymerase chain reaction and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing (RNA-seq). We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16. RESULTS: The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt-villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode Delta-like canonical Notch ligand and other secretory-specific transcription factors. CONCLUSIONS: In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1.

Endoplasmic Reticulum Stress-Induced Upregulation of STARD1 Promotes Acetaminophen-Induced Acute Liver Failure.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose is a major cause of acute liver failure (ALF). Mitochondrial SH3BP5 (also called SAB) and phosphorylation of c-Jun N-terminal kinase (JNK) mediate the hepatotoxic effects of APAP. We investigated the involvement of steroidogenic acute regulatory protein (STARD1), a mitochondrial cholesterol transporter, in this process and sensitization by valproic acid (VPA), which depletes glutathione and stimulates steroidogenesis. METHODS: Nonfasted C57BL/6J mice (control) and mice with liver-specific deletion of STARD1 (Stard1ΔHep), SAB (SabΔHep), or JNK1 and JNK2 (Jnk1+2ΔHep) were given VPA with or without APAP. Liver tissues were collected and analyzed by histology and immunohistochemistry and for APAP metabolism, endoplasmic reticulum (ER) stress, and mitochondrial function. Adult human hepatocytes were transplanted into Fah-/-/Rag2-/-/Il2rg-/-/NOD (FRGN) mice to create mice with humanized livers. RESULTS: Administration of VPA before administration of APAP increased the severity of liver damage in control mice. The combination of VPA and APAP increased expression of CYP2E1, formation of NAPQI-protein adducts, and depletion of glutathione from liver tissues of control mice, resulting in ER stress and the upregulation of STARD1. Livers from control mice given VPA and APAP accumulated cholesterol in the mitochondria and had sustained mitochondrial depletion of glutathione and mitochondrial dysfunction. Inhibition of ER stress, by administration of tauroursodeoxycholic acid to control mice, prevented upregulation of STARD1 in liver and protected the mice from hepatoxicity following administration of VPA and APAP. Administration of N-acetylcysteine to control mice prevented VPA- and APAP-induced ER stress and liver injury. Stard1ΔHep mice were resistant to induction of ALF by VPA and APAP, despite increased mitochondrial levels of glutathione and phosphorylated JNK; we made similar observations in fasted Stard1ΔHep mice given APAP alone. SabΔHep mice or Jnk1+2ΔHep mice did not develop ALF following administration of VPA and APAP. The ability of VPA to increase the severity of APAP-induced liver damage was observed in FRGN mice with humanized liver. CONCLUSIONS: In studies of mice, we found that upregulation of STARD1 following ER stress mediates APAP hepatoxicity via SH3BP5 and phosphorylation of JNK1 and JNK2.

Zinc mitigates renal ischemia-reperfusion injury in rats by modulating oxidative stress, endoplasmic reticulum stress, and autophagy.

Oxidative stress is a major factor involved in the pathogenesis of renal ischemia/reperfusion (I/R). Exogenous zinc (Zn) was suggested as a potent antioxidant; however, the mechanism by which it strengthens the organ resistance against the effects of reactive oxygen species (ROS) is not yet investigated. The present study aims to determine whether acute zinc chloride (ZnCl2 ) administration could attenuate endoplasmic reticulum (ER) stress, autophagy, and inflammation after renal I/R. Rats were subjected to either sham operation (Sham group, n = 6), or 1 hr of bilateral ischemia followed by 2 hr of reperfusion (I/R groups, n = 6), or they received ZnCl2 orally 24 hr and 30 min before ischemia (ZnCl2 group, n = 6). Rats were subjected to 1 hr of bilateral renal ischemia followed by 2 hr of reperfusion (I/R group, n = 6). Our results showed that ZnCl2 enhances renal function and reduces cytolysis (p < 0,05). In addition, it increased significantly the activities of antioxidant enzymes (SOD, CAT, and GPX) and the level of GSH in comparison to I/R (p < 0,05). Interestingly, ZnCl2 treatment resulted in significant decreased ER stress, as reflected by GRP78, ATF-6,p-eIF-2α, XPB-1, and CHOP downregulaion. Rats undergoing ZnCl2 treatment demonstrated a low expression of autophagy parameters (Beclin-1 and LAMP-2), which was correlated with low induction of apoptosis (caspase-9, caspase-3, and p-JNK), and reduction of inflammation (IL-1ß, IL-6, and MCP-1) (p < 0,05). In conclusion, we demonstrated the potential effect of Zn supplementation to modulate ER pathway and autophagic process after I/R.

Mitochondrial Cholesterol and the Paradox in Cell Death.

Mitochondria are considered cholesterol-poor organelles, and obtain their cholesterol load by the action of specialized proteins involved in its delivery from extramitochondrial sources and trafficking within mitochondrial membranes. Although mitochondrial cholesterol fulfills vital physiological functions, such as the synthesis of bile acids in the liver or the formation of steroid hormones in specialized tissues, recent evidence indicates that the accumulation of cholesterol in mitochondria may be a key event in prevalent human diseases, in particular in the development of steatohepatitis (SH) and its progression to hepatocellular carcinoma (HCC). Mitochondrial cholesterol accumulation promotes the transition from simple steatosis to SH due to the sensitization to oxidative stress and cell death. However, mitochondrial cholesterol loading in HCC determines apoptosis resistance and insensitivity to chemotherapy. These opposing functions of mitochondrial cholesterol in SH and HCC define its paradoxical role in cell death as a pro- and anti-apoptotic factor. Further understanding of this conundrum may be useful to modulate the progression from SH to HCC by targeting mitochondrial cholesterol trafficking.

Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy.

Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor-acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nmol/L to 50 µmol/L) in the presence of melatonin (1 and 2 mmol/L) was assessed in HCC cell lines HepG2, HuH7, and Hep3B. Cell viability was reduced by sorafenib from 1 µmol/L in HepG2 or HuH7 cells, and 2.5 µmol/L in Hep3B cells. Co-administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co-administration of 2.5 µmol/L sorafenib and 1 mmol/L melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin-2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage, and BAX expression. These results demonstrate that the pro-oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib.

Endoplasmic reticulum stress mediates amyloid ß neurotoxicity via mitochondrial cholesterol trafficking.

Disrupted cholesterol homeostasis has been reported in Alzheimer disease and is thought to contribute to disease progression by promoting amyloid ß (Aß) accumulation. In particular, mitochondrial cholesterol enrichment has been shown to sensitize to Aß-induced neurotoxicity. However, the molecular mechanisms responsible for the increased cholesterol levels and its trafficking to mitochondria in Alzheimer disease remain poorly understood. Here, we show that endoplasmic reticulum (ER) stress triggered by Aß promotes cholesterol synthesis and mitochondrial cholesterol influx, resulting in mitochondrial glutathione (mGSH) depletion in older age amyloid precursor protein/presenilin-1 (APP/PS1) mice. Mitochondrial cholesterol accumulation was associated with increased expression of mitochondrial-associated ER membrane proteins, which favor cholesterol translocation from ER to mitochondria along with specific cholesterol carriers, particularly the steroidogenic acute regulatory protein. In vivo treatment with the ER stress inhibitor 4-phenylbutyric acid prevented mitochondrial cholesterol loading and mGSH depletion, thereby protecting APP/PS1 mice against Aß-induced neurotoxicity. Similar protection was observed with GSH ethyl ester administration, which replenishes mGSH without affecting the unfolded protein response, thus positioning mGSH depletion downstream of ER stress. Overall, these results indicate that Aß-mediated ER stress and increased mitochondrial cholesterol trafficking contribute to the pathologic progression observed in old APP/PS1 mice, and that ER stress inhibitors may be explored as therapeutic agents for Alzheimer disease.

ASMase regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH. METHODS: Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase(-/-) mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD. RESULTS: ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase(-/-) mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase(-/-) mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase(-/-) mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase(+/+) mice but not in ASMase(-/-) mice. ASMase(-/-) PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. CONCLUSIONS: These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH.

Loss of ARID3A perturbs intestinal epithelial proliferation-differentiation ratio and regeneration.

Intestinal stem cells at the crypt divide and give rise to progenitor cells that proliferate and differentiate into various mature cell types in the transit-amplifying (TA) zone. Here, we showed that the transcription factor ARID3A regulates intestinal epithelial cell proliferation and differentiation at the TA progenitors. ARID3A forms an expression gradient from the villus tip to the upper crypt mediated by TGF-ß and WNT. Intestinal-specific deletion of Arid3a reduces crypt proliferation, predominantly in TA cells. Bulk and single-cell transcriptomic analysis shows increased enterocyte and reduced secretory differentiation in the Arid3a cKO intestine, accompanied by enriched upper-villus gene signatures of both cell lineages. We find that the enhanced epithelial differentiation in the Arid3a-deficient intestine is caused by increased binding and transcription of HNF1 and HNF4. Finally, we show that loss of Arid3a impairs irradiation-induced regeneration with sustained cell death and reprogramming. Our findings imply that Arid3a functions to fine-tune the proliferation-differentiation dynamics at the TA progenitors, which are essential for injury-induced regeneration.

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading.

BACKGROUND & AIMS: The pathogenesis of alcohol-induced liver disease (ALD) is poorly understood. Here, we examined the role of acid sphingomyelinase (ASMase) in alcohol induced hepatic endoplasmic reticulum (ER) stress, a key mechanism of ALD. METHODS: We examined ER stress, lipogenesis, hyperhomocysteinemia, mitochondrial cholesterol (mChol) trafficking and susceptibility to LPS and concanavalin-A in ASMase(-)(/-) mice fed alcohol. RESULTS: Alcohol feeding increased SREBP-1c, DGAT-2, and FAS mRNA in ASMase(+/+) but not in ASMase(-/-) mice. Compared to ASMase(+/+) mice, ASMase(-/-) mice exhibited decreased expression of ER stress markers induced by alcohol, but the level of tunicamycin-mediated upregulation of ER stress markers and steatosis was similar in both types of mice. The increase in homocysteine levels induced by alcohol feeding was comparable in both ASMase(+/+) and ASMase(-/-) mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca(2+) homeostasis. Moreover, alcohol-induced mChol loading and StARD1 overexpression were blunted in ASMase(-/-) mice. Tunicamycin upregulated StARD1 expression and this outcome was abrogated by tauroursodeoxycholic acid. Alcohol-induced liver injury and sensitization to LPS and concanavalin-A were prevented in ASMase(-/-) mice. These effects were reproduced in alcohol-fed TNFR1/R2(-/-) mice. Moreover, ASMase does not impair hepatic regeneration following partial hepatectomy. Of relevance, liver samples from patients with alcoholic hepatitis exhibited increased expression of ASMase, StARD1, and ER stress markers. CONCLUSIONS: Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.

USP7 inactivation suppresses APC-mutant intestinal hyperproliferation and tumor development.

Adenomatous polyposis coli (APC) mutation is the hallmark of colorectal cancer (CRC), resulting in constitutive WNT activation. Despite decades of research, targeting WNT signaling in cancer remains challenging due to its on-target toxicity. We have previously shown that the deubiquitinating enzyme USP7 is a tumor-specific WNT activator in APC-truncated cells by deubiquitinating and stabilizing ß-catenin, but its role in gut tumorigenesis is unknown. Here, we show in vivo that deletion of Usp7 in Apc-truncated mice inhibits crypt hyperproliferation and intestinal tumor development. Loss of Usp7 prolongs the survival of the sporadic intestinal tumor model. Genetic deletion, but not pharmacological inhibition, of Usp7 in Apc+/- intestine induces colitis and enteritis. USP7 inhibitor treatment suppresses growth of patient-derived cancer organoids carrying APC truncations in vitro and in xenografts. Our findings provide direct evidence that USP7 inhibition may offer a safe and efficacious tumor-specific therapy for both sporadic and germline APC-mutated CRC.

Engineering transplantable jejunal mucosal grafts using patient-derived organoids from children with intestinal failure.

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.

Mitochondrial-Lysosomal Axis in Acetaminophen Hepatotoxicity.

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure and a major indication for liver transplantion in the United States and Europe. Although significant progress has been made in understanding the molecular mechanisms underlying APAP hepatotoxicity, there is still an urgent need to find novel and effective therapies against APAP-induced acute liver failure. Hepatic APAP metabolism results in the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which under physiological conditions is cleared by its conjugation with glutathione (GSH) to prevent its targeting to mitochondria. APAP overdose or GSH limitation leads to mitochondrial NAPQI-protein adducts formation, resulting in oxidative stress, mitochondrial dysfunction, and necrotic cell death. As mitochondria are a major target of APAP hepatotoxicity, mitochondrial quality control and clearance of dysfunctional mitochondria through mitophagy, emerges as an important strategy to limit oxidative stress and the engagement of molecular events leading to cell death. Recent evidence has indicated a lysosomal-mitochondrial cross-talk that regulates APAP hepatotoxicity. Moreover, as lysosomal function is essential for mitophagy, impairment in the fusion of lysosomes with autophagosomes-containing mitochondria may compromise the clearance of dysfunctional mitochondria, resulting in exacerbated APAP hepatotoxicity. This review centers on the role of mitochondria in APAP hepatotoxicity and how the mitochondrial/lysosomal axis can influence APAP-induced liver failure.

The effect of zinc acexamate on oxidative stress, inflammation and mitochondria induced apoptosis in rat model of renal warm ischemia.

AIM: Zinc has proved its efficacy in many models of ischemia reperfusion (I/R) injury. In this study, we used zinc acexamate (ZAC) as an exogenous source of zinc against renal I/R injury and we investigated whether its protective effects are mediated by the decrease of oxidative stress, inflammation, and mitochondria induced-apoptosis. METHODS: Rats were orally pretreated with vehicle or ZAC (10 or 100 mg/kg) 24 h and 30 min prior to 1 h of bilateral renal warm ischemia and 2 h of reperfusion. RESULTS: Our data showed that 10 mg/kg of ZAC, but not 100 mg/kg, improved renal architecture and function. Also, the low dose of ZAC up-regulated antioxidant enzymes activities and glutathione level and decreased lipids and proteins oxidation. Interestingly, the use of ZAC resulted in a significant reduce of pro-inflammatory cytokines (IL-1ß, IL-6 and MCP-1), enhanced mitochondria integrity and decreased expression of the pro-apoptotic protein caspase-9. CONCLUSION: We conclude that renal I/R induced oxidative stress, inflammation and apoptosis and that the use of ZAC at 10 mg/kg, but not 100 mg/kg, protects rat kidneys from I/R injury by down-regulating these processes.

The 2-oxoglutarate carrier promotes liver cancer by sustaining mitochondrial GSH despite cholesterol loading.

Cancer cells exhibit mitochondrial cholesterol (mt-cholesterol) accumulation, which contributes to cell death resistance by antagonizing mitochondrial outer membrane (MOM) permeabilization. Hepatocellular mt-cholesterol loading, however, promotes steatohepatitis, an advanced stage of chronic liver disease that precedes hepatocellular carcinoma (HCC), by depleting mitochondrial GSH (mGSH) due to a cholesterol-mediated impairment in mGSH transport. Whether and how HCC cells overcome the restriction of mGSH transport imposed by mt-cholesterol loading to support mGSH uptake remains unknown. Although the transport of mGSH is not fully understood, SLC25A10 (dicarboxylate carrier, DIC) and SLC25A11 (2-oxoglutarate carrier, OGC) have been involved in mGSH transport, and therefore we examined their expression and role in HCC. Unexpectedly, HCC cells and liver explants from patients with HCC exhibit divergent expression of these mitochondrial carriers, with selective OGC upregulation, which contributes to mGSH maintenance. OGC but not DIC downregulation by siRNA depleted mGSH levels and sensitized HCC cells to hypoxia-induced ROS generation and cell death as well as impaired cell growth in three-dimensional multicellular HCC spheroids, effects that were reversible upon mGSH replenishment by GSH ethyl ester, a membrane permeable GSH precursor. We also show that OGC regulates mitochondrial respiration and glycolysis. Moreover, OGC silencing promoted hypoxia-induced cardiolipin peroxidation, which reversed the inhibition of cholesterol on the permeabilization of MOM-like liposomes induced by Bax or Bak. Genetic OGC knockdown reduced the ability of tumor-initiating stem-like cells to induce liver cancer. These findings underscore the selective overexpression of OGC as an adaptive mechanism of HCC to provide adequate mGSH levels in the face of mt-cholesterol loading and suggest that OGC may be a novel therapeutic target for HCC treatment.

Intestinal Stem Cell Niche: The Extracellular Matrix and Cellular Components.

The intestinal epithelium comprises a monolayer of polarised columnar cells organised along the crypt-villus axis. Intestinal stem cells reside at the base of crypts and are constantly nourished by their surrounding niche for maintenance, self-renewal, and differentiation. The cellular microenvironment including the adjacent Paneth cells, stromal cells, smooth muscle cells, and neural cells as well as the extracellular matrix together constitute the intestinal stem cell niche. A dynamic regulatory network exists among the epithelium, stromal cells, and the matrix via complex signal transduction to maintain tissue homeostasis. Dysregulation of these biological or mechanical signals could potentially lead to intestinal injury and disease. In this review, we discuss the role of different intestinal stem cell niche components and dissect the interaction between dynamic matrix factors and regulatory signalling during intestinal stem cell homeostasis.

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease.

Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.